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cardiovascular disease is the leading cause of mortality in many economically developed nations accounting for about 30% of all deaths and its incidence is still increasing . ongoing research aims to investigate and prevent the early development of cardiovascular risk factors such as atherosclerosis , hypertension , dyslipidemia , chronic inflammation , and insulin resistance . the beneficial effects of n-3 polyunsaturated fatty acids ( n-3 pufas ) were proved in several observational and experimental studies . the lipid lowering action of n3-pufas was detected at the beginning , so these nutrients were used for the treatment of dyslipidemic disorders . their anti - inflammatory , antithrombotic , antiatherosclerotic , and antiarrhythmogenic effects were observed later . low - grade chronic inflammation is now recognized as a prominent process in the development of atherosclerosis and coronary heart disease . the induction of inflammation may well provide a link between hyperlipidemia and atherogenesis [ 2 , 3 ] . atherosclerosis is now considered a systemic disease featured by low - grade arterial inflammatory lesions that can develop through the disease progression . in physiological conditions , endothelial cells synthesize and release adequate amounts of nitric oxide ( no ) and prostaglandins ( such as pge2 and pge3 ) and maintain a downstream balance between pro- and anti - inflammatory molecules . however , in the presence of atherosclerosis this balance disrupts leading towards an increase production of proinflammatory cytokines as interleukins 1 , 2 , and 6 ( il-1 , 2 , and 6 ) and tumor necrosis factor ( tnf- ) , with further progression of the disease . these pro - inflammatory cytokines can induce oxidative stress by enhancing the production of reactive oxygen species ( ros ) by monocytes , macrophages , and leukocytes . pufas and their eicosanoid derivatives may play a significant role modulating the inflammatory response ( figure 1 ) . unsaturated fatty acids are referred to as pufas when two or more double bounds are present . there are two pufas families , omega-3 ( n-3 ) and omega-6 ( n-6 ) fatty acids . they differ in location of the last double bond relative to the terminal methyl end of the molecule . the human body can produce almost all fatty acids , except linoleic acid ( la , c18:2 n-6 , precursor to the n-6 series of fatty acids ) and -linoleic acid ( ala , c18:3n-3 , precursor to the n-3 series of fatty acids ) . these two pufas are named essential fatty acids because the body can not synthesize them . endogenous conversion ( elongation and desaturation ) of the initial c18 pufa precursors results in the synthesis of longer - chain counterparts such as eicosapentaenoic acid ( epa ) and docosahexaenoic acid ( dha ) in the n-3 family and dihomo--linoleic acid ( dgla ) and arachidonic acid ( aa ) in the n-6 family . in humans the biochemical pathways converting ala to epa and epa to dha are limited : 0.28% of ala is converted to epa ( generally more in women ) and 04% of ala to dha [ 812 ] . results from pilot studies suggest that ala conversion is also limited to function as a surrogate for fish consumption . thus , tissue and circulating epa and dha levels are primarily related to their dietary intake . fish is the major food source of long - chain n-3 pufas , including epa , dha , and docosapentaenoic acid ( dpa ) , while ala is a plant n-3 fatty acid mainly found in seeds , nuts , and their oils . thus , plant sources of n-3 fatty acids can not currently be considered as a replacement for seafood - derived n-3 pufas . this suggests that n-3 fatty acids derived from different sources might have their own specific effects on cardiovascular risk markers . linoleic acid is thought to decrease the conversion of ala into epa and dha by competing for the n-6-desaturase enzyme . previous studies showed that genetic variations in this enzyme may be related to cardiovascular disease . furthermore , because the two fatty acid pathways are mutually exclusive ( i.e. , n-3 fatty acid can not become n-6 fatty acid and vice versa ) , a balanced intake of ala and la as precursors or of their longer - chain products epa , dha , and aa is required . there is a vast amount of epidemiologic evidence of a cardioprotective effect of fish oil - derived epa and dha and it was confirmed in randomized controlled trials [ 1720 ] . several intervention studies , such as the gruppo italiano per lo studio della sopravvivenza nell'infarto miocardico ( gissi)-prevenzione trial and the cardiovascular health study , have shown that an increased intake of eicosapentaenoic acid ( epa , c20:5n-3 ) and docosahexaenoic acid ( dha , c22:6n-3 ) lowers the risk of coronary heart disease ( chd ) [ 21 , 22 ] . the first gissi - prevenzione trial , a randomized open label study in 11324 italian patients with recent myocardial infarction , demonstrated that patients had a 15% lower combined risk of mortality , nonfatal myocardial infarction , and stroke upon supplementation for 3.5 years with 850 mgday-1 of lc n-3 pufa . the relative risk of cardiovascular mortality was also decreased by 30% and that of sudden death by 45% . the gissi - prevenzione trial demonstrated that a significant protective effect could be obtained with doses much lower than those previously considered necessary for significant beneficial effects . the second gissi - hf study , a randomized double - blind placebo - controlled trial in 6975 italian patients with chronic heart failure , revealed a moderate decrease in both all - cause mortality admissions to hospital for cardiovascular disease upon supplementation for an average of 3.9 years with 1 g of lc n-3 pufa daily . again , the beneficial effects were seen in a population already treated with recommended therapies . the japan eicosapentaenoic acid ( epa ) lipid intervention study , ( jelis ) trial , was performed on 18645 japanese men and women with hypercholesterolaemia treated with statins . supplementation with 1.8 g epa daily decreased major coronary events by 19% over 4.6 years . this may be evident in japanese population where a consistent reduction of chd is observed probably because of a high background seafood intake [ 25 , 26 ] . in summary , a modest intake of lc however , higher doses and longer duration of intervention , as reported for the jelis study , have the potential to protect from non - fatal chd events . presently , there is evidence to suggest that the two lc n3-pufas epa ( eicosapentaenoic acid , 20:5n3 ) and dha ( docosahexaenoic acid , 22:6n3)exert pleiotropism ( akin to those effects which have been reported for certain cardiovascular drugs ) by modulating a range of diverse target mechanisms involved in cardiovascular disease development . [ 2730 ] . since the appearance of purified forms of dha in the market in the 1990s , researchers have started to investigate the differential effects of epa and dha on cardiovascular health . however , the number of human studies is still limited in this field and the independent effects of epa and dha on various cardiovascular outcomes are yet to be firmly established . furthermore , the incorporation of epa and dha into the cell membrane influences its organisation , fluidity , and permeability , as well as the activity of transmembrane proteins , including receptors , enzymes , and ion channels . both epa and dha modulate k , na , and ca channel activities in myocardial cells , regulating myocyte electrical excitability and contractility [ 3234 ] . these effects are concentration dependent and appear to be mediated by the action of epa and dha on membrane fluidity , although other mechanisms , such as a direct binding of n-3 lcp to the channel , may be involved . furthermore , animal studies support the increasing evidence that dha rather than epa is preferentially incorporated into the myocardial cell membrane . collectively , these findings help to explain the antiarrhythmic and heart rate ( hr)-lowering effects observed by dha but not by epa in humans . in addition , incorporation of dha into the membrane of cardiomyocytes influences the beta adrenergic system to a greater extent than epa , that is , potentially another important mechanism involved in the hypotensive and anti - arrhythmic action of dha . furthermore , dha incorporation into the membrane of endothelial cells stimulates atp release from the endothelium , increasing vasodilation by stimulating nitric oxide ( no ) release . the induction of no release , together with the decrease in noradrenaline levels , may also account for the bp - lowering effect of dha . a recent meta - analysis including thirty randomized controlled trials showed that prolonged fish oil intake may reduce hr , especially in populations with a high baseline hr . in dyslipidemic males and postmenopausal women , this decrease appears to be mediated by dha rather than epa [ 37 , 41 , 42 ] . in contrast , no significant effect of either epa or dha on hr was observed in healthy males for similar dosage and treatment duration . hr variability ( hrv ) is a strong predictor of cvd , including sudden cardiac death , arrhythmic chd , and atrial fibrillation . fish oils have shown anti - arrhythmic properties in animal studies , and several clinical and epidemiological studies have reported an association between hrv and n-3 lcp blood cell levels and/or fish oil intake [ 4446 ] . short - term trials ( 612 week ) in healthy individuals showed no or low effect of a daily intake of 1.23.6 g of ala on blood lipids , ldl - oxidation , lipoprotein ( a ) , and apolipoproteins a - i and b [ 8 , 47 ] . long - term treatment with high - dose ala ( 40 g / daily ) showed a beneficial effect by reduction of body weight and blood ldl / total cholesterol ratio [ 47 , 48 ] . previous evidence favored recommendation for modest dietary consumption of ala ( 2 - 3 g daily ) in the primary and secondary prevention of chd . the recently published case - control study by campos et al . shows a strong inverse associations between ala status - intake and nonfatal mi . recent data support the assumption that ala may protect against atherosclerosis [ 50 , 51 ] . however , data from two recent epidemiologic studies suggest that high tissue ala is related to an increased rather than decreased risk of fatal cardiovascular events and sudden death . also , n-3 pufas may interfere with cvd with other physiological effects : the reduction of triglyceride ( tg ) synthesis is well recognized . moreover , the reduction of hepatic vldl synthesis may contribute to this protective effect reducing the availability of fatty acids for triglyceride synthesis due to the decreased de novo lipogenesis ( dnl ) , increasing the beta - oxidation of fatty acids , reducing the delivery of nonesterified fatty acids to the liver , reducing the activity of tg synthesis by hepatocytes , and finally increasing the hepatic synthesis of phospholipids [ 5459 ] . in both experimental models and human studies , a different effect was reported in some studies between epa and dha : both epa and dha produced a significant reduction of tgs and ldl - cholesterol , raising effects of fish oil [ 61 , 62 ] . recently , buckley and coworkers showed that a four - week supplementation with dha significantly reduced tg levels in normolipidemic human subjects by 22% , while epa decreased tg levels by 15% without reaching statistical significance . in another four - week interventional study , both epa and dha reduced postprandial tg without affecting fasting tg levels in healthy human subjects . however , epa and dha seemed to reduce triglyceridaemia to the same extent when given for a long - enough period [ 60 , 61 , 6772 ] . studies in humans show that an average intake of n-3 pufas 3 - 4 g / daily decreases serum triacylglycerol concentration by 2530% in a dose - dependent manner . the same intake does not affect total cholesterol but increases ldl cholesterol by 510% and hdl cholesterol by 13% . the increase in ldl cholesterol is mainly due to a rise of the larger and the potentially less atherogenic ldl particles . hypertension ( ah ) and dyslipidemia ( dlp ) are the most important and frequent risk factors for cvd in the general population , with a prevalence of 5080% according to different authors [ 7579 ] . on the other hand almost half of the patients with increased total cholesterol ( tc ) have systo - diastolic hypertension [ 8083 ] . modifications of erythrocyte - membrane fatty acids ( fa ) composition are an early indicator of the development of ah and lipid disorders . thus , modifications of erythrocyte - membrane fa are fairly subtle indicators of lipid metabolism pathology which manifest themselves much earlier than changes in plasma lipoproteins . modifications of fatty acids composition of the cell membrane lipid matrix play an important role in the ah pathogenesis . changes of the fatty acids ( fa ) composition with decrease of essential pufas may result in increase of membrane microviscosity , activation of pro - inflammatory eicosanoids synthesis and increased sensitivity of the smooth muscle cells in artery walls to the influence of vasoconstrictors . that is the reason of the great interest in the study of the spectrum of erythrocyte lipids in hypertensive dyslipidemic patients . deficiency of n-3 pufa is a distinctive feature of the modification of erythrocyte - membrane fa in hypertensive dyslipidemic patients . deficiency of endogenous n-3 pufas may lead to changes in physicochemical properties of cell membranes , activation of the synthesis of proinflammatory and vasoconstrictive eicosanoids , and finally induction of a systemic inflammatory syndrome . thus , structural and functional changes in erythrocyte cell membranes develop in hypertension as a part of the common systemic disorder of lipid metabolism . the fa disorganization of the cell membrane may cause the development of hyperlipidemia and the progression of hypertensive disease . thus , an important role of fa in the pathogenesis of cardiovascular disease seems clear . a large part of the cardioprotective actions of lc n-3-pufas is likely to be mediated by the vascular endothelium [ 28 , 8688 ] . this thin monolayer of cells plays a central role in cardiovascular homeostasis and function via the production of a range of potent autocrine and paracrine biochemical mediators that control the tone of vascular smooth muscle cells [ 31 , 89 , 90 ] . vascular endothelium is also the site for the inception , progression , and clinical manifestations of atherosclerosis [ 9193 ] . multifactorial endothelial dysfunction induced by dlp , toxins , cigarette - smoking , and so on may be the early event in the development of atherosclerosis . the endothelium becomes proadhesive and induces an increased adhesion of circulating monocytes that subsequently infiltrate the arterial intima . at this level endothelial cells or macrophages release reactive oxygen species that oxidize circulating ldl into oxidized - ldl and create the lipid streak . in the evolution from the lipid streak to the atherosclerotic plaque , numerous cytokines that cause infiltration of smooth muscle layer by leukocytes and fibroblasts and promote platelet adhesion are involved in turn ( figures 2 and 3 ) . during the migration over the vessel wall , monocytes are exposed to various proinflammatory stimuli that contribute to their differentiation into macrophages . other macrophages induce a local inflammatory compartment within the vassel wall , that is , continuously powered by cytokines , activated t - helper cells and scavanger receptor activators . the inhibition of this self - maintaining process may reduce atherogenesis as demonstrated in mutant mice deficient for macrophage colony stimulating factor ( m - csf ) or macrophage migration inhibitory factor ( mif ) [ 95 , 96 ] . in more advanced lesions , cell debris , apoptotic cells , and free cholesterol particles accumulate in the lipid - rich necrotic core of the plaque , are sometimes separated from the blood only by a thin fibrous cap . the number of inflammatory cells within the lesion is strongly associated with the risk of rupture of the so - called vulnerable plaque . at last , the process may propagate intraluminal thrombosis , thereby distal ischemia of heart or brain tissue [ 97 , 98 ] . through their positive antithrombotic , lipid - lowering , proendothelial functional activities , n-3 in fact supplementation of n-3 pufas improves flow - mediated arterial dilatation by an increased endothelial synthesis of nitric oxide [ 86 , 99111 ] . several , although not all , clinical trials have also found that consumption of n-3 pufas lowers circulating markers of endothelial dysfunction , such as e - selectin , vascular cell adhesion molecule-1 , and intracellular adhesion molecule-1 [ 74 , 112 , 113 ] . thus , normalization of endothelial function could partly mediate the protective effects of n-3 pufa against cvd . a recent randomized clinical trial established the role of pufas on plaque stability . patients with carotid artery atherosclerosis were randomized to take placebo , fish oil ( n-3 pufas ) , or seed oil ( n-6 pufas ) . subjects treated with n-3 pufas showed higher epa and dha concentration , reduction of monocytes and macrophages , and a thicker fibrous cap compared to controls and group treated with n-6 pufas , all changes that can enhance the stability of atherosclerotic plaques . by contrast , increased assumption of n-6 pufas did not affect carotid plaque fatty acid composition or stability . plaques stability may explain the reductions in nonfatal and fatal cardiovascular events , associated with increased n-3 pufas intake . the triglyceride - lowering , antiatherogenic , antithrombotic , and anti - inflammatory effects of n-3 pufas were clearly demonstrated in human studies . conversely , their anti - arrhytmogenic effect was showed only in vitro and in animal experiments , as confirmation in humans has been limited by the absence of reliable physiological measures or biomarkers to quantify antiarrhythmic potential . inflammation is a physiological response to tissue trauma or infection , but leukocytes , which are the effector cells of the inflammatory process , have powerful capabilities of tissue remodelling . thus , the passage of leukocytes from the bloodstream into inflamed tissue is tightly regulated to ensure their precise localization . recruitment of circulating neutrophils into the tissue stroma occurs during the early phases of inflammation . in this process , peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells , through the vessel wall and out into the tissue stroma . although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells , stimulated by the inflammatory cytokine ( tnf- ) , transit across the endothelial monolayer is regulated by additional stimuli . a new step in the neutrophil recruitment process that relies upon a lipid - mediated signal to regulate the migration of neutrophils across endothelial cells this signal is supplied by the metabolism of arachidonic acid into the eicosanoid prostaglandin - d2 ( pgd2 ) by cyclooxygenase ( cox ) enzymes . this step in the neutrophil recruitment process was revealed when the epa was used as an alternative substrate for cox enzymes , leading to the generation of prostaglandin - d3 ( pgd3 ) [ 117120 ] . this alternative eicosanoid inhibited the migration of neutrophils across endothelial cells competing for the pgd2 receptor [ 121 , 122 ] . pgd2 signalling is subordinate to the chemokine - mediated activation of neutrophils , but without the sequential delivery of this signal , neutrophils fail to penetrate the endothelial cell monolayer . the ability of dietary epa to inhibit this process reveals an unsuspected level of regulation in the migration of inflammatory leukocytes . this may contribute to clarify the interactions between pufas and the inflammatory system and direct research on novel therapeutic agents that target the inflammatory system with greater affinity and/or specificity than dietary supplements of n-3-pufas . the inflammation healing mechanisms are of main interest , and in recent years , new endogenous anti - inflammatory and proresolving lipid mediators generated from pufas ( lipoxins , resolvins , protectin , and maresin ) were uncovered . lipid mediator metabolomics of self - resolving inflammatory exudates recently highlighted a new family of potent anti - inflammatory and proresolving mediators . serhan and coworkers identified families of novel bioactive mediators derived from arachidonic acid ( aa - derived lipoxins ) , eicosapentaenoic acid ( epa - derived e - series resolvins ) , and docosahexaenoic acid ( dha - derived d - series resolvins , protectin , and maresin ) [ 124135 ] . these lipid mediators promote resolution through enhanced clearance of apoptotic pmns , chemokines , cytokines , and microbial products by macrophages [ 136 , 137 ] . n-3 pufas may act through several mechanisms , for instance , by preventing conversion of the n-6 pufa arachidonic acid to pro - inflammatory eicosanoids and by their conversion to potent anti - inflammatory mediators such as resolvins [ 124126 , 138 ] . a possible biological mechanism underlying the beneficial effects of lc n-3 pufas on inflammation and endothelial function is that they may compete with n-6 fatty acids for prostaglandin and leukotriene synthesis at the cyclooxygenase and lipoxygenase levels . lc n-3 pufas from fish or fish oil modulate prostaglandin metabolism by increasing the active vasodilator and inhibitor of platelet aggregation prostaglandin e3 , the weak platelet aggregator and vasoconstrictor thromboxane a3 , and the weak inducer of inflammation leukotriene b5 . otherwise , the production of thromboxane a2 , a potent platelet aggregator and vasoconstrictor , and leukotriene b4 , an inducer of inflammation and a powerful inducer of leukocyte chemotaxis and adherence is reduced . another suggested mechanism is that lc n-3 pufas may bind reactive oxygen species because of their multiple double bonds and lead to a decreased production of hydrogen peroxide . hydrogen peroxide is a critical activator of the nuclear factor-b system of transcription factors that controls the coordinated expression of adhesion molecules and of leukocyte - specific chemoattractants upon cytokine stimulation . he and coworkers found independent inverse associations of lc n-3 pufas and nonfried fish with il-6 , crp and mmp3 . the relevance of these biomarkers of inflammatory and endothelial activation in the atherogenic process is well recognized . previous investigations suggest that both crp and il-6 , two systemic inflammatory markers , are independent predictors of cvd and may play an important role in atherogenesis . in addition , mmp3 is suggested as an independent prognostic factor in stable coronary artery disease . soluble intercellular adhesion molecule-1 ( sicam-1 ) is thought to be a key factor in the adherence of monocytes to the endothelium and subsequent transmigration into the intima ( figure 2 ) . the role of sicam-1 in the pathogenesis of inflammation and atherosclerosis has been confirmed in experimental models . previous investigations also indicate that sicam-1 is an independent predictor of cvd apart of other traditional risk factors . . observed that fried fish but not non - fried fish consumption was significantly inversely related to sicam-1 . this finding is not expected since the frying process may reduce the content of lc n-3 pufas and produce trans - fatty acids . one possible explanation is that fried fish consumption may be a marker of relatively unhealthy lifestyle ; those who had high fried fish consumption were more likely to suffer from dyslipidemia and were likely to be under treatment with medications ( e.g. , statins ) , which may lower sicam-1 . however , the inverse association between fried fish intake and sicam-1 level remained significant when patients taking cholesterol - lowering medications were excluded . lc n-3 pufas are believed to affect inflammatory processes mainly through two pathways : endothelial activation and changes in eicosanoid production , or a combination of the two . the first , predicated on the observation that n-3 pufas regulate the transcription of endothelial cell inflammatory genes by downregulating the activity of the nuclear factor - kb , predicts changes in the levels of adhesion receptor and chemokine expression after supplementation with epa [ 149151 ] . the second hypothesis proposes that upon endothelial cell activation , epa may compete with the n-6-pufa arachidonic acid ( aa ; 20:4n-6 ) for cyclooxygenase enzymes ( cox1 and cox2 ) after both fatty acids are released from membrane phospholipids by endogenous phospholipases . a large amount of investigations suggest , the cardioprotective effects of lc n-3 pufas epa and dha intake in human subjects , because of their lipid lowering , hypotensive , anti - arrhythmic , and anti - thrombotic properties . moreover , studies performed in the last twenty years showed heterogeneous effects of different n-3 pufas on various cardiovascular outcomes , which may be of paramount relevance in primary and secondary prevention of cardiovascular disease . recent in vitro investigations as well as clinical studies also demonstrated that lc - n3-pufas significantly interact with inflammation - related mechanisms , such as endothelial activation , modification of eicosanoid metabolism , and resolution of the inflammatory process . low - grade chronic inflammation is present in several diseases and is characterized by abnormal circulating levels of pro- and anti - inflammatory cytokines . n-3 pufas may modulate inflammation , that is , suggested by the reduction of plasma inflammatory cytokines ( tnf- , il-6 ) and inflammatory markers as high sensitive c reactive protein , observed after the intake of epa and dha . low - grade chronic inflammation plays a key role in the induction and progression of atherosclerosis and consequently of cardiovascular disease . taking into consideration the pleiotropic nature of their actions , we suggest that dietary intake of lc n-3 pufas may lead to improvements in cardio - metabolic health parameters of their antioxidant , anti - inflammatory , and antiarrhytmic actions .

phospholipids play an essential role in cell membrane structure and function . the length and number of double bonds of fatty acids in membrane phospholipids are main determinants of fluidity , transport systems , activity of membrane - bound enzymes , and susceptibility to lipid peroxidation . the fatty acid profile of serum lipids , especially the phospholipids , reflects the fatty acid composition of cell membranes . moreover , long - chain n-3 polyunsatured fatty acids decrease very - low - density lipoprotein assembly and secretion reducing triacylglycerol production . n-6 and n-3 polyunsatured fatty acids are the precursors of signalling molecules , termed eicosanoids , which play an important role in the regulation of inflammation . eicosanoids derived from n-6 polyunsatured fatty acids have proinflammatory actions , while eicosanoids derived from n-3 polyunsatured fatty acids have anti - inflammatory ones . previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis : actually , atherosclerosis is predominantly a chronic low - grade inflammatory disease of the vessel wall . several studies suggested the relationship between long - chain n-3 polyunsaturated fatty acids and inflammation , showing that fatty acids may decrease endothelial activation and affect eicosanoid metabolism .

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our understanding of h pylori induced inflammation leading to cancer is built on the work of 3 pioneering pathologists ( figure 1 ) . rudolf virchow , a 19th century prussian physician - scientist , is widely regarded as the father of modern pathology . among his many contributions to outlining the scientific basis of disease was the idea , based on many of his own observations , that cancer arose from initially normal cells in response to chronic irritation , or inflammation . numerous examples of inflammation - induced cancers are now appreciated , including many of the common gastrointestinal tract and hepatobiliary malignancies , such as acid reflux induced esophageal adenocarcinoma , inflammatory bowel disease associated colon cancer , and hepatocellular neoplasms associated with chronic viral hepatitis . throughout the 20th century , evidence accrued that gastric cancers tended to arise in stomachs already affected by chronic inflammation , especially atrophic gastritis with its accompanying hypochlorhydria , and that gastric cancer was a consequence and not a mere accompaniment of the gastritis . these ideas set the stage for pelayo correa , a pathologist from colombia , a country with a particularly high gastric cancer prevalence . after training in pathology in colombia and in the united states ( emory university ) , correa devoted his professional career at home and subsequently in the united states ( at the national cancer institute , louisiana state university , and now vanderbilt university ) to understanding the etiology of gastric cancer . in 1975 , correa et al , from the national cancer institute , massachusetts institute of technology , and from his home country , published a model for gastric cancer development . in this landmark report , it was hypothesized that the development of the more common intestinal subtype of gastric cancer resulted from a stepwise process , beginning with chronic atrophic gastritis and progressing to intestinal metaplasia and cancer over the next 3050 years . the initial changes were postulated to occur in the first decade of life , which we now know to be when h pylori colonization occurs . a more detailed model , published in 1988 , included what was known of the phenotypic markers accompanying these sequential changes . correa initially thought that the agent(s ) responsible for promoting this slow progression from gastritis to cancer were environmental , based on studies of migrants from high gastric cancer risk areas . for example , japanese immigrants to hawaii and european immigrants to the united states had been shown to have lower gastric cancer rates than their parents and grandparents , more similar to those of the native population where they settled.7 , 8 the prime environmental culprit originally was thought to be a diet high in salt and n - nitroso - compounds and low in micronutrients from fresh fruits and vegetables . it was postulated that this led to the promotion of gastric mutagenesis and , together with hypochlorhydria , bacterial overgrowth , thereby contributing to further nitrosamine formation . interestingly , in his 1988 publication , correa briefly discussed a possible role for campylobacter pylori , a newly discovered gastric bacterium , in the initiation of the disease . correa s subsequent work has focused primarily on the role of h pylori in gastric cancer , and this model has stood the test of time ( figure 2 ) . for his outstanding contributions to the field of gastric carcinogenesis , the third pathologist of note is robin warren from australia who , together with barry marshall , was awarded the nobel prize in physiology or medicine in 2005 for the discovery of h pylori and its role in gastritis and peptic ulcer disease . in the 1970s , the widespread use of gastrointestinal endoscopy allowed pathologists the opportunity to view gastric tissue that had been removed during a biopsy and fixed rapidly , without the artifacts inherent to the ischemia and autolysis of surgical specimens . he then recruited marshall , a medical resident looking for a research project , to correlate the pathologic findings with the endoscopic features . together , they discovered that these bacteria ( initially termed campylobacter pyloridis , then c pylori , and , subsequently , h pylori ) were very common in peptic ulcer patients . eventually , they successfully cultured these formerly elusive bacteria and showed that they caused gastritis and ulcer disease.11 , 12 , 13 , 14 in retrospect , other investigators also had observed such bacteria over the preceding century , but their clinical significance had not been appreciated and they had even been proven to be a post mortem artifact before their rediscovery in australia . although warren and marshall did not investigate the role of h pylori in gastric cancer directly , they were aware of the relationship between gastritis and cancer . indeed , marshall speculated with amazing prescience in their very first publications ( in unusual side - by - side , single - author letters in 1983 ) that if these bacteria are truly associated with antral gastritis , as described by warren , they may have a part to play in other poorly understood , gastritis associated diseases ( ie , peptic ulcer and gastric cancer ) . initial investigations into a possible association of h pylori with gastric cancer that were conducted in the 1980s and early 1990s provided only weak evidence for a link between h pylori and gastric cancer . a positive correlation between h pylori seroprevalence and gastric cancer in cross - sectional sampling was reported in a study conducted among 13 european nations . however , although some other publications reported similar trends,17 , 18 several others did not find a positive correlation between the presence of h pylori antibodies and either gastric cancer or precancerous gastric lesions.19 , 20 , 21 , 22 around this time , clinicopathologic studies performed to look for evidence of h pylori infection ( by serum antibodies ) or in gastric tissue directly in cases of gastric cancer also yielded inconsistent results . some studies showed h pylori infection rates no higher than those observed in noncancer controls . this is most likely because by the time gastric cancer has developed , extensive intestinal metaplasia and hypochlorhydria had rendered the stomach less hospitable to persistent h pylori colonization , thus also explaining some of the negative findings in the cross - sectional studies discussed earlier . much more convincing evidence for the role of h pylori in gastric cancer came from 3 large cohorts with nested case - controls , in which serum had been banked from cancer - free subjects and the cohort had been followed up for approximately a decade.24 , 25 , 26 in each cohort , evidence of prior h pylori infection ( evaluated by enzyme - linked immunosorbent assay [ elisa ] in the banked serum ) was found to be significantly more common in those subjects who subsequently developed gastric cancer compared with a sample of those who had not ( table 1 ) . in a meta - analysis , these 3 studies provided an overall odds ratio for gastric cancer development in h pylori infected vs -uninfected persons of 3.8 ( 95% confidence interval [ ci ] , 2.36.2 ) . based on the compelling results of these cohort studies , the world health organization s international agency for research on cancer declared in 1994 that there was sufficient evidence to classify h pylori as a definite ( group 1 ) carcinogen . this was despite the fact that at that time there was no significant supporting evidence from either animal models or mechanistic basic science research . subsequent research worldwide has confirmed and expanded upon the early epidemiologic studies , with the key additional clinical findings as follows:1.cardia cancers are not associated strongly with h pylori infection , supporting the idea that cardia cancers arise via alternative mechanisms , with risk factors more similar to cancers of the gastroesophageal junction and lower esophagus.2.in pooled meta - analysis , the odds ratio for h pylori in gastric cancer was no different when considering intestinal vs diffuse histologic cancer subtypes . thus , in addition to promoting the well - established correa cascade of intestinal - type cancers , h pylori is just as great a risk factor for the much less well - defined pathway leading to diffuse - type gastric cancer.3.measuring h pylori exposure by elisa underestimates prior exposure and , therefore , the attributable h pylori risk in noncardia cancer , because of false - negative results . for example , in a retrospective swedish case - control study , measuring prior exposure through a western blot against h pylori cytotoxin - associated gene a ( caga ) , rather than a conventional igg elisa , increased the odds ratio from 2.2 ( 95% ci , 1.43.6 ) to 21.0 ( 95% ci , 8.353.4).4.in noncardia gastric cancers , caga - positive h pylori strains ( which are the more common strains worldwide ) increase the risk of gastric cancer to a greater extent than do infections by the more rare caga - negative strains . this is consistent with the oncogenic effects of the caga protein that have been evaluated mainly in vitro.5.h pylori eradication can reduce the risk of developing gastric cancer ( see later ) . cardia cancers are not associated strongly with h pylori infection , supporting the idea that cardia cancers arise via alternative mechanisms , with risk factors more similar to cancers of the gastroesophageal junction and lower esophagus . in pooled meta - analysis , the odds ratio for h pylori in gastric cancer was no different when considering intestinal vs diffuse histologic cancer subtypes . thus , in addition to promoting the well - established correa cascade of intestinal - type cancers , h pylori is just as great a risk factor for the much less well - defined pathway leading to diffuse - type gastric cancer . measuring h pylori exposure by elisa underestimates prior exposure and , therefore , the attributable h pylori risk in noncardia cancer , because of false - negative results . for example , in a retrospective swedish case - control study , measuring prior exposure through a western blot against h pylori cytotoxin - associated gene a ( caga ) , rather than a conventional igg elisa , increased the odds ratio from 2.2 ( 95% ci , 1.43.6 ) to 21.0 ( 95% ci , 8.353.4 ) . in noncardia gastric cancers , caga - positive h pylori strains ( which are the more common strains worldwide ) increase the risk of gastric cancer to a greater extent than do infections by the more rare caga - negative strains . this is consistent with the oncogenic effects of the caga protein that have been evaluated mainly in vitro . h pylori eradication can reduce the risk of developing gastric cancer ( see later ) . in a prolonged observational study following patients who had attended a japanese endoscopy unit , uemura et al showed that those who were h pylori infected ( and in whom the infection was not treated ) had a much higher rate of progression to gastric cancer over the subsequent 12 years than the patients who had no initial evidence of h pylori . however , to prove conclusively that h pylori is responsible for the increased gastric cancer risk , prospective interventional studies were necessary . recruitment into such studies was hampered by the 1994 declaration of h pylori as a definite carcinogen , after which it became ethically problematic to enter patients into the control arms of eradication studies designed to determine the gastric cancer risk reduction . consequently , an appropriately powered , definitive study of a large number of subjects followed up for many years to the end point of gastric cancer was never completed . nevertheless , recent meta - analyses of the many underpowered studies that were conducted are conclusive , indicating that h pylori eradication decreases the risk of gastric cancer development by approximately 40% in studies of primary prevention ( asymptomatic individuals ) , and by 54% as a tertiary prevention strategy ( preventing the occurrence of a second gastric malignancy after endoscopic resection of an early gastric cancer).36 , 37 it is not known whether or not there is a point of no return along the correa pathway , beyond which h pylori eradication will not prevent progression to gastric cancer . in the analysis by lee et al , which included 24 publications of more than 48,000 individuals followed up for more than 340,000 person - years ( 14 studies of primary prevention , 10 studies of tertiary prevention ) , the benefit of h pylori eradication was , not surprisingly , most evident in subjects living in areas with the highest gastric cancer prevalence . because all investigations were conducted in parts of the world where gastric cancer is most prevalent ( mostly in south - east asia ) , it is uncertain if similar reductions can be extrapolated to regions where noncardia gastric cancer is less common , such as the united states . however , it may be reasonable to suppose that high - risk populations in low - risk countries , including immigrants from central and south america and south - east asia to the united states who remain h pylori infected from childhood , should benefit greatly from h pylori eradication too . however , with the exception of endoscopic screening in south korea , and radiologic and endoscopic programs in japan , there had been little effort made previously to control this burden with public health programs . the results of the recent intervention studies reported earlier have now prompted renewed interest in screening for h pylori and eradicating the organism when found as a potentially cost - effective strategy , especially in parts of the world that have a high prevalence of h pylori and gastric cancer.39 , 40 , 41 a pilot study of mass population screening and eradication started in the matsu islands of taiwan in 2004 , and the initial results are very promising . gastric cancer incidence has decreased by approximately 25% , peptic ulcers by two thirds , and gastric atrophy by 77% compared with historical data . several other population - based trials are underway , including one with almost 100,000 chinese subjects from linqu county , a region of high gastric cancer prevalence . the eradication of h pylori from a community should help prevent much gastric cancer , as well as greatly diminish the morbidity of peptic ulcers too . it has been calculated that the number needed to treat to prevent a case of gastric cancer may be as low as 15 in chinese men , to as high as 245 in women in the united states . although there is a concern regarding the ecologic consequences of using multiple antibiotics in a large proportion of the population to prevent h pylori related disease , the recent exciting results of vaccination against h pylori with an oral urease b construct suggest that a nonantibiotic strategy may be possible in the future , with potentially less impact on the general human microbiome . further investigation of this vaccine should be a priority , particularly because promising preclinical models with a variety of vaccine candidates since the 1990s had never resulted previously in impressive results in clinical trials . if preventive or therapeutic vaccination emerges as a viable alternative for h pylori eradication , it will help avert widespread antibiotic administration and the consequent problems of microbiome perturbation and the generation of microbial antibiotic resistance among h pylori and other innocent bystander bacteria . in the midst of growing optimism for implementing h pylori eradication strategies in high gastric cancer risk regions , it is important to recognize that there is considerable hesitation to adopt similar approaches in the developed world , mainly out of concern for a beneficial effect of h pylori on esophageal diseases . the results of multiple studies have indicated that h pylori is significantly less common in patients with esophageal adenocarcinoma than in matched controls , although h pylori infection has no association , positive or negative , with esophageal squamous cancer47 , 48 ( figure 3 ) . this places h pylori in a unique position among the world health organization s list of biological agents that cause cancer , in that its presence is associated positively with certain cancers ( gastric noncardia adenocarcinoma , gastric marginal zone b - cell lymphoma ) although related inversely to another cancer type ( esophageal adenocarcinoma ) . the absence of h pylori may promote more severe esophageal mucosal damage , barrett s metaplasia , and the consequent development of esophageal adenocarcinoma . a postulated mechanism is through a greater gastric acid secretory capacity from a pristine , uninflamed , parietal cell mass in the absence of h pylori gastritis , although there is little evidence that this actually leads to enhanced esophageal acid exposure . an inverse relationship between h pylori and esophageal adenocarcinoma does not necessarily imply that h pylori is protective any more than a positive association can be taken to be evidence of causation . h pylori has been an almost universal inhabitant of the human stomach from the dawn of mankind until the 1980s . its recent disappearance from the 21st human microbiome , one of the missing microbes , has been linked to extragastric immune dysregulation ( for which there is good evidence from animal models ) and a multitude of other contemporary maladies ( eg , asthma , obesity , and inflammatory bowel disease ) , for which the evidence is questionable and the biological plausibility equally uncertain.55 , 56 , 57 the decrease in noncardia gastric cancer in north america and western europe over the past 100 years that coincided with ( or maybe even preceded ) decreasing h pylori prevalence , was famously described as the epidemiology of an unplanned triumph . the rapid increase in esophageal adenocarcinoma in the same population more recently ( such that it is now approaching the prevalence of noncardia gastric cancer ) perhaps may be considered the unplanned downside of the disappearance of h pylori from western stomachs . it is this unexpected accompaniment of h pylori eradication that largely has tempered enthusiasm for population - based h pylori eradication efforts in the developed world . whether to pursue population - based h pylori screening and treatment strategies therefore will need to be individualized according to the current and projected future medical needs of each community , and these differ quite markedly around the globe . the declaration of h pylori as a definite gastric carcinogen in 1994 and the subsequent realization that the vast majority of noncardia cancers are attributable to h pylori infection has led to a fundamental change in the way that we view gastric cancer pathogenesis in the funderburg era . considering gastric cancer as the consequence of an infection has led to enthusiasm for screening for and eradicating h pylori in areas of high gastric cancer prevalence , but the fact that gastric cancer remains a rare consequence of h pylori infection provides plenty of opportunity for investigating the co - factors that promote gastric neoplastic development after h pylori colonization . recent research in this field has focused on genetic susceptibility ( such as polymorphisms in genes governing gastric inflammatory responses ) , h pylori heterogeneity , and on other environmental influences , such as dietary salt or the presence of non - helicobacter species within the gastrointestinal microbiome that may explain why only a small proportion of individuals who are colonized by h pylori go on to develop gastric cancer . this knowledge ultimately may have clinical utility in stratifying individuals with h pylori infection into high vs low gastric cancer risk , and to the creation of personalized surveillance , chemoprevention , and dietary intervention programs focused on patients at highest risk . as an example , an early study reported enormous differences ( up to 90-fold ) in gastric cancer susceptibility in h pylori infected cases from portugal , when stratified by polymorphisms in interleukin 1 and the interleukin 1receptor antagonist , together with h pylori vacuolating cytotoxin a ( vaca ) and caga typing . investigating the precise molecular and cellular mechanisms of gastric cancer development associated with h pylori infection is likely to provide additional insights into gastric cancer , as well as stimulate ideas about the pathogenesis of other cancers that are known to be associated with inflammation , although not necessarily with specific constituents of the microbiome . since 1994 , considerable progress has been made on dissecting the role of h pylori role in the molecular pathogenesis of inflammation - associated gastric cancer ( as outlined in the review by crowe in this issue ) . the multiplicity of animal models to study h pylori associated gastric carcinogenesis ( reviewed in tsukamoto et al and krueger et al , as well as by wang in this issue ) also has provided many important mechanistic insights that were not conceivable back in 1994 , when rodent infection studies were in their infancy and the evidence for h pylori as a carcinogen was based on a limited number of clinical studies . support from the funderburg family has been instrumental in promoting new ideas about gastric cancer and its origins , as well as opening new therapeutic possibilities . the work that started in our laboratory in 2002 as a result of this funding has allowed us to investigate how h pylori misregulates the expression of p27 , a cyclin - dependent kinase inhibitor that is an important constitutive endogenous cell - cycle regulator.67 , 68 we also subsequently explored the p27-deficient mouse as a model with which to study the mechanisms of h pylori induced gastric carcinogenesis,69 , 70 and to examine its reversibility after h pylori eradication . our understanding of gastric carcinogenesis continues to grow rapidly . for many years , pathologists were at the cutting edge of this knowledge , based on their meticulous observations of the unfortunate patients who died of this largely untreatable disease . the realization that a single bacterial species was the inciting cause of the majority of gastric cancers went against the prevailing clinical and scientific dogma . as a result of this initially uncomfortable discovery , gastric cancer , which was a disease that formerly was principally the focus of pathologists and surgeons , has become widely embraced by the scientific and clinical community as a model of bacterial carcinogenesis and a window to inflammation - associated cancers in general .

gastric cancer has long been recognized to be accompanied and preceded by chronic gastritis , lasting decades . arguably , the most important development in our understanding of gastric cancer pathogenesis over the past 50 years has been the realization that , for most cases of gastric cancer , helicobacter pylori is the cause of the underlying gastritis . gastritis can promote gastric carcinogenesis , typically via the correa cascade of atrophic gastritis , intestinal metaplasia , and dysplasia . nested case - control studies have shown that h pylori infection increases the risk of gastric cancer significantly , both of the intestinal and diffuse subtypes , and that h pylori is responsible for approximately 90% of the world s burden of noncardia gastric cancer . based largely on randomized studies in high gastric cancer prevalence regions in east asia , it appears that primary and tertiary intervention to eradicate h pylori can halve the risk of gastric cancer . some public health authorities now are starting screening and treatment programs to reduce the burden of gastric cancer in these high - risk areas . however , there is currently much less enthusiasm for initiating similar attempts in the united states . this is partially because gastric cancer is a relatively less frequent cause of cancer in the united states , and in addition there are concerns about theoretical downsides of h pylori eradication , principally because of the consistent inverse relationship noted between h pylori and esophageal adenocarcinoma . nevertheless , establishing a link between chronic h pylori infection and gastric cancer has led to novel insights into cancer biology , the gastrointestinal microbiome , and on individual and population - based gastric cancer prevention strategies .

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neovascular age - related macular degeneration ( namd ) is a leading cause of visual loss among elderly population [ 1 , 2 ] . before the introduction of intravitreal antivascular endothelial growth factor ( anti - vegf ) agents for the treatment of namd , only prevention from visual acuity loss might have been achieved in a limited number of patients with different treatment options like laser photocoagulation , photodynamic therapy , and vitreoretinal surgery [ 39 ] . intravitreal treatments with bevacizumab ( full length antibody against vegf - a ) and ranibizumab ( fab part of antibody against vegf - a ) have led the majority of the patients to prevent the baseline visual acuity ( va ) and even to achieve visual improvement in some of the patients [ 10 , 11 ] . the multicenter studies with ranibizumab , like marina , anchor , pronto , and excite , and the comparative study of ranibizumab and bevacizumab , the catt study , showed that ranibizumab and bevacizumab were effective to prevent va loss up to 95% of the patients and is effective to make an improvement in va up to 40% of the patients [ 1115 ] . today , the catt trial answered the questions about the efficacy of bevacizumab versus ranibizumab and showed that both drugs had similar effects in the treatment of namd . however , the safety of the drugs still remains unclear , and there is an ongoing debate about this issue . in this study , we aimed to compare the functional and anatomical outcomes of bevacizumab therapy to ranibizumab therapy on as - needed treatment regimen for namd in turkish patients . the clinical data of the patients who were treated with either bevacizumab or ranibizumab between 2009 and 2011 were retrospectively evaluated for this study . inclusion criteria were age of 50 years or more , newly diagnosed namd , no treatments received other than bevacizumab alone or ranibizumab alone , and a minimum follow - up period of 12 months . patients were not included if they had any of the following criteria : retinal diseases other than namd , previous intravitreal injections , or a history of photodynamic therapy or laser photocoagulation . the tenets of the declaration of helsinki were followed throughout the study , and written informed consent was obtained from all patients before the treatments were started . data collected from the patients ' records included age , gender , type of choroidal neovascularization ( cnv ) ( predominantly or minimally classic or occult ) , bcva , and central retinal thickness ( crt ) before treatment , at months 3 , 6 , 9 , and 12 during treatment , and at the most recent follow - up examination . all patients underwent a standardized examination including measurement of bcva via the early treatment diabetic retinopathy study ( etdrs ) chart at 4 meters , slit - lamp biomicroscopy and fundus examination and measurement of intraocular pressure ( iop ) via applanation tonometry . fundus photography , fluorescein angiography ( hra-2 , heidelberg engineering , heidelberg , germany ) , and optical coherence tomography ( oct ) imaging ( stratus oct tm , carl zeiss meditec inc . , all examinations were repeated monthly , except fluorescein angiography , which was repeated only when the cause of visual acuity deterioration could not be clarified with the clinical examination and other imaging methods . oct was used for detecting subretinal fluid and measurement of crt , the latter being defined as the mean thickness of the neurosensory retina in the central 1 mm diameter region , computed via oct mapping software provided with the device . all injections were performed under sterile conditions after topical anesthesia , and 10% povidone - iodine scrub ( betadine , purdue pharma , stamford , ct , usa ) was used on the lids and lashes , and then 5% povidone - iodine was administered on the conjunctival sac . intravitreal bevacizumab ( avastin , genentech , south san francisco , ca , usa ) or ranibizumab ( lucentis , novartis , basel , switzerland ) was injected with a 30-gauge needle through the pars plana at 3.5 mm to 4 mm posterior to the limbus . patients were then instructed to consult the hospital if they experienced decreased vision , eye pain , or any new symptoms . for the first three months of treatment , all patients received monthly doses of bevacizumab , or ranibizumab ( 1.25 mg/0.05 ml for bevacizumab , 0.5 mg/0.05 ml for ranibizumab ) . the patients were then examined monthly and were retreated if they met any of the following criteria : visual loss of 1 or more lines , newly developed macular hemorrhage , evidence of cnv enlargement on examination or fluorescein angiography , any amount of persistent subretinal fluid one month after an injection . visual loss of 1 or more lines , newly developed macular hemorrhage , evidence of cnv enlargement on examination or fluorescein angiography , any amount of persistent subretinal fluid one month after an injection . visual acuity was converted to logarithm of minimum angle of resolution ( logmar ) for statistical analysis . the changes in bcva and crt over time were analyzed with paired sample t - test . chi - square test was used to compare nominal parameters between the groups , and independent sample t - test was used for continuous parameters . the statistical analysis was performed using spss version ( version 15.0 , spss inc . , the bevacizumab group consisted of 79 patients , and the ranibizumab group consisted of 74 patients . the general characteristics of the patients were similar between the two groups ( table 1 ) . the mean bcva of the patients in the bevacizumab group at baseline , months 3 , 6 , 9 , 12 , and at the most recent followup was 1.02 0.46 logmar ( range 0.12.1 logmar ) , 0.86 0.46 logmar ( range 0.22.1 logmar ) , 0.83 0.43 logmar ( range 0.22.1 logmar ) , 0.82 0.43 logmar ( range 0.22.1 logmar ) , 0.82 0.42 ( range 0.22.1 logmar ) , and 0.83 0.43 ( range 0.22.1 logmar ) , respectively . the mean bcva of the patients in the ranibizumab group at baseline , months 3 , 6 , 9 , 12 , and at the most recent followup was 0.97 0.46 logmar ( range 0.12.1 logmar ) , 0.83 0.47 logmar ( range 0.11.8 logmar ) , 0.81 0.47 logmar ( range 0.01.8 logmar ) , 0.81 0.42 logmar ( range 0.11.8 logmar ) , 0.82 0.43 ( range 0.01.8 logmar ) , and 0.82 0.47 ( range 0.02.1 logmar ) , respectively ( figure 1 ) . the change in mean bcva from baseline to months 3 , 6 , 9 , 12 , and the most recent followup was statistically better in both of the groups ( p < 0.05 , for all ) . however , there was not a statistically significant difference between the two groups in regards of change in bcva at all of the time points ( p = 0.7 for month 3 , p = 0.6 for month 6 , p = 0.5 for month 9 , p = 0.5 for month 12 , and p = 0.6 for the most recent followup ) ( table 2 ) . at the most recent followup , 34 of the 79 patients ( 43.0% ) in the bevacizumab group and 30 of the 74 patients ( 40.5% ) in the ranibizumab group gained va 3 lines ( p = 0.4 ) . sixty - eight of the 79 patients ( 86.1% ) in the bevacizumab group and 64 of the 74 patients ( 86.5% ) in the ranibizumab group had stable or improved vision ( loss of < 3 lines , remained stable , or gained 1 lines ) ( p = 0.5 ) . eleven of the 79 patients ( 13.9% ) in the bevacizumab group and 10 of the 74 patients ( 13.5% ) in the ranibizumab group had va loss 3 lines ( p = 0.5 ) . the mean crt in the bevacizumab group at baseline , and at months 3 , 6 , 9 , and 12 , and at the most recent followup was 335 120 m ( range 180758 m ) , 289 118 m ( range 106796 m ) , 266 79 m ( range 140558 m ) , 260 93 m ( range 148713 m ) , 266 100 ( range 150789 m ) , and 262 124 ( range 103979 m ) , respectively . the mean crt in the ranibizumab group at the baseline , and at months 3 , 6 , 9 , and 12 , and at the most recent followup was 315 88 ( range 139681 m ) , 250 68 m ( range 133487 m ) , 243 63 m ( range 135410 m ) , 254 72 m ( range 135540 m ) , 247 60 ( range 140415 m ) , and 256 73 ( range 130483 m ) , respectively ( figure 2 ) . the mean crt at months 3 , 6 , 9 , and 12 , and at the most recent followup was statistically different compared to baseline in both of the groups ( p < 0.05 for all ) . however , the change in mean crt from the baseline to months 3 , 6 , 9 , and 12 , and at the most recent follow - up was not statistically different between the two groups ( p = 0.2 , p = 0.8 , p = 0.3 , p = 0.9 , and p = 0.2 , resp . ) ( table 3 ) . the mean number of injections at month 12 was 4.8 1.2 ( range 37 ) in the bevacizumab group and 4.7 1.4 ( range 38 ) in the ranibizumab group , and the difference was not statistically significant between the two groups ( p = 0.8 ) . the mean baseline intraocular pressure ( iop ) was 15.5 2.3 mm hg ( range 919 mm hg ) and 15.4 1.7 ( range 1220 mm hg ) in the bevacizumab and ranibizumab groups , respectively ( p = 0.7 ) . the mean iop at the most recent followup was 15.3 2.4 mm hg ( range 1022 mm hg ) and 15.6 2.1 ( range 1122 mm hg ) , in the bevacizumab and ranibizumab groups , respectively ( p = 0.4 ) . no serious ocular or systemic complications were observed in any of the patients . only mild complications like punctate keratitis , subconjunctival hemorrhage , and transient mild anterior uveitis functional and anatomical outcomes of namd patients treated with bevacizumab and ranibizumab in a population of turkish patients were reported in this study . in both of the groups , the mean bcva and crt were significantly better than that in baseline at every time point . the two groups were parallel to each other with respect to change in bcva and crt , injection numbers , and complications . in previous clinical studies , these two drugs , bevacizumab or ranibizumab , have been found to provide similar improvements in visual and anatomical outcomes to our study [ 13 , 1620 ] . a short term efficacy and safety study by landa et al . reported that bevacizumab and ranibizumab treatments resulted in similar improvements in visual and anatomical outcomes one month after an intravitreal injection in patients with namd . they also stated that bevacizumab was as safe as ranibizumab . in a retrospective study by feng et al . , 371 namd patients who were treated with intravitreal bevacizumab or ranibizumab on an as - needed treatment regimen were evaluated . they reported that , after a median follow - up time of 12 months , 24.5% of patients in the bevacizumab group and 25.8% of patients in the ranibizumab group had a gain of 15 or more letters ; in addition , 79.5% of the patients in the bevacizumab group , and 84.9% of the patients in the ranibizumab group lost fewer than 15 letters in visual acuity . they stated that there was not a statistical difference in regards of visual acuity changes between the two groups . we also did not find any difference in visual acuity change at different time points between the bevacizumab and ranibizumab groups . and the rate of the patients who were stable in regards of visual acuity results was very similar to the study by feng et al . reported that bevacizumab and ranibizumab were both effective in stabilizing visual acuity in namd patients after 12 months of followup . in the aforementioned study , it was reported that visual acuity outcomes were similar between the two drugs ; 27.3% of the patients in the bevacizumab and 20.2% of the patients in the ranibizumab group were reported to have an improvement of three or more lines in visual acuity , and the difference between the groups was not found to be statistically significant . the mean injection numbers at month 12 were 4.4 in the bevacizumab group and 6.2 in the ranibizumab group which were statistically different between the groups . the mean injection numbers at month 12 were similar to our study ; however , we did not find a difference between the two groups . in a retrospective study concerning the efficacy of bevacizumab and ranibizumab in namd by bellerive et al . , the mean visual acuity improvement at month 12 was greater in the ranibizumab group than the bevacizumab group . they stated that the proportion of occult lesions were greater in the bevacizumab group than the ranibizumab group and postulated that this trend might be explained by this variability . also the proportion of the patients who lost fewer than 0.3 logmar was reported to be 83% in the bevacizumab group and 92 in the ranibizumab group , with a mean number of injections of 4.7 in the bevacizumab group and 4.9 in the ranibizumab group at month 12 . the visual acuity outcomes and the mean injection numbers of the study were comparable with our study . however , the visual and anatomical outcomes were similar between the groups in our study . the catt study was designed as a multicenter , noninferiority trial to compare the efficacy and safety of bevacizumab and ranibizumab . in the study , both monthly and as - needed treatment regimens were evaluated within and between the two drugs . at first year , it was reported that there was not a significant difference between the two drugs in regards of visual acuity and anatomical outcomes and the mean injection numbers . at month 12 , as - needed bevacizumab was found to be equivalent to as - needed ranibizumab with a gain of 5.9 etdrs letters and 6.8 etdrs letters in visual acuity , respectively . at month 12 , the eyes treated with as - needed bevacizumab and ranibizumab showed a significant decrease in crt , and the difference was not statistically significant between the bevacizumab groups and ranibizumab groups . the mean injection numbers of as - needed bevacizumab group and as - needed ranibizumab group were 7.7 and 6.9 at month 12 , respectively . the two - year results of the study were similar to the first - year results ; however the mean gain in visual acuity was found to be greater in monthly groups than as - needed groups . the study also addressed the safety issue and adverse events between the two drugs . at 2 years , it was reported that 6.1% of the patients in the bevacizumab group and 5.3% of the patients in the ranibizumab group had died . the proportion of the patients with atherothrombotic events and venous thrombotic events were similar between the two groups . the rate of serious adverse events was found to be greater in the bevacizumab groups than the ranibizumab groups with a cumulative risk ratio of 1.30 ; however , this difference was not found to be statistically significant . in addition , the studies which addressed the safety issues of bevacizumab and ranibizumab showed that this issue still remains as a debate [ 2123 ] . strengths of the study include the fact that patients in the bevacizumab and ranibizumab groups had received no other forms of treatment and similar baseline characteristics of the two groups . in summary , in the present study we found that 86.1% of the patients in the bevacizumab group and 86.5% of the patients in the ranibizumab group had a stable or improved visual acuity after a mean follow - up time of approximately 18 months . the visual and anatomical outcomes and the number of injections were similar between the two drugs . the patients with namd treated with as - needed bevacizumab or as - needed ranibizumab showed significant improvements in visual acuity throughout at least 1.5 years compared to baseline visual acuity .

purpose . to compare the efficacy of intravitreal bevacizumab versus ranibizumab in the treatment of neovascular age - related macular degeneration ( namd ) . methods . retrospective , comparative study . the newly diagnosed namd patients who were treated with intravitreal bevacizumab or ranibizumab on an as - needed treatment regimen were included in the study . main outcome measures were the change in best corrected visual acuity ( bcva ) , and central retinal thickness ( crt ) . secondary outcome measures were the number of injections , and complications . results . a total of 154 patients were included in the study . bevacizumab group consisted of 79 patients , and ranibizumab group consisted of 74 patients . mean follow - up time was 18.9 months , and 18.3 months in the bevacizumab and ranibizumab groups , respectively . there was not a significant difference between the two groups regarding the change in bcva and crt at all time points ( p > 0.05 for all ) . the mean number of injections at month 12 was 4.8 and 4.7 in bevacizumab and ranibizumab groups , respectively ( p > 0.05 ) . no serious complications were detected in any of the groups . conclusion . both of the bevacizumab and ranibizumab found to be effective in the treatment of namd in regards of functional and anatomical outcomes with similar number of treatments and similar side effects .

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preeclampsia ( pe ) is a leading cause of maternal and neonatal mortality and morbidity , complicating 25% of all the pregnancies . classically , the presence of proteinuria in women with gestational hypertension is clinically diagnosed as pe and is associated with increased maternal or neonatal morbidity and mortality compared with gestational hypertension . however , several studies have shown that proteinuria is not a significant predictor of adverse maternal and neonatal outcomes . the incidence of the disease increased in china , as the opening of the two - child policy , but there was not an indicator of the severe pe . severe pe is often combined with hypoalbuminemia ; we hypothesize that hypoalbuminemia may serve as an indicator of the severity of pe . hypoalbuminemia occurs mainly due to systemic small vessel spasm , increased secretion of angiotensin and damaged and increased permeability of vascular endothelial cells , thereby leading to a large number of proteins and liquid leaking in tissue clearance and a loss of a large number of plasma proteins ( especially serum albumin ) , causing intravascular dehydration . this intravascular dehydration may accelerate the appearance of intravascular lesions , which is thought to be a predisposing factor of the hemolysis , elevated liver enzymes and low platelet ( hellp ) syndrome and acute fatty liver of pregnancy . in addition , the reduction of hepatic blood flow due to the production of albumin is decreased . in two recent studies , hypoproteinemia during pregnancy was observed in the pe group in the second trimester before the onset of clinical symptoms , and the authors suggest the possibility of preventing pe by maintaining adequate blood protein levels during early pregnancy . in the present study , we asked whether hypoproteinemia was effective at predicting perinatal risk in women with pe . the aim of our study was to evaluate maternal and perinatal outcomes in women with pe , according to the different value of albumin ( 25 g / l and < 25 in this retrospective cohort study , 299 women from the maternal and child health hospital of anhui province were enrolled between january 2014 and june 2015 . the anhui medical university ethics committee provided ethics approval , and written informed consent was obtained from all participants . g / l ( mild hypoproteinemia [ mhp ] , n = 220 ) , pe with an albumin value of 25 g / l ( severe hypoproteinemia [ shp ] , n = 79 ) . for the study serum albumin was measured at the laboratory of the maternal and child health hospital of anhui province , using the unicel dxc800 of beckman coulter ( the united states ) . pe was defined according to the guidelines of the international society for the study of hypertension in pregnancy : ( 1 ) hypertension : blood pressure 140/90 mmhg on at least two occasions and at least 46 h apart ; ( 2 ) the severe features , such as severe hypertension , platelet count below 100,000/l , serum creatinine concentration of 1.1 mg / dl , or doubling of the serum creatinine concentration in the absence of other renal disease , levels of liver transaminases elevated to twice the normal concentration , pulmonary edema , new - onset cerebral or visual disturbance ; ( 3 ) proteinuria : more than 300 mg protein in a 24-h urine collection , or a urinary protein - to - creatinine ratio of 0.3 , or a urine dipstick protein grade of 1 + or greater if other methods are unavailable . pe was defined by ( 1 ) + ( 2 ) or ( 1 ) + ( 3 ) . moreover , exclusion criteria were : kidney diseases , preexisting proteinuria , essential hypertension or other maternal chronic conditions ( autoimmune diseases , diabetes , etc . ) , and twin pregnancies . maternal outcomes were evaluated : delivery mode ( spontaneous vaginal , instrumental vaginal , cesarean section [ cs ] ) , eclampsia , hellp syndrome , hypertension , abnormal liver function , abnormal renal function , ascites , oligohydramnios , thrombocytopenia , and abruptio placenta . for these complex cases , the trial management team reviewed the case and a diagnosis was made by two senior deputy chief physicians or chief physicians , acting independently . neonatal outcomes included : perinatal deaths , preterm birth ( < 37 and 28 weeks gestation , spontaneous or iatrogenic ) , neonatal asphyxia , and fetal growth restriction ( fgr , defined as actual birth weight below 10% for gestational age ) . in the meantime , birth weight was also assessed . the comparisons of the continuous variables between the two groups were estimated using the t - test or mann whitney test . the comparisons of the categorical variables between the groups were estimated using the chi - square test or fisher s exact test . a univariate logistic regression was used to analyze the associations between shp pe with poor maternal and neonatal outcomes . pe was defined according to the guidelines of the international society for the study of hypertension in pregnancy : ( 1 ) hypertension : blood pressure 140/90 mmhg on at least two occasions and at least 46 h apart ; ( 2 ) the severe features , such as severe hypertension , platelet count below 100,000/l , serum creatinine concentration of 1.1 mg / dl , or doubling of the serum creatinine concentration in the absence of other renal disease , levels of liver transaminases elevated to twice the normal concentration , pulmonary edema , new - onset cerebral or visual disturbance ; ( 3 ) proteinuria : more than 300 mg protein in a 24-h urine collection , or a urinary protein - to - creatinine ratio of 0.3 , or a urine dipstick protein grade of 1 + or greater if other methods are unavailable . pe was defined by ( 1 ) + ( 2 ) or ( 1 ) + ( 3 ) . moreover , exclusion criteria were : kidney diseases , preexisting proteinuria , essential hypertension or other maternal chronic conditions ( autoimmune diseases , diabetes , etc . ) , and twin pregnancies . maternal outcomes were evaluated : delivery mode ( spontaneous vaginal , instrumental vaginal , cesarean section [ cs ] ) , eclampsia , hellp syndrome , hypertension , abnormal liver function , abnormal renal function , ascites , oligohydramnios , thrombocytopenia , and abruptio placenta . for these complex cases , the trial management team reviewed the case and a diagnosis was made by two senior deputy chief physicians or chief physicians , acting independently . neonatal outcomes included : perinatal deaths , preterm birth ( < 37 and 28 weeks gestation , spontaneous or iatrogenic ) , neonatal asphyxia , and fetal growth restriction ( fgr , defined as actual birth weight below 10% for gestational age ) . in the meantime , birth weight was also assessed . the comparisons of the continuous variables between the two groups were estimated using the t - test or mann whitney test . the comparisons of the categorical variables between the groups were estimated using the chi - square test or fisher s exact test . a univariate logistic regression was used to analyze the associations between shp pe with poor maternal and neonatal outcomes . a total of 309 women with singleton pregnancies were identified ; ten women were excluded due to incomplete outcome data . there was no significant difference in age and body mass index between the two groups of pregnant women ( p = 0.17 and 0.23 , respectively ) , shown in table 1 . maternal characteristics in mild hypoproteinemia and severe hypoproteinemia preeclampsia the analysis shows that women with shp were delivered almost 1.5 weeks earlier than the mhp group ( p 0.01 ) . in the shp group , the percentage of cs was higher , and the rate of spontaneous delivery was lower ( p = 0.04 and 0.04 , respectively ) . severe hypertension was more frequent in the shp group than in the mhp group ( p = 0.01 and < 0.01 , respectively ) . women with shp were more likely to present with abnormal liver or renal function , ascites , and abruptio placenta than women in the mhp group ( p < 0.05 ) . pregnancy outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia neonatal outcomes are presented in table 3 ; the short - term neonatal outcome was worse in the shp group . the average birth weight was significantly lighter ( 2498.1 866.5 g ) in the shp group than in the mhp group ( 2940.1 768.0 g ) . neonates of the women in the shp group were more likely to have fgr , neonatal intensive care unit admission , and preterm deliveries ( p < 0.01 ) . short - term neonatal outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia to further clarify the associations between shp pe with cs , poor maternal and neonatal outcomes , a univariate logistic regression was performed to analysis . the results show that shp was a significant risk factor for cs ( odds ratio [ or ] 2.99 , 95% confidence interval [ ci ] 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women [ table 4 ] . a total of 309 women with singleton pregnancies were identified ; ten women were excluded due to incomplete outcome data . there was no significant difference in age and body mass index between the two groups of pregnant women ( p = 0.17 and 0.23 , respectively ) , shown in table 1 . maternal characteristics in mild hypoproteinemia and severe hypoproteinemia preeclampsia the analysis shows that women with shp were delivered almost 1.5 weeks earlier than the mhp group ( p 0.01 ) . in the shp group , the percentage of cs was higher , and the rate of spontaneous delivery was lower ( p = 0.04 and 0.04 , respectively ) . severe hypertension was more frequent in the shp group than in the mhp group ( p = 0.01 and < 0.01 , respectively ) . women with shp were more likely to present with abnormal liver or renal function , ascites , and abruptio placenta than women in the mhp group ( p < 0.05 ) . neonatal outcomes are presented in table 3 ; the short - term neonatal outcome was worse in the shp group . the average birth weight was significantly lighter ( 2498.1 866.5 g ) in the shp group than in the mhp group ( 2940.1 768.0 g ) . neonates of the women in the shp group were more likely to have fgr , neonatal intensive care unit admission , and preterm deliveries ( p < 0.01 ) . short - term neonatal outcome in mild hypoproteinemia and severe hypoproteinemia preeclampsia to further clarify the associations between shp pe with cs , poor maternal and neonatal outcomes , a univariate logistic regression was performed to analysis . the results show that shp was a significant risk factor for cs ( odds ratio [ or ] 2.99 , 95% confidence interval [ ci ] 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women [ table 4 ] . the associations between severe hypoproteinemia preeclampsia with cesarean section , poor maternal and neonatal outcomes the pathophysiologic mechanisms of pe are unclear ; the leading theory is that defects in placental implantation and function ultimately display as the development of pe . scholars believe that gestational hypertension and preeclampsia are isolate disease processes with different mechanisms , research evidence includes the differential risk factors between hypertension and pe , specific histologic changes in the placenta and kidneys in pe only , the levels of antiangiogenic peptides of placental origin are elevated in pe but not in gestational hypertension , and a far lower irculating volume in pe compared with gestational hypertension . pe patients often complicated with multiple organ hypoperfusion , the reduction of hepatic blood flow due to the production of albumin is decreased , thus lead to the gradual development of hypoproteinemia . in the prognosis , women with chronic and gestational hypertension usually have good outcomes . however , pe represents one - third of cases of severe obstetric morbidity ; the mortality associated with pe and eclampsia was about 0.83 per 100,000 maternities . simultaneously , pe is also strongly related with intrauterine growth restriction , neonatal asphyxia , low birth weight , preterm delivery , and neonatal respiratory distress syndrome . however , a common robust biomarker has not been detected . in this study , we evaluate maternal and perinatal outcomes in women with pe according to the different value of albumin ( 25 g / l and < 25 hypoalbuminemia is considered a factor of poor maternal and neonatal outcomes . in this study , shp was a significant risk factor for cs ( or 2.99 , 95% ci 1.137.91 ) , poor maternal outcomes ( or 5.83 , 95% ci 3.3210.24 ) , and poor neonatal outcomes ( or 4.43 , 95% ci 2.577.62 ) in pe women . the systemic small artery of women with pe was in spasm ; protein absorption and utilization in the gastrointestinal tract were affected , often causing the emergence of hypoproteinemia . hypoalbuminemia indicates that the conditions are further exacerbated in pe and further affects the function of various organs , such as retinal damage , white matter lesions , pericardial effusion , ascites , and liver and kidney dysfunction . in our study , the presence of shp has been associated with increased risk of cs , severe hypertension , abnormal liver or renal function , ascites , and abruptio placenta . the percentage of cs in shp was 93.7% , higher than in the mhp group . the incidence of abnormal liver or renal function in shp ( 13.9% ) was significantly higher than in mhp ( 5.5% and 1.8% , respectively ) , and the difference was statistically significant ( p = 0.03 and < 0.01 , respectively ) . placental vascular spasm in pe increases blood flow resistance from mother to the fetus , resulting in chronic fetal hypoxia . with a high number of the mother s protein lost and protein and other nutrient deficiencies , these factors can lead to intrauterine growth restriction and low birth weight . women with shp pe were more likely to deliver preterm infants ( 41 , 51.9% , p < 0.01 ) , lower average birth weight ( 2498.1 866.5 vs. 2940.1 768.0 g , p < 0.01 ) , fgr ( 15 , 19.0% , p < 0.01 ) than those with mhp pe . it seems likely that many unknown risk factors may be present in women with pe , and it is unclear whether this finding occurred as a pathophysiology of latent pe or was responsible for it . however , we show here that shp is a risk factor for women with pe . shp pe is associated with a higher risk of adverse maternal and neonatal outcomes than mhp pe , deserving closer surveillance during pregnancy . hbc and xtw performed the design and coordinated the study , data collecting , agreed for all aspects of the work , and prepared the manuscript . ft provided assistance in the design , data collecting , analysis , and interpretation of data of the study and participated in manuscript preparation .

background : the aim of this study was to evaluate maternal and perinatal outcomes in preeclampsia ( pe ) , according to the value of albumin.materials and methods : preeclamptic women were retrospectively divided into mild hypoproteinemia ( mhp , n = 220 ) and severe hypoproteinemia ( shp , n = 79 ) pe according to the value of albumin . the maternal and perinatal outcomes were evaluated in both groups.results:two hundred and ninety - nine single pregnancies complicated by pe were included in this study . gestational age at delivery was earlier in shp than mhp ( p < 0.01 ) . severe hypertension , abnormal liver function , abnormal renal function , ascites , and abruption occurred more frequently in shp than in mhp ( p < 0.01 , 0.03 , < 0.01 , 0.01 , and 0.04 , respectively ) . women in shp had a higher rate of cesarean section than those in mhp ( p = 0.04 ) . fetal growth restriction infants were more frequent in shp than in mhp ( p < 0.01 ) . the occupancy rate of the neonatal intensive care unit was higher in shp than in mhp ( p < 0.01).conclusion : shp pe is associated with a higher risk of adverse pregnancy outcome than mhp pe , deserving closer surveillance during pregnancy .

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the generation of a humanized monoclonal antibody ( mab ) that inhibits the interaction of vegf - a with its receptors vegfr-1 and vegfr-2 and the development of small - molecule protein tyrosine kinase ( ptk ) inhibitors to the cytoplasmic regions of the vegf receptors have provided avenues to explore the efficacy of targeting the vegf - a signaling pathway in cancer . extensive mouse tumor models developed through the 1990s led to the success of the vegf - a mab known as avastin ( or bevacizumab ) in treating a range of human cancers including colorectal cancer , lung cancer , and renal cell carcinoma . however , the recent withdrawal of avastin for treating breast cancer in the united states has highlighted the limitations of anti - angiogenic treatments , as well as the need for a careful cost - benefit analysis to ensure that scarce healthcare resources are well spent in the treatment of cancer . while avastin is clearly beneficial in a subset of cancer patients , the development of resistance to avastin and the pre - existing refractoriness of some tumors suggest the role of other growth factors in sustaining tumor angiogenesis . alternative ligands ( other than vegf - a ) for the angiogenic receptor vegfr-2 ( i.e. , vegf - c and vegf - d ; see below ) and other angiogenic factors from distinct growth factor families [ e.g. , fibroblast growth factor ( fgf ) ] are logical candidates for promoting resistance to avastin . in addition , other mechanisms , such as vascular mimicry , the involvement of endothelial precursor cells ( epcs ) or altered tumor metabolism leading to reduced oxygen requirement , may also contribute to the development of resistance . pigf is a ligand for vegfr-1 , which has been reported to play a dual role in cancer as a stimulator of angiogenesis and as an autocrine or paracrine factor promoting proliferation of tumor cells expressing vegfr-11 , . it has also been proposed that pigf may modulate the effect of vegf - a by displacing vegf - a from vegfr-1 expressed on endothelial cells . a range of animal tumor models have provided supportive or contradictory data on the role of pigf in driving tumor angiogenesis and tumor growth , . these findings suggest a context - dependent activity for pigf in cancer , either due to the presence of other vegf family members or the diverse expression of vegf receptors on various cell types including endothelial cells , tumor cells , or associated immune cells , . while vegf - b , a ligand for vegfr-1 , has been shown to be expressed in human cancer , its functions in arteriogenesis and in fatty acid uptake by cells have highlighted its role in other pathologies , predominantly cardiovascular disease . recently , vegf - b was shown to regulate endothelial cell uptake and transport of fatty acids in muscle , leading to the hypothesis that vegf - b antagonists could be used as novel pharmacologic agents in the treatment of type 2 diabetes . although inhibitory antibodies against vegf - b have been developed , so far there is little data on the role of this growth factor in tumor biology ; it was reported , however , that overexpression of vegf - b in a mouse model of pancreatic cancer suppressed tumor growth , highlighting the differences in biological functions between the vegf family members . vegf - c and vegf - d form a subfamily within the vegf family , consisting of highly related polypeptides that require post - translational cleavage of the n- and c - terminal propeptides to generate mature forms with enhanced binding affinities for vegfr-2 and vegfr-3,. notably , vegfr-3 is expressed on lymphatic endothelium , where it can promote lymphangiogenesis , and it is also expressed on angiogenic blood vessels . vegf - c is expressed in a range of human cancers , including solid tumors such as nasopharyngeal , liver and gastric cancers , with a preponderance in asian populations . mouse tumor studies have displayed the ability of vegf - c to promote tumor angiogenesis and lymphangiogenesis in vivo and to drive tumor growth and metastatic spread. given that the mature form of vegf - c is a high affinity ligand for vegfr-2 and that vegf - c is expressed in many human cancers , vegf - c is likely to be an alternative ligand to vegf - a for vegfr-2binding , which could in turn promote tumor angiogenesis . therefore , vegf - c , in combination with anti - angiogenic drugs such as avastin , may be a viable target for anti - cancer therapy . the growth factor vegf - d is closely related in structure to vegf - c , and it includes a central vegf homology domain ( vhd ) related to other vegf family members , with n- and c - terminal propeptides that can be proteolytically cleaved by enzymes such as proprotein convertases and plasm in , , . vegf - d is expressed in a range of human cancers , , and has been associated with poor patient outcome in some tumor types , . importantly , animal models of cancer have demonstrated that vegf - d can promote tumor angiogenesis and lymphangiogenesis , solid tumor growth , dilation of collecting lymphatic vessels , and lymphatic and distant organ metastasis. the proteolytic processing of vegf - d is required for promoting tumor growth and spread . opportunities for targeting vegf - d signaling in cancer could involve mabs specific to the vhd of vegf - d that are capable of inhibiting binding to vegfr-2 and vegfr-3 , , . alternatively , ptk inhibitors that block vegfr-2 and vegfr-3 signaling would interfere with vegf - d - mediated signal transduction . further , mabs to vegfr-2 and vegfr-3 , that would prevent the binding of vegf - d , or a soluble form of vegfr-3 that could sequester both vegf - c and vegf - d , could be employed . targeting the vegf - d signaling pathway would likely have the merit of inhibiting both tumor angiogenesis and lymphangiogenesis , which could , in turn , restrict both solid tumor growth and metastatic spread . what have the past 20 years taught us about targeting vegf - a signaling ? clearly , this period has provided biochemists , biologists , and clinicians the time to design and evaluate a variety of agents that modify or inhibit these signaling pathways . the agents that were developed have included a broad range of molecules targeting different components of the pathways , including vegf - a itself , vegf receptors , vegf co - receptors , ptks and signaling intermediates , and transcription factors . during this period , other vegf family members were also identified , enhancing the diversity of signaling induced by the vegf family of ligands . avastin , a humanized mab to vegf - a , has been widely used in a range of prevalent human cancers over the past 8 years , typically in combination with cytotoxic chemotherapy . although this agent has provided significant benefit to cancer patients , there is a need for other drugs that could be combined with avastin to deliver improved clinical outcomes . use of agents targeting other vegf family members , in combination with avastin , may be a potential approach . further , small - molecule ptk inhibitors of vegf receptors ( that are not highly specific ) have been employed although dose - limiting toxicity in combination with cytotoxic chemotherapy has restricted their widespread use . further insights into the structure and function of the ptk domains of vegf receptors may allow development of more specific small - molecule ptk inhibitors . further studies delving into the complex network of signaling cascades that drive angiogenesis , lymphangiogenesis , and resistance to anti - angiogenic drugs such as avastin are clearly required . genome - wide functional approaches such as those using small interfering rna ( sirna ) technology , supported by bioinformatics , could help strategize effective targeting of growth factors , receptors , and ptks . agents specifically targeting vegf family members and their receptors are currently in various stages of development ; mabs to vegf - b , vegf - c , vegf - d , pigf , vegfr-1 , vegfr-2 , and vegfr-3 are being evaluated by the biotechnology and pharmaceutic industries for their efficacy as anti - cancer agents . for these mabs , a key challenge will be identifying specific cancer indications in which clinical benefit can be achieved . given that many cancers are resistant to avastin or develop resistance over the course of treatment , these mabs may be tested in patients whose cancers are avastin - resistant and/or whose cancers express the appropriate target growth factor . hence , assays that quantitate the levels of these growth factors or other relevant biomarkers will be required to identify the appropriate patients , although assessing these biomarkers may not be straightforward . vegf family members are secreted proteins , which may easily allow testing in blood - drawn samples ; however , some family members ( e.g. , vegf - c and vegf - d ) are proteo - lytically processed to generate forms with a different bioactivity . hence , it will be important to fully appreciate which forms are most bioactive and clinically relevant . it is important to note that inhibiting signaling by vegf family members could potentially have pro - tumor effects . recent studies have demonstrated that anti - angiogenic treatments could cause increased tumor metastasis through mechanisms such as the induction of hypoxia ( caused by vegf - a inhibition ) , resulting in increased tumor cell motility , . nonetheless , these issues have been addressed in various tumor models and by comparing the effect of antibodies and small - molecule inhibitors to show that anti clearly , other signaling pathways ( i.e. , not directly involving vegf family members ) such as those driven by members of the platelet - derived growth factor ( pdgf ) , insulin - like growth factor ( igf ) , and fgf growth factor families , exert control on tumor angiogenesis and may be important in promoting tumor growth . other alternative molecular targets , such as c - met , may also be able to affect multiple tumor properties such as metastasis , angiogenesis , tumor growth , and cellular motility upon hypoxia. although not the subject of this review , such pathways may provide valid therapeutic targets for inhibiting angiogenesis and/or lymphangio - genesis in cancer , thereby restricting tumor growth and spread . furthermore , recent studies from our own laboratory have indicated the role of known inflammatory pathways in enhancing the spread of cancer , opening avenues for the use of existing anti - inflammatory drugs in cancer . this possibility is further exemplified by the recent studies published by rothwell et al . , demonstrating the anti - cancer effect of non - steroidal anti- inflam matory drugs ( nsaids ) such as aspirin . while expensive drugs such as avastin allow the treatment of specific cancers in the western world , the capacity to employ relatively inexpensive off - patent small molecules ( such as aspirin ) with a defined chemistry and known biological effects could provide a cost - effective and population - based preventative approach to cancer in asian countries .

the vascular endothelial growth factor ( vegf ) family of soluble protein growth factors consists of key mediators of angiogenesis and lymphangiogenesis in the context of tumor biology . the members of the family , vegf - a ( also known as vegf ) , vegf - b , vegf - c , vegf - d , and placenta growth factor ( pigf ) , play important roles in vascular biology in both normal physiology and pathology . the generation of a humanized neutralizing antibody to vegf - a ( bevacizumab , also known as avastin ) and the demonstration of its benefit in numerous human cancers have confirmed the merit of an anti - angiogenesis approach to cancer treatment and have validated the vegf - a signaling pathway as a therapeutic target . other members of the vegf family are now being targeted , and their relevance to human cancer and the development of resistance to anti - vegf - a treatment are being evaluated in the clinic . here , we discuss the potential of targeting vegf family members in the diagnosis and treatment of cancer .

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malaria is still one of the main sources of consideration for public health in many countries including iran , particularly in south and southeast regions of the country . deliberate implementation of control measures has resulted in a substantial reduction of malaria incidence in iran in recent years ( 1 ) , therefore malaria cases were reduced from 11,460 cases in 2008 to 3,239 in 2011(iranian center for disease management and control , cdmc , unpublished data ) . in iran , p. vivax is responsible for the majority of malaria cases while plasmodium falciparum accounts for 1015% of the cases ( cdmc , unpublished ) . iran entered the malaria pre - elimination program since 2009 and interventions such as preventing strategies , active case detection and timely appropriate treatment of patients have been performed in all endemic areas of the country . in order to achieve malaria elimination , which is a global concept to reduce the incidence of locally acquired infection into zero , early recognition of all parasite carriers bearing various disease symptoms and parasite rates is crucial . in this situation , active surveillance by precise laboratory methods in endemic areas provides an appropriate tool for detection of all kinds of infections particularly asymptomatic ones containing low parasite densities ( 2 ) . formerly asymptomatic malaria infections were described in hyper - endemic regions following frequent infective bites and repetitive immune system response toward the parasite attenuating parasitemia to undetectable levels in blood smear ( 3 , 4 ) . the prevalence of this kind of malaria infection was determined by means of different laboratory techniques in various endemic countries previously ( 59 ) , in parallel with other counties there are a few studies in iran which assessed asymptomatic malaria prevalence in different low endemic areas of the country ( 1012 ) . based on the controversial results of previous studies which show the fluctuation of asymptomatic infection among the plenty of endemic regions , the importance of regional acquiring of symptomless infection another highly important issue that has to be taking account in asymptomatic malaria investigation is using accurate detection methods to find all carriers , principally who does not have clinical malaria symptoms . this highlights the vitality of evaluating diagnostic method s delicacy for further decisions on malaria elimination interventions . to date , light microscopy of giemsa - stained thick and thin blood smears has been remained as the standard laboratory method of malaria diagnosis due to specificity and ability to quantify the level of parasitemia ( 13 ) . however , the reliance to the results of light microscopy is restricted due to the limitations associated with this method ( 14 ) . although microscopy is the most commonly used method of malaria diagnosis as the gold standard in iran , chromatographic rapid diagnostic tests ( rdts ) are introduced in remote settings , where microscopic diagnosis is either impractical or unfeasible and as a complementary method for confirming the parasite - infected cases . besides the mentioned methods , the usefulness of molecular methods such as nested - polymerase chain reaction ( pcr ) has been emphasized as highly sensitive means of malaria detection , particularly in sub - clinical parasitemia levels ( 1517 ) . the aim of this study was to detect low parasite and asymptomatic malaria infections by means of three malaria diagnostic tests ( microscopy , rdt and nested - pcr ) , in a low transmission region of bagh - e - malek in minab district , hormozgan province , southern iran . the results of this investigation would help further case surveillance strategies in the region and additionally all endemic settings of iran , as an essential approach toward achieving malaria elimination all over the country . the present study was performed as a cross - sectional survey in bagh - e - malek region , which is located in minab district , southeast of hormozgan province . hormozgan is one of the endemic areas in southern iran , containing about 10.8% of all iranian malaria patients in 2011 ( 349 cases reported based on the iranian cdmc unpublished report ) ( fig . the higher rate of transmission has been reported from bagh - e malek area , therefore this region was selected for this project . bagh - e - malek is a lowland area surrounded by mountains , which isolate it from the neighboring regions in minab , which has a population of 1,451 people . the annual average temperature of minab is 28 c ; pluviometer index of 249 mm and an annual relative humidity of 55.5% . iran s map , depicting the study area the total malaria cases in this area were 15 in 2011 ( iranian cdmc ) , while plasmodium vivax is the predominant species in bagh - e malek and whole province , also the chief anopheles vectors in minab are anopheles stephensi , an . . mainly two annual peaks of malaria are seen in this area ; which occurs in may and october . a total of 200 healthy volunteers were participated in this study from bagh - e malek area ( 62 males and 138 females ) . briefly 3 ml fresh blood sample were collected during july 2011 from the participants after getting informed consent , for further microscopic , rdt and molecular analysis . the study was approved by the ethics committee of hormozgan university of medical sciences ( approval no . inclusion criteria such as lack of having any malaria symptom or receiving anti - malarial drug in three weeks prior to the study and having no travel history to other malaria endemic areas in the past three months were considered in sample collection . thin films fixed using methanol then every slide stained with 10% giemsa and examined at 1000 with oil immersion to inspect malaria parasites by a skilled microscopist . further thick and thin blood smear preparation were carried out from all participants , 30 days following the sampling date as well as 60 days afterwards , in order to follow up the samples . all specimens were tested with the first response malaria pldh / hrp2 combo assay ( premier medical corporation ltd . the kit contains a two line strip pre - coated with two monoclonal antibodies , one against the pan - specific lactate dehydrogenase , pldh , of the plasmodium species ( p. falciparum , vivax , malariae , ovale ) and the other antibody was against p. falciparum species histidine - rich protein 2 ( hrp2 ) . briefly , 5 l of whole blood was dispensed onto sample well followed by two drops of assay buffer ( 60 l ) into developer well . the test control line was observed as an indicator , in order to validate the performance of the test . finally , differential diagnosis between p. falciparum and non - falciparum species were interpreted as described previously ( 19 ) . the molecular detection of plasmodium species was performed using nested - pcr amplification of 18 ssrrna of the parasite . to achieve this purpose , dna was extracted from whole blood samples using promega kit ( promega , madison , wi , usa ) and stored at 20c until further analysis with primers and cycling conditions that described previously ( 11,20 ) . in brief , 5 l of extracted dna was used in the first nest , by use of primers targeting an external region specific to the plasmodium genus followed by second nest using 2 l of the first nested - pcr product as template to amplify specific p. vivax and p. falciparum fragments using internal primers . amplification was performed in a total volume of 25 l including , 25 l of templates , 250 nm of primers , 10 mm tris hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 125 m of each of the four deoxynucleotide triphosphates and 0.4 u of taq polymerase ( invitrogen , carlsbad , ca ) . first and second nests repeated for 25 and 30 cycles , respectively while the annealing temperature was 72 c for both reactions . amplicons from the second nest of pcr were electrophoresed on 2% agarose gel and visualized under uv light after staining with ethidium bromide . a sample was considered positive for p. vivax and p. falciparum if a 120 and 419 base - pair fragment was detected , respectively . the present study was performed as a cross - sectional survey in bagh - e - malek region , which is located in minab district , southeast of hormozgan province . hormozgan is one of the endemic areas in southern iran , containing about 10.8% of all iranian malaria patients in 2011 ( 349 cases reported based on the iranian cdmc unpublished report ) ( fig . the higher rate of transmission has been reported from bagh - e malek area , therefore this region was selected for this project . bagh - e - malek is a lowland area surrounded by mountains , which isolate it from the neighboring regions in minab , which has a population of 1,451 people . the annual average temperature of minab is 28 c ; pluviometer index of 249 mm and an annual relative humidity of 55.5% . iran s map , depicting the study area the total malaria cases in this area were 15 in 2011 ( iranian cdmc ) , while plasmodium vivax is the predominant species in bagh - e malek and whole province , also the chief anopheles vectors in minab are anopheles stephensi , an . . mainly two annual peaks of malaria are seen in this area ; which occurs in may and october . a total of 200 healthy volunteers were participated in this study from bagh - e malek area ( 62 males and 138 females ) . briefly 3 ml fresh blood sample were collected during july 2011 from the participants after getting informed consent , for further microscopic , rdt and molecular analysis . the study was approved by the ethics committee of hormozgan university of medical sciences ( approval no . inclusion criteria such as lack of having any malaria symptom or receiving anti - malarial drug in three weeks prior to the study and having no travel history to other malaria endemic areas in the past three months were considered in sample collection . thin films fixed using methanol then every slide stained with 10% giemsa and examined at 1000 with oil immersion to inspect malaria parasites by a skilled microscopist . further thick and thin blood smear preparation were carried out from all participants , 30 days following the sampling date as well as 60 days afterwards , in order to follow up the samples . all specimens were tested with the first response malaria pldh / hrp2 combo assay ( premier medical corporation ltd . , the kit contains a two line strip pre - coated with two monoclonal antibodies , one against the pan - specific lactate dehydrogenase , pldh , of the plasmodium species ( p. falciparum , vivax , malariae , ovale ) and the other antibody was against p. falciparum species histidine - rich protein 2 ( hrp2 ) . briefly , 5 l of whole blood was dispensed onto sample well followed by two drops of assay buffer ( 60 l ) into developer well . the test control line was observed as an indicator , in order to validate the performance of the test . finally , differential diagnosis between p. falciparum and non - falciparum species were interpreted as described previously ( 19 ) . the molecular detection of plasmodium species was performed using nested - pcr amplification of 18 ssrrna of the parasite . to achieve this purpose , dna was extracted from whole blood samples using promega kit ( promega , madison , wi , usa ) and stored at 20c until further analysis with primers and cycling conditions that described previously ( 11,20 ) . in brief , 5 l of extracted dna was used in the first nest , by use of primers targeting an external region specific to the plasmodium genus followed by second nest using 2 l of the first nested - pcr product as template to amplify specific p. vivax and p. falciparum fragments using internal primers . amplification was performed in a total volume of 25 l including , 25 l of templates , 250 nm of primers , 10 mm tris hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 125 m of each of the four deoxynucleotide triphosphates and 0.4 u of taq polymerase ( invitrogen , carlsbad , ca ) . first and second nests repeated for 25 and 30 cycles , respectively while the annealing temperature was 72 c for both reactions . amplicons from the second nest of pcr were electrophoresed on 2% agarose gel and visualized under uv light after staining with ethidium bromide . a sample was considered positive for p. vivax and p. falciparum if a 120 and 419 base - pair fragment was detected , respectively . this investigation was carried out in order to assay the concordance between the outcomes of routine malaria diagnostic techniques with nested - pcr detecting method in finding asymptomatic cases in elimination phase of malaria control . hence , blood samples and smears were collected from 200 individuals residing in the endemic region of bagh - e - malek located in minab district , hormozgan province of iran . all of specimens were evaluated using microscopic , rdt and nested - pcr analysis in hormozgan provincial health center laboratory . all of participants were healthy individuals and age range was from 3 to 60 years old ( mean age 27.2 ) ( table 1 ) . details of demographic data and the frequency of asymptomatic infection detected by use of nested - pcr the entire 200 thick and thin giemsa - stained blood smears were inspected to detect plasmodium parasite by well - trained microscopist in hormozgan provincial health center laboratory and no plasmodium parasite were detected in any examined sample . additionally none of samples was diagnosed to have any malaria parasite during the 60-days follow up protocol , while all of samples remained symptomless . rdts were tested as a complementary analysis for all of 200 samples using monoclonal antibody coated sticks . no positive result was discovered for either p. falciparum and/or non - falciparum species in the performed tests . in this cross sectional study , nested - pcr amplification was performed as a sensitive and specific method to find parasite genome in extracted dna from asymptomatic samples . interestingly 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr using specific primers , although they were considered uninfected using microscopic and rdt tests ( table 1 ) . these cases were considered as asymptomatic malaria patients since they remained symptomless in follow up evaluations ( i.e. thick blood smear and rdt ) prior to treatment . all positive cases were female and only one of them had a malaria history 20 years prior to the time of this investigation . moreover , no plasmodium falciparum infection was detected by this technique for all examined samples . all infected subjects were treated after diagnosis by standard anti - malarial drugs according cdmc of iran protocol and reevaluated after treatment by means of molecular method . the entire 200 thick and thin giemsa - stained blood smears were inspected to detect plasmodium parasite by well - trained microscopist in hormozgan provincial health center laboratory and no plasmodium parasite were detected in any examined sample . additionally none of samples was diagnosed to have any malaria parasite during the 60-days follow up protocol , while all of samples remained symptomless . rdts were tested as a complementary analysis for all of 200 samples using monoclonal antibody coated sticks . no positive result was discovered for either p. falciparum and/or non - falciparum species in the performed tests . in this cross sectional study , nested - pcr amplification was performed as a sensitive and specific method to find parasite genome in extracted dna from asymptomatic samples . interestingly 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr using specific primers , although they were considered uninfected using microscopic and rdt tests ( table 1 ) . these cases were considered as asymptomatic malaria patients since they remained symptomless in follow up evaluations ( i.e. thick blood smear and rdt ) prior to treatment . all positive cases were female and only one of them had a malaria history 20 years prior to the time of this investigation . moreover , no plasmodium falciparum infection was detected by this technique for all examined samples . all infected subjects were treated after diagnosis by standard anti - malarial drugs according cdmc of iran protocol and reevaluated after treatment by means of molecular method . in the elimination phase of malaria control , monitoring all cases particularly hidden parasites is of major importance . this highlights choosing of an acceptable detecting method , which should be , applied in all incoming asymptomatic investigation afterwards in order to detect every single carrier which continue disease transmission in the population ( 21 ) . currently in malaria endemic areas of iran , microscopic and rdt assays are used for active case surveillance , although these methods could detect nearly all of symptomatic infections , in case of asymptomatic and mixed infections a more reliable technique would be helpful especially in elimination program as a public health concern in the country . although , microscopy is still the diagnosis gold standard , limitations such as requiring well skilled microscopists , technical equipment maintenance , discrepancy in microscopist training and insufficient quality control are associated with this method ( 22 ) . although expert microscopy could theoretically detect 10 p/l parasitemia , average microscopy has proven to be unable in detecting lower than 100 p/l parasitemia ( 23 ) . however , it should be mentioned that microscopy is the primary and easiest to establish method in the field situation . alternatively , despite the simplicity and being easy to use without particular training which highlight the role of rdts in malaria detection , they may have some disadvantages ; for instance , the intensity of test band is subject to amount of antigen particularly low parasite densities , and this may lead to reader variation in test results . furthermore , the short shelf life of rdts emphasizes requiring of proficient transportation , storage and distribution systems , additionally , diagnose of asymptomatic malaria infection realized to be unreliable using rdts ( 2325 ) . in addition to the importance of microscopy and rdt , mainly in field settings , in a number of recent investigations molecular methods were nominated as valuable and robust approaches particularly in detecting sub - clinical parasitaemia i.e. asymptomatic carriers , mixed infections and confirmation of plasmodium species in low - transmission areas ( 2629 ) . however , regarding pcr expenses and equipment requirements in contrast to usual diagnosis methods , the performance of nested - pcr in identifying asymptomatic carriers was compared to conventional microscopic and rdts in symptomless population of bagh - e - malek , a malaria endemic region , in southern iran . interestingly nested - pcr found three p. vivax carriers while microscopy and rdt missed the cases , one of the patients was a 12-year - old symptomless girl , who experienced a period of fever in the year prior to the study , and her disease has not been diagnosed since her thick and thin blood smears analyzed negative by light microscopy . the blood films of this patient remained negative even in re - sampling after identifying a positive pcr result . the current results emphasize the importance of using molecular methods in order to interrupt malaria transmission in iran besides microscopy and rdts as routine methods . it should be noted that toward eliminating malaria from the country and prevention of re - introduction , all the three types of malaria detection tools ( microscopy , rdt and molecular methods such as nested - pcr ) should be used to improve sensitivity , specificity and reliability for active case surveillance in endemic settings .

background : the aim of this study was to detect low parasite and asymptomatic malaria infections by means of three malaria diagnostic tests , in a low transmission region of minab district , hormozgan province , southern iran.methods:blood samples of 200 healthy volunteers from bagh - e - malek area were evaluated using microscopic , rapid diagnostic tests ( rdt ) and nested - pcr to inspect malaria parasite.results:the results showed no plasmodium parasite in subjects by means of microscopy and rdt . however , 3 p. vivax positive samples ( 1.5% ) were discovered by nested - pcr while microscopy and rdt missed the cases.conclusion:microscopy as the gold standard method and rdt correctly identified 98.5% of cases , and molecular analysis is sensitive and reliable , especially in the detection of asymptomatic infections for active case surveillance . regarding the existence of asymptomatic malaria in endemic area of hormozgan , iran , nested - pcr could be considered as a sensitive tool to interrupt malaria transmission in the country , beside the microscopic and rdt methods .

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over the last two decades many new immunotherapy approaches to the treatment of cancer have entered clinical development due to the increased understanding of the mechanisms of antigen presentation , lymphocyte recognition , functions of the innate immune system , and the means of regulation of these responses and the means by which tumors can circumvent these responses . many of these investigations have led to agents approved for standard clinical use , including infusional cytokine therapies for melanoma or renal cell cancer , intravesical bcg therapy for bladder cancer , and most recently an active cellular therapy targeting prostatic acid phosphatase ( pap , sipuleucel - t , provenge , dendreon ) for patients with advanced metastatic prostate cancer . many other agents have demonstrated benefit in large clinical trials , and approval is anticipated in the case of a monoclonal antibody targeting a t - cell checkpoint inhibitor targeting ctla-4 ( ipilimumab , bristol - myers squibb ) for advanced melanoma . ultimately , for these agents to be clinically approved there needs to be a demonstration that these treatments are relatively safe and patient care and outcome are positively affected . however , there is also an increasing recognition that some of these agents , while likely safe , may best be used in combination with other immune - activating or conventional therapies . this has presented challenges for evaluating these agents using traditional paradigms for clinical development . consequently there is a need to identify markers of biological response , ideally associated with clinical outcome , but permitting an evaluation of biological effect of these agents used in combination . in the case of antigen - specific vaccines , it has been relatively straightforward to evaluate immune responses to the target antigen as a biomarker of immunological efficacy . unfortunately , there are few instances in which target antigen immune response has been associated with clinical benefit . the situation is more difficult for broadly active immune modulating agents such as t - cell checkpoint inhibitors , including antibodies targeting ctla-4 or pd-1 , or tlr agonists , in which appropriate biomarkers of response have been more elusive . studies with anti - ctla-4 monoclonal antibodies , in particular , have sought to identify whether amplification of other t - cell costimulatory molecules , or antibodies to defined antigenic tumor - associated proteins [ 2 , 3 ] , might be useful as biomarkers . for whole cell tumor vaccines where there is not a specific , defined antigen being targeted , surrogate antigens known to be expressed by the tumor vaccine have been used as a means of monitoring immune responses from the vaccine . the use of immunologically recognized surrogate antigens , including her-2/neu , muc1 , and p53 , has been possible in the case of breast cancer where t - cell and igg responses to these antigens have been identified . however it is unknown whether responses to these antigens can be useful to study agents in combination or whether changes in responses to these antigens are associated with clinical outcome . over the last several years we have used serex- ( serological analysis of recombinant cdna expression libraries ) based studies to identify immunologically recognized proteins expressed by normal and malignant prostate tissue that might serve as targets for anti - tumor vaccines . in particular , we have evaluated the targets of igg responses in patients with chronic prostatitis or autoimmune disorders [ 8 , 9 ] , patients with prostate cancer treated with immune - modulating therapies , and igg responses to cancer - testis antigens in patients with prostate cancer [ 11 , 12 ] . over the course of these studies we have effectively identified hundreds of immunologically recognized proteins associated with prostate tissue and/or recognized by patients with prostate cancer . while the identification of hundreds of proteins presents challenges in prioritization for the development of antigen - specific vaccines , we previously questioned whether these antigens might also have diagnostic value with igg responses being able to distinguish individuals with prostate cancer ( or other inflammatory conditions of the prostate ) from men without prostate disease . other groups have similarly reported that igg responses to tissue - associated antigens might have diagnostic value in identifying patients with prostate cancer or nonsmall cell lung cancer . we have previously reported that a subset of 23 of these antigens were recognized in patients with prostate cancer as well as individuals with symptomatic prostatitis , suggesting that such autoantibody signatures might be useful to identify inflammatory conditions of the prostate , and potentially in a premalignant setting . in the current report , we hypothesized that this same panel of previously identified prostate - associated antigens might be used as a monitoring tool to assess immune responses elicited following immune - modulating therapy . while b - cells or igg production might not be an intended target of a particular therapeutic approach , igg responses are often elicited with concurrent t - cell activation . we reasoned that igg responses are easier to measure compared with antigen - specific t cells , and might be more stable over time in the peripheral blood compared with t - cell frequencies . moreover , the identification of off - target igg immune responses might further serve as an indication of antigen spread with secondary antigens recognized following immunological targeting and thus be more relevant to developing biomarkers associated with favorable clinical responses . to detect antibody responses to previously defined antigens , we applied a similar phage immunoblot approach evaluating igg responses to multiple antigens simultaneously . these types of complex biomarker data sets are historically very difficult to work with for two reasons : first is the complexity associated with biological networks ; second is the challenge of infrequent observation of immune biomarkers in a complex system . as such , the identification of useful biomarkers in data sets such as this study can be very challenging . in this paper , we sought to evaluate the use of machine - learned bayesian belief networks ( ml - bbns ) as a method for identifying potentially promising biomarkers and potential biomarkers networks [ 16 , 17 ] . we sought to train several ml - bbns to identify promising biomarkers and then use these networks to select a subset of features to train a network of immune biomarkers as they related to observed declines in serum prostate - specific antigen ( psa ) . our objective was to demonstrate the feasibility of this method to identify promising early biomarkers of immune response to vaccine therapies in our data . for the current studies , sera samples were collected prior to treatment and after three months of treatment from three separate trials , one in which patients ( n = 34 ) were treated with androgen deprivation ( adt ) therapy only , a standard therapy known to elicit prostate - associated immune responses [ 1820 ] , a trial in which patients with castrate - resistant prostate cancer ( n = 31 ) were treated with a viral vaccine encoding psa ( psav ) , and one in which patients with early recurrent prostate cancer ( n = 21 ) were treated with a plasmid dna vaccine encoding pap ( papv ) . patients treated with vaccines were subclassified as immunologic or clinical responders based on previously reported criteria to distinguish these groups . we report here that igg immune responses could be detected to individual antigens , and as long as one year after therapy the recognition of specific antigens was associated with individual treatments . the evaluation of igg responses to groups of antigens at three months suggests that predictive models might be developed with diagnostic potential . these findings support the concept of using measures of antigen spread as biomarkers of immunological efficacy for immune - active therapies , and igg responses to panels of tissue - associated antigens as measures of this antigen spread . sera used for the studies had been previously collected with irb - approved , written consent as part of three separate clinical trials ( figure 1 ) . these samples were all obtained prior to study treatment and after 3 months of treatment in the following settings : ( 1 ) a trial ( adt ) in which patients ( n = 34 ) with psa - recurrent , or newly metastatic prostate cancer , who had never previously received androgen deprivation , received 22.5 mg leuprolide by intramuscular injection with or without daily oral bicalutamide ; ( 2 ) a trial ( psav ) in which patients ( n = 31 ) with castrate - resistant , metastatic prostate cancer were vaccinated at 2-week intervals with a poxviral vaccine ( prostvac , vaccinia virus encoding psa priming immunization followed by fowlpox virus encoding psa booster immunizations ; ( 3 ) a trial ( papv ) in which patients ( n = 21 ) with psa - recurrent nonmetastatic prostate cancer , not receiving androgen deprivation , were vaccinated at 2-week intervals with a plasmid dna vaccine encoding pap . from the majority of patients treated with adt ( n = 24 of 34 ) or papv ( n = 19 of 21 ) , serum samples were also available 12 months after the baseline sample . from the vaccine studies , patients were grouped as clinical responders or non - responders as simply defined by a decrease in serum psa level at the 3-month time point relative to the baseline value . 100,000 pfu lambda phage encoding 126 unique antigens were spotted manually in triplicate in a 1624 array onto xl-1 blue e. coli . these individual antigens included 29 cancer - testis antigens , 40 proteins identified in patients with chronic prostatitis , and 57 antigens identified in individual patients , some of whom were treated with androgen deprivation or other immunomodulatory therapies [ 9 , 10 , 20 ] . a listing of antigens and their genbank accession numbers is included in supplemental table 1 in supplementary material available online at doi:10.1155/2011/454861 . plates were allowed to air - dry after which 10-mm isopropyl - d - thiogalactopyranoside- ( iptg ) suffused nitrocellulose membranes were overlain , and plates incubated at 37c overnight to allow recombinant protein expression . membranes were then washed , blocked , and probed with sera from patients pre- or post - treatment , diluted 1 : 100 in isotonic buffer . human igg was then detected with an igg - specific secondary antibody conjugated to alkaline phosphatase and immunoreactivity detected by development with 0.3 mg / ml nitro blue tertazolium chloride ( nbt ) ( fisher biotech ) and 0.15 mg / ml 5-bromo 4-chloro 3-indoylphosphate ( bcip ) ( fisher biotech ) . membranes were scanned and the digital format was assessed visually , with individual plaques scored positive or not by four independent observers , blinded to the treatment , timing of sample acquisition and membrane layout , as previously reported [ 8 , 15 ] . all of the membranes for the entire study were evaluated by the same observers at the same time . triplicate samples were evaluated for each antigen , and immunoreactivity to individual antigens was scored positive if there was concordance among 3 of 4 observers , and if immunoreactivity was scored positive in at least two of the three replicates . heatmap builder software ( version 1.1 , stanford university ) was used to generate heatmaps displaying changes ( gain , loss , or no change ) of antibody immune responses following treatment . our statistical analysis consisted of using a commercially available machine - learning software package ( fasteranalytics , decisionq corporation , washington , dc ) . machine learning is a field of computer science that uses intelligent algorithms to allow a computer to mimic the process of human learning . machine learning algorithms allow the computer to learn dynamically from the data that resides in the training dataset , detecting associations between features without human supervision . decisionq software generates several networks simultaneously and then continues to generate new hypotheses for each network . the software promotes the network with the best score as determined by goodness of fit relative to compactness . this allows for de novo exploration of associations in complex data sets . in preprocessing our data , we compared the pre- and post - treatment status of biomarkers and encoded the change in each biomarker as a feature . we then used these encoded features and clinical response ( psa decline ) to train models . the output of our machine - learning algorithms is a bayesian belief network ( bbn ) . a bbn encodes the joint probability distribution of all the variables in the domain by building a hierarchical network of conditional dependence . the graphical nature of the network allows the user to query the structure of conditional dependence to identify those features which provide the most information content in the network . in order to select a subset of features for inclusion in a final model , we used a stepwise process and trained a series of machine - learned ( ml)bbns for feature selection . we used this stepwise process as a means of identifying nodes with relatively high information content given our statistically challenging biomarker data sets . because these data sets have a very high degree of dimensionality ( features ) relative to evidence ( number of subjects ) , finding those features with the highest information content can be very challenging . to address this challenge , we trained multiple bbn - ml models and identified those features which recurred across multiple models as evidence of high information content . we modeled each of our study cohorts ( adt , psav , papv ) and then compared to the model structures between individual cohorts to identify shared nodes . we also identified high - content nodes ( greater than 10 associations ) and combined these with the shared nodes to create a selected subset or training a final model to evaluate a network of biomarkers to evaluate clinical response ( psa decline ) . we used our selected markers to then train three additional models : ( i ) a final subset model including clinical response ( psa decline ) on the vaccine cohorts , ( ii ) a model of subjects in the vaccine studies who were immune responders , and ( iii ) a model of subjects who were not immune responders in the vaccine studies . finally , we performed tenfold cross - validation on our clinical response subset model and used receiver operating characteristic ( roc ) curve analysis to calculate an area - under - the - curve ( auc ) metric for the feature psa decline , to determine if the subset model could robustly classify clinical response given immune biomarkers . the frequencies of igg responses to individual antigens were compared between treatment study populations using a chi - square test . we have previously reported that antibody responses to prostate antigens can be detected in patients with prostate cancer or other inflammatory conditions of the prostate . moreover , a subset of these prostate - associated antigens was commonly recognized in patients , relative to men without prostate disease , suggesting that the detection of igg responses to specific prostate - associated antigens might have diagnostic value . in the current analysis , we wished to determine whether the detection of igg responses to a panel of prostate - associated antigens might have utility in the evaluation of vaccine or other immunomodulatory therapies aimed specifically at eliciting immune responses to the prostate . for this , we obtained sera from men with prostate cancer prior to and following three months of therapy with standard androgen deprivation therapy ( n = 34 ) , and from men with prostate cancer ( n = 52 ) prior to and following three months of therapy with one of two different antigen - specific vaccines ( figure 1 ) . sera from these individuals were used to screen for igg responses to a panel of 126 antigens by immunoblot , as previously described . responses to all antigens were evaluated in blinded fashion at both time points , and in figure 2 , changes in igg responses ( gain or loss of response ) after 3 months were determined . as demonstrated , androgen deprivation elicited immune responses to multiple antigens , and in particular to antigens previously identified as antigens recognized in patients with chronic prostatitis [ 8 , 15 ] . responses to these prostatitis antigens were uncommon over a similar 3-month period in patients treated with either of the vaccines . gain or loss of igg responses to some antigens appeared to be shared by these different treatments , while responses to some appeared more specific for individual treatments . of note , gain or loss of igg responses to psa , while detected in one individual treated with adt , were not detected in patients receiving the psa - tricom vaccine . similarly igg responses to pap were not detected in any of the patients , including those receiving the pap - targeted vaccine , as previously reported . we next wanted to determine whether igg responses observed were generally stable , or increased over time , and also identify more specifically whether responses to some antigens were more generally associated with different therapies . in the majority of patients treated with adt and papv , evaluation of igg responses gained or lost after 12 months of adt , or 12 months after pap vaccine treatment compared with baseline demonstrated overall an increased number of antigens recognized ( figure 3 ) . interestingly , responses to individual antigens were observed to be highly specific for the treatment . for example , igg responses were elicited to the ribosomal l5 protein in 8/24 patients receiving adt , and 0/19 patients after receiving the papv ( p = .005 , chi - square test ) . similarly , igg responses elicited to the neuronal pas domain protein 2 ( npas2 ) antigen were observed more frequently in patients receiving the pap vaccine ( 5/19 ) compared with patients receiving androgen deprivation ( 0/24 , p = .0075 , chi - square test ) . even after one year , responses gained or lost to antigens previously identified as prostatitis antigens were not detected in patients treated with the vaccine . while we did not have access to a control population of sera from untreated men , given that these represented populations of subjects with nearly identical stage of disease , collected at the same institution , the differences in igg response patterns to individual antigens appears most related to the difference in treatment . moreover , these findings suggest that igg responses are elicited to off - target antigens by means of prostate - directed therapies , and the patterns of igg responses differ with respect to therapy . the results above demonstrated that , indeed , igg immune responses are elicited as a result of prostate - directed immune - active therapies . moreover , antigen - specific igg immune responses were highly associated with specific treatments , suggesting that the generation of immune responses to these off - target antigens might be associated with other measures of immune response or clinical outcome . these responses , however , were most detectable at 12 months after therapy initiation , a time relatively late to be useful in most circumstances as a predictive biomarker . responses detectable at three months would be more useful . however , the sample sizes for each individual trial were small , and multiple comparisons made by evaluating igg responses to multiple antigens present difficulties in statistically assessing the importance of an individual marker . consequently , we evaluated igg responses to multiple antigens by training a ml - bbn model to determine whether we could identify groups of igg responses that are associated with clinical response , using data obtained at three months . because the vaccine trials were conducted in different patient populations where different definitions of clinical response were used , we defined it simply for this purpose as a serum psa value at the 3-month time point lower than the baseline time point ( n = 1 for the papv trial , and n = 4 for the psav trial subjects ) . we identified nine ( 9 ) biomarkers that were shared between one or more model structures , as described in table 1 , as well as two high - content nodes in the all - cohort model . this resulted in a final subset of biomarkers to include in the final ml - bbn model : chromosome 20 gene contig change , rp11 - 738b7 dna on chromosome 7 change , chromosome 1 gene contig change1 , prolactin - induced protein change , acetyl - coenzyme a acyltransferase 1 change , bac rp11 - 321g3 change , cutaneous t - cell lymphoma tumor antigen sel-1 change , neuronal pas domain protein 2 change , o - fucosyltansferase change , page 1 change , and recombination signal binding protein ( rbpjk ) change . this indicates that there are two first - degree associates of psa decline , igg responses to chromosome 1 gene contig 1 and bac rp11 - 321g3 , and three immune biomarkers features which can be used to estimate psa decline : igg responses to chromosome 1 gene contig 1 , bac rp11 - 321g3 , and rp11 - 738b7 dna on chromosome 7 . further , these biomarkers are associated with igg responses to chromosome 20 gene contig , o - fucosyltansferase , page 1 , and cutaneous t - cell lymphoma tumor antigen sel-1 . to evaluate the robustness of this model , we performed tenfold cross - validation and calculated an auc for clinical response ( psa decline ) of 0.357 . this indicates that our first model is not a robust classifier , but is rather an exploratory model . in the current report , we sought to determine whether serum antibody responses to a panel of prostate tissue- and prostate cancer - associated antigens might be developed as a diagnostic tool to evaluate immune responses elicited following immune - active therapies , and further to determine whether this might be developed in the future as a biomarker of clinical response . using sera obtained from patients treated with three different therapies , we found that antigen - specific igg responses could be detected , likely elicited as a result of therapy . the patterns of response differed with respect to the individual therapy , and recognition of specific antigens was most evident at a later ( 12 months following treatment ) than at an earlier time point ( 3 months following treatment ) . using a ml - bbn model to evaluate groups of igg responses detected three months after treatment , we prioritized a cohort of antigens , immune responses to which were most associated with psa decline . these findings suggest that , with data from larger populations of subjects , models could be developed to assist in the detection of potentially therapeutic immune responses resulting from immune - based therapies . our results demonstrate that immune - active therapies , including androgen deprivation , elicit igg responses to individual prostate - associated antigens . this has already been demonstrated in previous studies [ 18 , 20 ] ; however , the igg responses from androgen deprivation therapy were most obvious many months after treatment , where responses to individual antigens were common and predominantly induced rather than lost . it is conceivable that some immune responses wax and wane over time , and in fact the detection of frequent gains and losses of immune responses to individual antigens , common across treatment groups detected earlier at three months , suggests that this can happen with some antigens . ideally , to control for this , we would have preferred to have sera samples from men without prostate cancer and/or not undergoing active treatment over the same periods of time . in the absence of this , however , we did have cohorts of subjects treated with different therapies . given that different individual and sets of antigens were specifically recognized following these different therapies suggests that the responses observed were not purely by chance or due to the waxing and waning of responses to individual antigens . the antigens recognized following androgen deprivation , in particular , were ones previously demonstrated to be commonly recognized by igg in patients with prostate cancer or inflammatory conditions of the prostate [ 8 , 15 ] . we did not observe igg responses to pap in patients receiving the pap - targeted vaccine , nor igg responses to psa in patients receiving the psa - targeted vaccine . this was actually not unexpected , as we have previously reported that these vaccines , while able to elicit antigen - specific t - cell responses , do not elicit robust antigen - specific igg in patients as detected by more sensitive elisa methods [ 6 , 22 ] . the observation of igg responses elicited with these treatments to other antigens suggests that they may be presented by cross - presentation following immune - mediated tumor cell targeting . of interest was the observation that the antigens recognized following androgen deprivation were different from those recognized following vaccine treatment . theoretically , the recognition of other nonvaccine target antigens represents antigen spread induced by immune targeting and presentation of other tissue - associated antigens . the recognition of different antigens suggests different mechanisms of antigen spread , or potentially recognition of other tissue - derived antigens , since most of the antigens in this panel are not prostate specific in expression . at present it is unclear whether the generation of such responses is favorable or not ; at least one report has suggested that the generation of igg responses to non - target antigens might be associated with a worse outcome . future studies will explore whether the antigens recognized are shared among different vaccine approaches , suggesting common mechanisms of antigen spread , or whether different vaccine therapies elicit specific responses to different off - target antigens . with larger group sizes , we also hope to address whether responses to these antigens are associated with measures of t - cell immune responses to the target antigen , further implicating antigen spread as the mechanism of their recognition . in addition , with larger group sizes we hope to answer whether these are clearly associated with improved clinical benefit or not , or whether this is dependent on treatment context and the specific antigen(s ) recognized , as we expect . given the small sample size and the multitude of igg response data points , we sought to identify if the use of ml - bbn modeling was feasible to identify biomarker cohorts in our study data . we were able to use a stepwise process and bbn model structures to identify those biomarkers which had high information content for use in a selected subset for ml - bbn modeling . we were subsequently able to use this subset to train an ml - bbn including clinical response , however on cross - validation , our auc for clinical response was poor . this is likely due to the fact that of the 52 vaccine subjects we only had 5 responders as defined . this resulted in a very small set of training outcomes , making models very sensitive to record deletion , as in the case of cross - validation . psa response has itself not been validated as a surrogate clinical endpoint , and adt itself elicits initial psa responses in the vast majority of patients . in addition , as further data are collected from additional subjects treated by vaccines , we expect this will produce a more robust predictive model . in any case , the use of ml - bbn modeling appears to provide a promising method for identifying biomarkers in complex data sets that can then be selected for further analysis , as the same subset of biomarkers appeared to produce high information content in models across different populations . further , once we have sufficient subjects to produce a robust model , tables of posterior estimates for clinical response given combinations of igg response biomarkers can be developed . an example inference table is provided in table 2 , where those biomarkers that are predictive of clinical response can provide a posterior estimate of response . this type of inference could support the translation of this research into a clinical application for determining whether an individual patient has responded from a particular vaccine therapy or potentially whether ongoing immunization should be performed . future modeling might further permit the selection of patients who would be appropriate to receive vaccine therapy based on pre - existing immunological response parameters .

we previously demonstrated that igg responses to a panel of 126 prostate tissue - associated antigens are common in patients with prostate cancer . in the current report we questioned whether changes in igg responses to this panel might be used as a measure of immune response , and potentially antigen spread , following prostate cancer - directed immune - active therapies . sera were obtained from prostate cancer patients prior to and three months following treatment with androgen deprivation therapy ( n = 34 ) , a poxviral vaccine ( n = 31 ) , and a dna vaccine ( n = 21 ) . changes in igg responses to individual antigens were identified by phage immunoblot . patterns of igg recognition following three months of treatment were evaluated using a machine - learned bayesian belief network ( ml - bbn ) . we found that different antigens were recognized following androgen deprivation compared with vaccine therapies . while the number of clinical responders was low in the vaccine - treated populations , we demonstrate that ml - bbn can be used to develop potentially predictive models .

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it is known that bone mass density and architecture are intimately dependent upon mechanical factors . several in vitro studies suggest that cell mechanical stimulation promotes proliferation and differentiation of osteogenic lineage [ 1 , 2 ] , in agreement with the results from several in vivo studies [ 35 ] . stress shielding causes bone loss through remodeling due to decreased loading , particularly in noncemented prostheses [ 6 , 7 ] . several solutions have been proposed for these two mechanical problems , namely , design variations and development of controlled - stiffness implants [ 8 , 9 ] . a new approach to this problem , with potential application to other orthopedic problems and other medical fields , would be the development of smart implants integrating systems for bone mechanical stimulation . the present in vivo study explores the use of the converse piezoelectric effect to mechanically stimulate bone with resource to polyvinylidene fluoride films ( pvdf ) ( measurement specialties inc company , usa ) . these films were used , after dip - coating to improve cell adhesion and assure electrical insulation , as a substrate for mc3t3 cells to explore the concept of using piezoelectric materials and converse piezoelectric effect to produce effective osteoblast mechanical stimulation . the coating was performed by dip - coating at constant velocity of 0,238 mm / s . the chosen material for coating was an acrylic , poly(methyl methacrylate ) ( pmma ) , ( perfex , international dental products , usa ) , used alone in the first three layers and a in forth layer along with 4% of bonelike ( 250500 m ) particles added ( kindly offered by inescporto ) . the displacement was higher where the cells were seeded , in the central area of the coated devices , in the order of 700 nm along the z - axis , in a semisinusoidal fashion . on the substrates submitted to dynamic conditions , stimulation was done with an alternating sinusoidal current ( ac ) , of 5 v , at 1 hz and 3 hz for 15 minutes at each frequency . in this exploratory in vivo study , the same coating procedures and daily stimulation regimens were applied . the biological response around the osteotomies was assessed through histology and histomorphometry in undecalcified sections , and through histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin ( opn ) , proliferating cell nuclear antigen ( pcna ) , and tartrate - resistant acid phosphatase ( trap ) . the actuator device was composed of a microboard containing an ultra - low power 16-bit microcontroller ( ez430-rf2500 , texas instruments , usa ) , powered by lithium battery and encapsulated in pmma and a set of six actuators composed of polyvinylidene fluoride ( pvdf ) and silver electrodes , electrically insulated by dip - coating as described elsewhere . each actuator measured 15 mm width and 40 mm length , with an active area of 12 31 mm . the actuator device was programmed to apply a 5 v square wave of 1 hz and 3 hz for 15 minutes at each frequency , once every 24 hours . finite numerical method studies and electronic speckle pattern interferometry process ( espi ) studies have shown the lower deformation levels occurred in the clamped region of the films , for a potential differential of 5 v , and the higher deformation occurred in the opposite extremity of the film . a control device , similar but static was also developed . a sterilization method was developed and validated based on iso 11737 - 1 and iso 14937 , using a series of decontaminating solutions ( 10% sodium hypochlorite for 20 minutes , without agitation , washing in sterile water , and immersion in 10% hydrogen peroxide for 30 minutes , without agitation , followed by rinse in sterile water and drying under laminar flux ) , before 2 hours of exposure to uv ( in two different positions ) . the actuator device was implanted in the left hind limb , and a control device was implanted in the right hind limb , of a 4-year - old merino ewe , with 45 kg body - weight . the animal was premedicated with atropine ( 0.7 mg / kg ) , xylazine ( 0.1 mg / kg ) and butorphanol ( 0.01 mg / kg ) , and anesthesia induced with thiopental sodium 5% ( 5 mg / kg ) by intravenous injection . the anesthesia was maintained through isoflurane with spontaneous ventilation . the stifle joint of both legs was prepared with a solution of povidone - iodine and ethanol 70% , after being shorn and shaved . two longitudinal incisions were made on the craniomedial border of the femur and at tibial crest level . the tibial bone was exposed by reflecting deep medial fascia , the vastus medialis and cranial part of the sartorius muscles and the periosteum , and two osteotomies were made using an especially metal designed guide to make two regular and well - orientated osteotomies using an oscillating saw . the bone was continuously irrigated with a sterile saline solution during the process of low - speed drilling and cutting . the same procedure was followed with a different design guide for the distal femoral physis , where four osteotomies were done . a stab wound was made in the fascia lata , and the extremities of the piezoelectric films passed through it , and then , the active area of each piezoelectric film was placed inside the slot osteotomy cavity . the actuator position inside the tibial bone can be clearly viewed in figure 1(a ) , obtained post - mortem . the portion of the device containing the microprocessor and the power supply was left in the nearby subcutaneous space . the actuator device was implanted in the left hind limb , and control on the right hind limb . after placing the implants , incisions were closed routinely with a continuous - pattern suture in the retinaculum and fascia , using absorbable surgicryl 2/0 ( smi ag , belgium ) and in the skin with silk 0 ( smi ag ) . postoperative x - ray images were obtained thirty days after implantation , before sacrifice , to ensure the proper evolution of the bone cuts and to detect signs of infection / inflammation ( figure 2 ) . one week after implantation calcein ( 15 mg / kg ; sigma , usa ) was injected subcutaneously and one week prior to sacrifice the same procedure was done with alizarin complexone ( 25 mg / kg ; sigma , usa ) . the present study was authorized by competent national authorities and conducted accordingly to felasa 's guidelines for animal care . both hind limbs were dissected , the implanted materials and surrounding tissue removed and fixed in 4% paraformaldehyde for two weeks . bone samples were cut transversally to the long axis of the bone , each including a piezoelectric film and the surrounding bone ( figure 1(a ) ) . soft tissues ( local lymph nodes and samples of the fibrous capsule surrounding the implants ) were routinely processed and embedded in paraffin . undecalcified bone samples of each of the implants were included in resin ( technovit 9100 , heraeus kulzer , germany ) according to the manufacturer 's instructions and 80 m thick sections cut with a saw microtome ( leica 1600 , germany ) parallel to the piezoelectric film long axis sections were then appropriately processed for routine staining ( giemsa eosin ) , mounted for fluorescence microscopy . for histomorphometric studies , the interface between the bone and implant was divided in four distinct areas : z1 , z2 , z3 , and z4 , from cortical towards bone surrounding the free extremity of the piezoelectric film ( figure 1(b ) ) . pictures were taken from the bone surrounding both sides of the film in areas z1 to z3 and z4 , using a 40x magnification ( figure 3 ) . for immunohistochemistry and histochemistry , bone sections were decalcified in formic acid 5% for 3 weeks , dehydrated in ethanol , cleared in xylene , and embedded in paraffin wax and 3 m sections cut . after deparaffinization and rehydration , immunohistochemistry sections were treated with 3% hydrogen peroxide for 10 minutes . primary antibodies for tartrate - resistant acid phosphatase ( trap ) ( thermo scientific , usa , mouse monoclonal antibody , ab-1 , clone 26e5 , cat . no.ms-1768 ) , proliferating cell nuclear antigen ( pcna ) ( neomarkers , usa , mouse monoclonal antibody , ab-1 , clone pc10 , cat . no.rb-9097 ) were diluted to 1 : 20 , 1 : 200 , and 1 : 50 , respectively . prior to immunostaining , the sections were pretreated for antigen retrieval at 100c in 10 mm citrate buffer , ph 6 , for 20 minutes in microwave oven , followed by cooling for 30 minutes at room temperature . for double staining of pcna and osteopontin , immunohistochemistry was done using a double staining kit picture ( zymed laboratories inc , usa ) , according with the manufacturer 's instructions . trap immunohystochemistry was performed with resource to kit picture - max polymer ( invitrogen , usa ) . slides were counterstained with mayer 's hematoxilin , dehydrated , mounted with entellan ( merck , germany ) , and coverslipped . for all sections , as negative controls adjacent sections were incubated : ( a ) without primary antibody and ( b ) with rabbit / rat normal serum ( similar concentration as that of primary ) . masson trichrome was done using a kit ( bio - optica , italy ) , followed by dehydration and mounting . observation and pictures were done using optic ( olympus bx41 ) and fluorescence microscope equipped with wb fluorescence cube ( olympus u - lh100hg ) . total bone area , immature bone area , and mature bone area were measured using sigma scanpro 5 ( systat software inc . the results , in percentage of total bone area , mature bone , and immature bone , were determined for each piezoelectric film in the pre - determined areas z1 to z4 . normal distribution of the results was verified using the kolmogorov - smirnov test , homogeneity of variance assessed through the levene test , and differences between groups tested using one - way anova ( at a 0.05 level ) . the statistical analysis was done using software originpro 8 ( originlab corporation , usa ) . total bone area around the actuators was significantly higher , when comparing to static controls ( 39.91 14.08% versus 27.20 11.98% ) ( figure 4 ) . the increment of bone occupied area was due to new bone formation , and in actuators the area occupied by woven bone and osteoid was 64.89 19.32% of the total bone area versus 31.72 14.54% in static devices ( figure 5 ) . the chosen staining technique , giemsa eosin , stains older bone in a darker shade of pink , whilst more recently deposed bone is of a lighter pink color and frequently adjacent to much less prominent osteoid ( dark blue ) areas ( figures 5(a ) and 5(b ) ) . taking into account the close relation in between osteoid and woven , recently formed bone , the authors chose not to make separated measurements that could be misleading . the significant differences arise from the bone areas surrounding the two distal thirds of the devices ; around the devices ' clamped area , no differences were found between actuated and static devices ( figure 6 ) . when assessing the mineral apposition rate in the distal third of the films , with the aid of the fluorochrome labeling , the mineral apposition rate was significantly higher around actuated devices ( 4.44 1.67 m / day ) than around static devices ( 2.70 0.95 m / day ) ( figure 5 ) . a fibrous capsule was obvious both in actuators and static devices , with no statistical significant differences in capsule thickness between the two groups ( 289.59 131.20 m in actuated films versus 293.93 84.79 m ) . immunohistochemistry failed to detect differences in trap expression between the two groups , but there is a marked elevation in opn detection around actuators , in z3 and z4 ( figure 7 ) . opn is also strongly labeled in the fibroblast layers closer to the pvdf films , both in actuators and static controls , and this is the area where positive pcna labeling was found more often . pcna labeling was also positive in some of the preosteoblastic cells in layers apposed to opn - positive columnar osteoblasts . the existence of micro - movements at the interface , depending on the magnitude , may enhance osteogenesis or impair it and promote the formation of fibrous tissue [ 11 , 12 ] . considering the deformation levels of the actuator devices , under the experimental conditions , the generated strains were low , but the results suggest they are enough to elicit response . mineral apposition rate and total bone mass are significantly higher around actuated films when compared to static controls , and these differences arise from around the areas of the films known to have higher displacement values . the increased opn expression we found around actuators when comparing to static controls is most likely associated with mechanical stress , and it was not accompanied by a proportional increase in trap . opn is a multifunctional noncollagenous glycoprotein present in bone matrix , expressed in various degrees by proliferating preosteoblasts , osteoblasts , and osteocytes but also by fibroblasts , osteoclasts , and macrophages [ 1315 ] . opn production is known to be increased in association with mechanical loading [ 15 , 16 ] , and its deficiency significantly decreases bone fracture toughness , leading to heterogeneous mineral distribution , since opn is known to bind strongly to hydroxyapatite , and cell surface as well [ 17 , 18 ] . we observed that all the devices were separated from neighboring bone by a fibrous capsule . this is probably due to the material itself , since there were no differences between actuators and static controls . it would be mandatory to develop and test a material with improved biocompatibility to evaluate accurately the bone material interface . one other issue arises from reflecting upon the material and the coating properties and behavior . the in vitro study previously developed shows evidence of adequate electric insulation for 48 hours in the experimental conditions . however , the in vivo environment is not as controlled and , in addition to a longer period of time , the implantation procedure itself may induce damage on the coating . if coating damage occurs , one should consider the possible effects of the material ( pvdf and silver nitrate particles from the electrode ) and electrical current . previous studies have shown cytotoxic silver nitrate activity , dependent on the concentration [ 19 , 20 ] . the clinical advantages of applying electrical and electromagnetic fields in the treatment of fractures and mal - unions have been long known although the mechanisms leading to the clinical response are not well understood [ 2123 ] . electric fields as low as 0.1 mv / cm promote osteoblast proliferation , with cell metabolic activity being influenced by the intensity of the electric field , and cell signaling based on the voltage - dependent calcium channels [ 24 , 25 ] . electric and electromagnetic fields also exert effects on fibroblasts , enhancing proliferation , protein synthesis and influencing cell alignment , by calcium - independent mechanisms [ 2629 ] . the fact that we found no significant differences in fibrous capsule thickness surrounding the devices suggests it is likely that the electric insulation remained satisfactory , but further studies would be needed to prove this . however , if an electric field was being created locally , increased bone formation along the whole actuator could also be expected . instead , new bone formation follows a pattern in accordance with what is known to be the areas of increased motion and deformation . this study was performed in one animal only but using an adequate number of devices ( total of twelve ) . the sheep was chosen as an animal model considering the bone biology similarities with the human species and the size of the devices . the results are very clear in evidencing qualitative and quantitative statistically significant differences when comparing static and actuated films , but it would be necessary to enlarge the animal study and to study response with longer implantation times . however , from an ethical point of view , it would be desirable to do so after developing a material other than pvdf , more biocompatible . in conclusion , the present in vivo study suggests that in fact piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth at the bone implant interface , eliciting an early response . piezoelectric materials have the advantages of enabling a wide range of deformation / displacement , frequencies , and regimens . therefore , they are excellent candidate components of actuators for clinical purposes . however , new materials should be developed , aiming better biocompatibility .

this in vivo study presents the preliminary results of the use of a novel piezoelectric actuator for orthopedic application . the innovative use of the converse piezoelectric effect to mechanically stimulate bone was achieved with polyvinylidene fluoride actuators implanted in osteotomy cuts in sheep femur and tibia . the biological response around the osteotomies was assessed through histology and histomorphometry in nondecalcified sections and histochemistry and immunohistochemistry in decalcified sections , namely , through masson 's trichrome , and labeling of osteopontin , proliferating cell nuclear antigen , and tartrate - resistant acid phosphatase . after one - month implantation , total bone area and new bone area were significantly higher around actuators when compared to static controls . bone deposition rate was also significantly higher in the mechanically stimulated areas . in these areas , osteopontin increased expression was observed . the present in vivo study suggests that piezoelectric materials and the converse piezoelectric effect may be used to effectively stimulate bone growth .

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the study was conducted in the department of oral medicine and radiology and a cancer centre . the total sample consisting of 90 individuals was divided into a study group of 60 individuals and a control group of 30 individuals . the study group had 60 oscc patients ( both male and female ) between the ages of 25 and 75 years with histologically proven oral cancer but before onset of any form of treatment . individuals with other malignancies , liver disease , tuberculosis , diabetes mellitus , cardiovascular diseases , or any other chronic systemic diseases were excluded from the study . a group of 30 healthy volunteers aged 2575 years without any history of debilitating systemic illness and no history of tobacco or alcohol consumption formed the control . details of the study are explained to the volunteers and written informed consent is obtained . serum l - fucose level was estimated using clinical chemistry autoanalyzer based on winzler method . chicago : spss inc .. to compare the mean values between two groups ( between genders ) , independent samples t - test was used . to compare the mean values between three or more groups ( stages of cancer ) , one - way anova was used . chicago : spss inc .. to compare the mean values between two groups ( between genders ) , independent samples t - test was used . to compare the mean values between three or more groups ( stages of cancer ) , one - way anova was used . a comparison of the overall serum l - fucose levels between the oscc patients and healthy controls showed the mean value of serum l - fucose levels of oscc patients to be 10.85 mg / dl and the healthy controls had 3.47 mg / dl , thereby showing a difference of 7.38 mg / dl . statistical analysis revealed a significant p value ( p < 0.001 ) [ graph 1 ] . serum level of levo - fucose between oral cancer and healthy individuals among the 60 oscc , 2 belong to stage i with a mean serum l - fucose level of 8.13 mg / dl . the serum l - fucose level progressively increased with its level being 9.18 mg / dl in stage ii , 10.53 mg / dl in stage iii , and 11.59 mg / dl in stage iv . stage iv showed the highest level with a maximum value of 12.54 mg / dl and minimum of 10.55 mg / dl . statistical analysis shows a significant p value ( p < 0.001 ) [ table 1 ] . serum level of l - fucose in various stages of oral cancer an attempt to correlate the serum l - fucose levels with histopathological grading between genders and ages yielded no significant results thereby inferring that serum l - fucose levels are directly indicative of the degree of tissue destruction . group of neoplasms affecting any region of the oral cavity is termed as oral cancer , among which oscc represents > 90% of all the neoplasm . recently , the knowledge about cancer biomarkers has increased , and numerous studies have been carried out regarding these . this has paved the way for improving the management of cancer patients by enhancing the accuracy of detection and efficacy of treatment . understanding the relevance of biomarkers before using them is very important for diagnosis , treatment , and proper follow - up . tumor markers are produced either by the tumor itself , as a tumor by product , by the body in response to the presence of cancer or certain benign ( noncancerous ) conditions . these can aid in the diagnosis of cancer and in the assessment of tumor burden . l - fucose is a monosaccharide , which is a common component of many n- and o - linked glycans and glycolipids produced by mammalian cells . lack of a hydroxyl group on the carbon at the 6-position ( c-6 ) and the l - configuration is the two structural features which help to distinguish fucose from other six - carbon sugars present in mammals . it is the most essential sugars that the body demands for normal cell - to - cell communication . physiologically , the concentrations in serum are less but are increased in cancer and other diseases . it is important in many of the signaling pathways which turns a healthy cell into a dysplastic cell . the protein diversity is achieved by varying sequence and structure of sugar moieties or glycan attachment . mammalian cells either express or mediate many of their properties through the cell surface . change in glycosylation of cell surface proteins is essential in tumor progression , especially the terminal epitopes of glycoproteins , which have been proposed to play an essential role in cell cell interactions , cell adhesion , malignant transformation , and metastasis . fucosylation of glycoproteins ( the addition of l - fucose at the terminal end of the oligosaccharide chain ) is one of the most important features that mediate several specific biologic functions . tumor cells alter their surface by increasing fucosylation levels to escape recognition ; this contributes to numerous abnormal characteristics of tumor cells such as decreased adhesion and uncontrolled tumor growth . several studies have suggested that estimating the serum / tissue fucose levels could be a promising approach for the early detection , diagnosis , and prognosis of various cancer types . group of enzymes catalyze incorporation of fucose from activated nucleotide donor guanosine diphosphate - fucose to the reducing end of complex glycans in a linkage - specific manner . these enzymes are expressed in many tissues and are elevated in serum and tumors of cancer patients . it has been mentioned in the article that increased fucosylation is associated with elevated fuct activity . cancer cells which are shed or released into circulation from the primary tumor often overexpress fucosylated glycans on their surface . the expression of fucosylated glycoproteins ( i.e. , fucoproteins ) has been detected by means of specific lectins . profound fucosylation of the serum microenvironment may be a reason that interrupts adhesion and induces the formation of metastases ( for example ) , several fucose - containing natural ligands increased expression of fucosylated cell surface antigens , such as lewis x / y ( lex / y ) or sialyl lex/ , and the upregulation of 1 , 3/4-fuct have been associated with malignant transformation and increased metastatic potential of tumors , which leads to poor prognosis of patients with cancer . -l - fucosidase is a lysosomal enzyme that catalyzes the hydrolytic cleavage of terminal fucose residue that is involved in maintaining the homeostasis of fucose metabolism . extensive studies on the nature of metastasis have shown that only a small subpopulation of cells in tumors possess the characteristics necessary for their release from the primary tumor and transport to and establishment of tumor foci in distant organs . carbohydrate moieties on cell surface glycoconjugates play an important role in this metastatic spread since it could be demonstrated that they are involved in adhesion processes . several studies have mentioned that estimation of serum l - fucose levels could be a reassuring approach for the early detection , diagnosis , and prognosis of various types of cancer including oral carcinoma . thus , the present study was undertaken to estimate the serum level of l - fucose ( tumor marker ) among various tnm stages in oral cancer patients and compare them with healthy individuals . present study suggested that serum level of l - fucose can not be correlated with age and gender . this was similar to other studies which were published in the literatures . in our study , out of 60 cancer patients , 31 were well differentiated , 27 - moderately differentiated , and 2 poorly differentiated and anaplastic - nil . however , there was a negative correlation between serum fucose levels and histopathological tumor differentiation in our study as well as in the literatures . in our study , the mean serum value of l - fucose in cancer group ( 10.85 mg / dl ) and control group ( 3.47 mg / dl ) shows a wide margin of difference ( 7.38 mg / dl ) . moreover , all the 60 patients in the cancer group were categorized according to tnm staging . comparison of all the four tnm stages in cancer group showed increase in fucose levels as the stages progressed . they assessed the levels of glycoprotein - associated carbohydrates such as hexose ; hexosamine , fucose , and sialic acid . they found a significant rise in the level in cases when compared to control subjects . furthermore , there was a progressive rise in these markers as the stages of oral cancer advances . in cancer patients , elevated levels have been observed more so in advanced stages and in cases with metastasis than in subjects without metastasis . some studies have concluded that it is the most effective of the essential sugars when it comes to slowing the growth of cancer cells . fall in serum fucose levels after treatment has been reported in many studies although it is emphasized that the follow - up period has to be long enough for significant serological alteration . unfortunately , rise in serum fucose level is not specific for cancers , as elevated serum fucose levels have also been reported in other pathological states such as cirrhosis liver , meningitis , rickets , osteomalacia , tuberculosis , cardiovascular disorders as well as in depressive disorders . furthermore , it has been observed that the serum fucose level is raised in different groups of malignancies such as breast cancer , ovarian cancer , colorectal adenocarcinomas , and leukemia as well as brain tumors . thus , it becomes important to exclude other degenerative and proliferative diseases , while estimating the serum fucose levels in oral cancer . literature is replete with studies on fucose levels in various malignancies including a few on oral cancer . based on the analysis of the results of our study , we can conclude that there was a positive correlation between the serum l - fucose levels and tnm stages of oscc . serum l - fucose levels remained constant among healthy individuals and was raised proportionately in oral cancer patients , commensurate with the stage of cancer . in conjunction with clinic - diagnostic procedures , serum l - fucose levels can be used as an easy , noninvasive , cost - effective , biochemical indicator of cancer detection , staging , therapeutic success , prognosis , and as a posttreatment evaluation tool . further investigation on a large scale would in all probability prove serum l - fucose as an effective tumor marker in the diagnosis and management of oral cancer .

background : oral cancer is a result of disordered cellular behavior initiated by various stimuli which is characterized by the alteration of serum glycoproteins consisting of different monosaccharides . one of these is levo - fucose ( l - fucose ) , a methyl pentose . elevated levels of protein - bound fucose have been reported in various malignancies.aim:the present study attempted to correlate levels of serum l - fucose as a biomarker with the various tumor node metastasis ( tnm ) stages of oral cancer.methodology:the study was carried out on 90 subjects consisting of 30 healthy controls and 60 histopathologically proven oral squamous cell carcinoma ( oscc ) cases . the serum fucose level estimation was done based on the method adopted by winzler . statistical analysis included independent sample 's t - test , one - way anova test , karl pearson correlation test , and tukey 's hsd post hoc test to evaluate the significance and variability of values between groups.results:significant elevation in serum fucose levels was noticed among oscc patients when compared with the controls and a progressive ascent of l - fucose levels were noted as the stage of severity increased . serum fucose levels were independent of histopathological grading , age , and sex.conclusion:serum l - fucose levels were increased in oscc patients , and a positive correlation was observed between serum l - fucose levels and tnm staging of oscc . thus , serum l - fucose can be used as an effective diagnostic and prognostic biomarker in oscc patients .

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alan aderem ( institute for systems biology , seattle , washington ) , which were originally generated by dr . shizuo akira ( research institute for microbial diseases , osaka university , osaka , japan ) . lcn2 mice were generated by gene targeting in embryonic stem ( es ) cells from mouse strain 129 , and targeted es cells were injected into c57bl/6 blastocysts as described previously ( 12 ) . lcn2-null mice were backcrossed onto the b6 background for 10 generations before mice were used for the experiments . heterozygous mating scheme was used to generate wild - type and lcn2 mice for the experiments . aminals were housed in a specific pathegon - free facility at the university of minnesota . animal handling followed the national institutes of health guidelines , and experimental procedures were approved by the university of minnesota animal care and use committee . age - matched male wild - type and lcn2 mice were allocated into groups ( 34 mice / cage ) and fed an hfd ( fat calories : 60% ) obtained from bio - serv ( f3282 ; new brunswick , nj ) or a regular chow diet ( rcd ) , with free access to water for all studies . in the adaptive thermogenesis study , age - matched wild - type and lcn2 mice on an rcd were exposed to 4c , with free acess to diet and water . rectal temperature of the mice was measured at the indicated time points using a microtherma thermometer with rectal probe for mice ( braintree scientific , braintree , ma ) . during the experiemental period of hfd feeding , glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . after mincing , epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . the range of cell sizes that can effectively be measured using this aperture is 20240 m . after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . during the experiemental period of hfd feeding , glucose and insulin tolerance tests were conducted by the intraperitoneal injection of glucose and insulin . mice were fasted overnight ( 12 h ) for the glucose tolerance test ( gtt ) and for 6 h for insulin tolerance tests ( itts ) . gtts and itts were conducted by intraperitoneal injection of glucose ( 1 mg / g body wt ) or insulin ( 0.75 units / kg body wt ) with blood collection via the tail vein at 0 , 15 , 30 , 60 , 90 , and 120 min . briefly , frozen liver tissue ( 100 mg ) was homogenized in 1 ml water . an aliquot of the organic phase was collected and dried with nitrogen and then dissolved in isopropanol alcohol containing 1% triton . preparation of isolated adipose cells from wild - type and lcn2 mice was performed as described previously ( 14,15 ) . after mincing , epididymal fat pads were digested with collagenase ( 2 mg / ml solution ) in digestion vials containing krebs - ringer bicarbonate hepes buffer , ph 7.4 ; 200 nmol / l adenosine ; and 3.5% bsa . after a 2-h digestion , adipose cells were separated by centrifugation at 1,200 rpm for 10 min and washed twice with krebs - ringer bicarbonate hepes buffer . after the final wash , adipose cells were collected for rna extraction . adipose tissue was obtained from the epididymal and inguinal fat pad of wild - type and lcn2 mice fed an rcd . tissue samples ( 2030 mg ) were immediately fixed in osmium tetroxide and incubated in a water bath at 37c for 48 h , as described previously ( 16 ) , and then adipose cell size was determined by a beckman coulter multisizer iii with a 400-m aperture . the range of cell sizes that can effectively be measured using this aperture is 20240 m . after collection of pulse sizes , the data were expressed as particle diameters and displayed as histograms of counts against diameter using linear bins and a linear scale for the x - axis . the figures were generated using graphpad prism 5.01 for windows ( graphpad software , la jolla , ca ) . differences in parameters between wild - type and lcn2 mice were evaluated using a two - group t test with a 0.05 two - sided significance level . at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . growth curve , weight of white and brown fat pads and organs in lcn2 mice . growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . epi : epididymal ; ing : inguinal . because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34 0.34 ; lcn2 , 38.2 0.30 ) . however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . . five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator activated receptor ( ppar ) coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . interestingly , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . 2d ) . to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . serum metabolic parameters in wild - type and lcn2 mice data are means se . measurements were performed on 6-h fasted mice fed an rcd and hfd ( n = 911 ) . * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . * p < 0.05 . to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . to elucidate hepatic glucose production in wild - type and lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . 6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . d : the mrna expression of lipogenic genes in liver ( n = 6 ) . the data are represented as means se . * p < 0.05 ; * * p < 0.01 . ( a high - quality digital color representation of this figure is available in the online issue . ) compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig . plasma lipid profile in wild - type and lcn2-null mice data are means se . measurements were performed on 6-h fasted mice fed an rcd and hfd ( n = 911 ) . * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . at 3 weeks of age , male wild - type and lcn2 mice were fed either an hfd for 12 weeks or rcd . throughout the experimental period , body weight was significantly different between wild - type and lcn2 mice fed an hfd ( fig . lcn2 mice were significantly heavier than wild - type mice on either an hfd or rcd . the weight of white and brown fat pads was significantly higher in hfd - fed ( fig . 1d ) mice than that in wild - type mice . both hfd - fed and aged lcn2 mice showed a significantly higher weight of liver , heart , kidney , and spleen ( fig . similar metabolic phenotypes were also observed in female lcn2 mice ( data not shown ) . however , the overall growth did not appear to be significantly affected , as the body length and bone length ( trabecular ) were not significantly different between the genotypes ( data not shown ) . figure 1 g and h demonstrated the fat - cell size distributions of wild - type and lcn2 mice on an rcd at 15 weeks of age , as measured by multisizer analysis using cells isolated from osmium - fixed epididymal and inguinal adipose tissue . the average peak diameter of the large - cell population was significantly larger for lcn2 mice compared with wild - type mice , indicating that lcn2 adipose tissue contains larger adipose cells . growth curve , weight of white and brown fat pads and organs in lcn2 mice . growth curve of lcn2 mice on an hfd ( n = 1014 ) ( a ) and rcd ( n = 13 ) ( b ) . fat pad weight of lcn2 mice on an hfd at 16 weeks of age ( c ) and on rcd at 30 weeks of age ( d ) . organ weight of lcn2 mice on hfd at 16 weeks of age ( e ) and on rcd at 30 weeks of age ( f ) . * p < 0.05 ; * * p < 0.01 . fat - cell size distribution of mice on rcd diet at 15 weeks of age ( g and h ) . because lcn2-deficient mice gained more body weight and increased adiposity , particularly when mice were fed an hfd or aged , we examined food intake and energy expenditure in lcn2 mice . an analysis of indirect calorimetry measurements showed that food intake , ambulatory activity , vo2 , and rq were not significantly different between rcd - fed wild - type and lcn2 mice at 18 weeks of age ( fig . we then assessed thermogenic activity of brown adipose tissue ( bat ) under thermoneutral and thermal stress conditions . body temperature was measured in an ambient temperature of 28c and 22c as well as during acute exposure to 4c . wild - type and lcn2 mice at 12 weeks of age exhibited a similar body temperature in an ambient temperature of 28c ( wild type , 38.34 0.34 ; lcn2 , 38.2 0.30 ) . however , female lcn2 mice had a significantly lower rectal temperature than wild - type mice ( fig . 2a ) , while male lcn2 mice had a trend toward decrease in rectal temperature when kept in an ambient temperature of 22c . more strikingly , lcn2 mice displayed cold sensitivity and could not survive when exposed to 4c for > 10 h. lcn2 mice ( seven of seven ) died after being exposed to 4c for 12 h , while all of the wild - type mice ( n = 7 ) in the experiment survived . when acutely exposed to 4c , rectal temperature of lcn2 mice dropped significantly within 3 h compared with wild - type mice ( fig . five - hour cold exposure caused a 10-fold increase in lcn2 gene expression in adipose tissue in wild - type mice ( fig . a : basal rectal body temperature of 8-week - old male and female wild - type and lcn2 mice on an rcd ( n = 710 ) measured during the daytime in an ambient temperature of 22c . b : body temperature curve of lcn2 mice ( n = 8) and wild - type mice ( n = 7 ) exposed to 4c . c : gene expression of lcn2 in wat of wild - type mice exposed to 4c for 5 h. d : gene expression of ucp-1 and pgc-1 in bat and ppar , cpt-1 , tfam , and nrf1 in skeletal muscle of hfd - fed mice ( n = 6 ) . g : gene expression of hsl in bat and wat of mice exposed to 4c for 5 h. h : gene expression of mitochondrial genes in bat of lcn2 mice under the hfd condition . * p < 0.05 ; * * p < 0.01 . to explore the possible mechanisms for cold intolerance in lcn2 mice , uncoupling protein ( ucp)-1mediated thermogenic activity and capacity of bat we observed that upon hfd feeding , lcn2 mice expressed significantly lower levels of ucp-1 and peroxisome proliferator activated receptor ( ppar ) coactivator ( pgc-1) genes in bat than wild - type mice mice ( fig . however , ucp-1 and pgc-1 expression in bat were markedly stimulated at an even higher level in lcn2 mice , as wild - type mice after exposure to 4c for 5 h ( fig . figure 2f showed that the protein expression levels of adipose triglyceride lipase ( atgl ) in white adipose tissue ( wat ) were slightly reduced in lcn2 mice under the hfd and cold condition compared with wild - type mice . however , cold - induced hsl gene expression in bat and wat was completely diminished in lcn2 mice ( fig . lastly , we investigated the mitochondrial oxidative capacity of bat , as this function of bat is critical for providing the energy for thermogenesis . interestingly , we observed that there was a significant reduction in the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function , such as nuclear respiratory factor ( nrf)1 , transcription factor a , mitochondrial ( tfam ) , atp synthase 5 ( atp5 ) , cytochrome c oxidase ( cox ) 4 , and carnitine palmitoyltransferase ( cpt)-1 in bat of lcn2 mice fed an hfd ( fig . in addition , the expression of transcription factors ppar , nrf1 and tfam , and cpt-1 gene in skeletal muscle was also significantly downregulated in lcn2 mice fed an hfd ( fig . to assess the effects of lcn2 deficiency on systemic insulin sensitivity , gtts and itts were performed . glucose clearance curves in response to glucose administration were significantly increased in hfd - fed ( fig . 3b ) , indicating that lcn2 mice are more glucose intolerant upon hfd feeding . in the itt , increased insulin - stimulated glucose disposal curve was observed in lcn2 mice , indicating that lcn2 mice are more insulin resistant than wild - type controls when fed an hfd ( fig . 3d ) . analysis of serum metabolic parameters , as shown in table 1 , demonstrated that levels of fasting serum glucose and insulin were significantly elevated , while adiponectin levels were reduced in hfd - fed lcn2 mice when compared with wild - type controls . serum levels of leptin and plasminogen activator inhibitor-1 were not significantly changed in lcn2 mice on an rcd , although serum leptin levels had a trend toward a decrease in hfd - fed lcn2 mice . after adjustment to body weight , the serum levels of glucose , insulin , and adiponectin were consistently different at the statistically significant level , indicating that the changes in glucose tolerance and insulin sensitivity after hfd in lcn2 mice is not simply due to body weight effects . to further confirm that lcn2 mice have decreased insulin sensitivity , in vivo insulin - stimulated akt phosphorylation was evaluated . wild - type and lcn2 mice on rcd were injected intraperitoneally with insulin , killed after 10 min , and tissues were collected for evaluation of akt phosohorylation . consistently , insulin - stimulated akt phosphorylation in liver , muscle , and adipose cells was significantly reduced in lcn2 mice as compared with wild - type mice ( fig . the examination of lcn2 effect on upstream signaling components of insulin signaling pathway demonstrated that the expression levels of insulin receptor substrate-1 protein were markedly reduced in wat of lcn2 mice on an hfd compared with wild - type mice ( fig . this data further support that lcn2 mice developed more insulin resistance and lcn2 regulates insulin signaling activity likely at the upstream level . gtts ( a ) and itts ( c ) conducted in lcn2 mice on an hfd ( n = 1012 , age 1415 weeks ) . gtts ( b ) and itts ( d ) conducted in lcn2 mice on an rcd ( n = 1012 , age 2829 weeks ) . serum metabolic parameters in wild - type and lcn2 mice data are means se . measurements were performed on 6-h fasted mice fed an rcd and hfd ( n = 911 ) . * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . in vivo insulin - stimulated akt phosphorylation in lcn2 mice . representative western blot analysis of serine - phosphorylated akt in muscle , liver , and isolated primary adipose cells from lcn2 ( n = 4 per group ) and wild - type mice ( n = 4 per group ) under basal and in vivo insulin - stimulated conditions . to test the role of lcn2 in inflammatory response , expression of lcn2 and inflammatory molecules was examined in adipose tissue and liver of hfd - induced wild - type and lcn2 mice . 5b ) . furthermore , the gene expression of proinflammatory cytokine tnf and monocyte chemoattractant protein ( mcp)-1 were upregulated , while expression of arginase 1 , an anti - inflammatory marker , was downregulated in adipose tissue of hfd - fed lcn2 mice compared with wild - type control mice ( fig . downregulation of arginase 1 and interleukin ( il)-10 was also observed in the liver of hfd - fed lcn2 mice ( fig . a : protein expression levels of lcn2 in adipose tissues of wild - type mice fed an rcd and hfd . b : protein expression levels of lcn2 in bone marrow macrophages ( bm - mac ) isolated from wild - type mice fed an rcd and hfd . c : gene expression of inflammatory molecules in adipose tissue and liver of wild - type and lcn2 mice fed on hfd . to investigate whether increased liver weight in lcn2 mice is associated with the development of fatty liver and dyslipidemia , liver triglyceride content and blood lipid profiles were measured . in comparison with wild - type controls , hfd - fed and aged lcn2 mice demonstrated a significant increase in lipid accumulation as detected by oil - red o staining of the liver section ( fig . the development of more severe fatty liver disease , together with elevated fasting blood glucose levels , suggests hepatic insulin resistance in lcn2 mice . to elucidate hepatic glucose production in wild - type and lcn2-deficient mice fed an hfd , mice were fasted for 18 h and gene expression of two key gluconeogenic enzymes pepck and glucose-6-phosphatase ( g6pase ) were detected by quantitative pcr . as illustrated in fig . 6c , expression levels of pepck1 , pepck2 , and g6pase genes were significantly higher in the liver of lcn2 mice than that in wild - type controls , suggesting that increased hepatic glucose production is attributed to hyperglycemia in lcn2 mice . we next examined the hepatic capabilities for fatty acid synthesis and oxidation to explore the possible mechanism for the development of fatty liver in lcn2 mice . 6d , the lipogenic genes sterol regulatory element - binding protein-1c ( srebp-1c ) , acetyl - coa carboxylase 1 ( acc1 ) , spot 14 ( s14 ) , and scd-1 were expressed at a significantly higher level in hfd - fed lcn2 mice compared with wild - type controls , while genes involved in fatty acid oxidation such as ppar , cpt-1 , and acetyl - coa carboxylase 2 were similarly expressed between genotypes ( data not shown ) . therefore , the development of fatty liver results primarily from the increased capability for fatty acid synthesis in the liver in lcn2 mice . b : liver triacyglyceride levels in lcn2 mice on an rcd ( n = 68 , age 30 weeks ) and an hfd ( n = 11 , age 1516 weeks ) . c : the mrna expression of gluocneogenic genes in liver ( n = 6 ) . d : the mrna expression of lipogenic genes in liver ( n = 6 ) . the data are represented as means se . * p < 0.05 ; * * p < 0.01 . ( a high - quality digital color representation of this figure is available in the online issue . ) compared with wild - type mice , lcn2 mice on an rcd had significantly higher serum levels of triglycerides , cholesterol , and free fatty acids . the hfd feeding did not induce a further increase in serum levels of triglyceride and cholesterol but significantly augmented the levels of ldl and -hydroxybutyrate in lcn2 mice compared with wild - type controls . the above data have suggested that lcn2-deficient mice became more insulin resistant upon hfd feeding and age . since adipose tissue is a key site for insulin - regulated glucose / lipid metabolism and glucose disposal , we examined the expression of genes involved in lipid metabolism in primary adipose cells isolated from lcn2 mice . compared with wild - type adipose cells , expression levels of genes involved lipid metabolism and adipokines , including ppar , lipoprotein lipase ( lpl ) , glut4 , adiponectin , and leptin were markedly reduced in adipose tissue of hfd - fed lcn2 mice ( online appendix fig plasma lipid profile in wild - type and lcn2-null mice data are means se . measurements were performed on 6-h fasted mice fed an rcd and hfd ( n = 911 ) . * p < 0.01 vs. wild - type on an rcd ; p < 0.01 vs. wild - type on an hfd . we show here that lcn2 plays a critical role in lipid metabolism , adaptive thermoregulation , and diet - induced obesity and insulin resistance in vivo . lack of lcn2 in mice potentiates hfd - induced obesity , dyslipidemia , fatty liver , glucose intolerance , and insulin resistance . we also discovered that lcn2 is a critical regulator of adaptive thermogenesis and mitochondrial oxidative function in bat . lcn2 deficiency impairs adaptive thermogenesis and increases the susceptibility of lcn2 mice to hfd - induced obesity . our data demonstrated that lcn2 mice gained more body weight when they were fed hfd or aged . increased body fat mass together with enlarged multiple organs in particular , increased organ weights may largely account for increased body weight in lcn2 mice under the rcd condition . in general , wild - type and lcn2 mice at a young age did not demonstrate significantly different metabolic phenotypes when they were fed rcd . however , lcn2 mice developed more severe systemic insulin resistance , hyperglycemia , hyperinsulinemia , and hypoadiponectinemia under the hfd condition . one of the most profound phenotypic features of lcn2 mice is cold sensitivity and lower body temperature under thermal stress conditions . it has been known for a long time that nonshivering thermogenesis is used to maintain constant body temperature and energy balance , controlling body weight , especially in small mammals ( 1719 ) . both neural and hormonal signals , including norepinephrine , epinephrine , thyroid hormones , glucagon , insulin , glucocorticoid , and leptin , are known to regulate thermogenesis ( 17,20 ) through different thermogenic mechanisms involving stimulating atp utilization , increasing ucp-1mediated proton leak and heat production and affecting substrate availability and utilization for energy production . in rodents , bat is the main tissue that is responsible for sympathetic nervous system regulated nonshivering themogenesis with the involvement of norepinephrine and epinephrine ( 17,20,21 ) . ucp-1 and pgc-1 are the key genes that mediate sympathetic thermogenic responses in bat . for example , ucp-1 gene expression in bat of ob / ob mice was reduced ( 23 ) . however , bat ucp-1 expression could be normally stimulated in response to cold environment in young ob / ob mice , albeit ob / ob mice show decreased capability of utilizing fat and cold intolerance ( 23 ) . in this study , we found that 5-h cold exposure leads to a significant increase in lcn2 expression in wat , which is in agreement with the result in a recent study ( 24 ) . in the absence of lcn2 , mice were cold intolerant and failed to maintain the body temperature when acutely exposed to cold temperature . interestingly , cold exposure could still significantly stimulate gene expression of ucp-1 and pgc-1 in bat in lcn2 mice , suggesting that cold - induced sympathetic thermogenesis remained intact in the absence of lcn2 . the role of leptin in thermogenesis has been well documented , and its thermogenic effect is considered to be primarily central ( 25 ) . for example , leptin administration has been reported to rescue cold - induced hypothermia in the absence of ucp-1 in leptin and ucp-1 double knockout mice ( 26,27 ) , while the opposite results were perceived in the other two independent studies ( 28,29 ) . in lcn2 mice on rcd , serum leptin levels were not different from that in wild - type mice , albeit serum leptin levels had a trend toward decreased in lcn2 mice in response to hfd feeding . additionally , wild - type and lcn2 mice on an rcd did not exhibit a difference in body temperature in thermoneutral conditions . therefore , it seems rather unlikely that leptin could be the major contributor to cold intolerance in lcn2 mice ; leptin may be only partially involved in lcn2-regulated thermogenesis . in addition to hormonal signals , defects in key metabolic regulators of substrate availability for energy production , atp synthesis and mitochondrial biogenesis and oxidative function could all possibly affect thermogenic function . indeed , studies in mice with the genetic disruption of key metabolic regulators have proved this assumption ; mice without expressing atgl could similarly result in cold intolerance ( 30 ) . in the present study , the disruption of lcn2 completely diminished cold - induced hsl gene expression in bat and wat , whereas atgl expression in wat was only slightly affected in lcn2 mice under both the hfd and cold conditions . since hsl mice are not cold sensitive ( 31 ) , hsl is not an important regulator for adaptive thermogenesis . therefore , the effect of changes in atgl levels on the supply of substrates for energy production for thermogenesis is minimal in lcn2 mice . on the other hand , the production of the energy necessary for thermogenesis requires normal mitochondrial oxidative function , and mitochondrial dysfunction could have a significant impact on adaptive thermogenesis . this hypothesis has been supported by a previous study in estrogen - related receptor ( eer ) knockout mice . in eer-deficient mice , mitochondrial biogenesis and oxidative function in bat was disturbed , and mice failed to maintain body temperature when exposed to cold ( 32 ) . to this end interestingly , we found that the expression levels of mitochondrial genes involved in mitochondrial biogenesis and oxidative function such as nrf1 , tfam , atp5b , cox 4 , and cpt-1 were significantly reduced in bat of lcn2 mice fed an hfd . in the skeletal muscle , the expression of -oxidation genes ppar and cpt-1 , as well as mitochondrial genes nrf1 and tfam , were also downregulated in hfd - fed lcn2 mice . all the above data together suggest that mitochondrial metabolism is dysfunctional in bat and muscle of lcn2 mice , which mainly contributes to decreased thermogenic capacity , decreased energy expenditure , and the high susceptibility to diet - induced obesity and insulin resistance during the thermal stress conditions ( mice have been living at 22c ) . in acute cold , muscle - shivering thermogenesis is considered to be the primary mechanism for a body temperature defense ( 21,33,34 ) . bat nonshivering thermogenesis participates in combating the cold temperature only when the extra capacity is already recruited for using during the acute cold exposure ( 35 ) . therefore , cold intolerance ( acute exposure to 4c for 5 h ) in lcn2 mice may largely result from decreased oxidative capacity in skeletal muscle and bat as well as decreased atgl expression in wat . our results clearly demonstrated that insulin responsiveness in muscle , adipose tissue , and liver was reduced , indicating systemic insulin resistance in lcn2 mice . furthermore , fasting for 18 h induces significantly higher levels of expression for pepck and g6pase in the liver of hfd - fed lcn2 mice , supporting that the increased hepatic glucose production is an important contributor to fasting hyperglycemia in lcn2 mice . this phenomenon is likely due to the impaired regulation of lipid metabolism in adipose tissue and liver in lcn2 mice . we showed that lcn2 mice on an rcd had higher serum levels of triglycerides but lower serum triglyceride levels when fed an hfd as compared with wild - type mice . lower serum triglycerides in hfd - fed lcn2 mice could be due to the increased triglycerides synthesis and decreased vldl ( or triglyceride ) secretion into the circulation resulting from the development of fatty liver in lcn2 mice . this explanation is strengthened by a previous study ( 36 ) in lirko ( insulin receptor knockout ) mice with hepatic insulin resistance and 50% decrease in serum triglyceride levels . as reported in our previous in vitro study ( 6 ) , lcn2 is capable of suppressing inflammatory response of macrophages to lps stimulation . we speculated that lcn2 possesses anti - inflammatory role in metabolic regulation . in this in vivo study , we show that lcn2 expression is downregulated in adipose tissue and bone marrow macrophages ( bm - mac ) in hfd - induced obese mice . in the absence of lcn2 , the expression of proinflammatory cytokines such as mcp-1 and tnf are upregulated in adipose tissue , while expression levels of anti - inflammatory markers arginase 1 and il-10 are decreased in adipose tissue and liver , suggesting an increased inflammatory state in lcn2 mice . these results further suggest that lcn2 mice are more susceptible to hfd - induced proinflammatory response than wild - type mice . in summary , herein we discovered a novel role for lcn2 in energy metabolism , adaptive thermogenesis , and insulin resistance . mice lacking lcn2 display impaired adaptive thermogenesis and deteriorated diet - induced obesity and insulin resistance . decreased mitochondrial oxidative capacity in bat and skeletal muscle may be the primary mechanisms for the development of diet - induced insulin resistance and cold sensitive in lcn2 mice . however , the mechanisms for how lcn2 regulates mitochondrial function and lipid metabolism remain to be further identified . it is likely that the binding of lcn2 to small hydrophobic molecules such as retinoic acids , fatty acids , and other unknown ligands or to the cell surface receptor such as megalin or ldl - related protein 2 ( 37 ) could potentially involve or mediate lcn2 effects in metabolism . our findings provide new insight into the role of lcn2 in lipid metabolism and metabolic homeostasis . in this manner , lcn2 may be a potential therapeutic target for controlling obesity - associated type 2 diabetes and metabolic complications .

objectivelipocalin ( lcn ) 2 belongs to the lipocalin subfamily of low molecular mass secreted proteins that bind small hydrophobic molecules . lcn2 has been recently characterized as an adipose - derived cytokine , and its expression is upregulated in adipose tissue in genetically obese rodents . the objective of this study was to investigate the role of lcn2 in diet - induced insulin resistance and metabolic homeostasis in vivo.research design and methodssystemic insulin sensitivity , adaptive thermogenesis , and serum metabolic and lipid profile were assessed in lcn2-deficient mice fed a high - fat diet ( hfd ) or regular chow diet.resultsthe molecular disruption of lcn2 in mice resulted in significantly potentiated diet - induced obesity , dyslipidemia , fatty liver disease , and insulin resistance . lcn2/ mice exhibit impaired adaptive thermogenesis and cold intolerance . gene expression patterns in white and brown adipose tissue , liver , and muscle indicate that lcn2/ mice have increased hepatic gluconeogenesis , decreased mitochondrial oxidative capacity , impaired lipid metabolism , and increased inflammatory state under the hfd condition.conclusionslcn2 has a novel role in adaptive thermoregulation and diet - induced insulin resistance .

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male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , tokyo , japan ) , and a strip of traction tape ( skin - trac , 3 40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . data were compared between wb and nwb groups using the chi - square test . a p - value of < 0.05 was considered to be significant . male wistar rats ( 10 weeks old ) were obtained from sankyo labo service , co. ltd . ( sapporo , japan ) . each animal was housed in an individual cage in a temperature- and humidity - controlled room with unlimited food and water and a 12-h light / dark cycle . all animals ( n = 22 ) were given 0.3-mg / kg lps ( from escherichia coli serotype 055 : b5 ; sigma , st . louis , mo , usa ) intravenously on days 1 and 2 . on days 3 , 4 and 5 , the animals were given 20-mg / kg methylprednisolone intramuscularly to promote development of non - traumatic onfh as described previously ( okazaki et al . all the injections were administered at 7:00 p.m. hindlimb unloading was accomplished as described by wronski and morey - holton ( morey - holton & globus 1998 ) with some modifications ( okazaki et al . , unpublished observation ) . the tail was then varnished with instant adhesive ( aron alpha ; toagosei co. , ltd . , tokyo , japan ) , and a strip of traction tape ( skin - trac , 3 40 in . ; zimmer , warsaw , in , usa ) was then attached to the tail beginning at the base of the tail above the hairline . to facilitate free movement about the cage , the cast was attached to a swivel anchored to the cage that allowed a 360 range of movement . the animals were then suspended in a head - down tilted position . over time , the suspension angle was readjusted as the body size increased to prevent any weight coming to bear on the hindlimbs . in nwb rats ( n = 11 ) , the tail was suspended from day 1 to sacrifice . the femur and humerus were harvested from each rat and fixed with 10% formalin-0.1 m phosphate buffer , ph 7.4 . bone samples were decalcified with kalkitox ( wako pure chemical industries , ltd . , osaka , japan ) and then neutralized with sodium sulphate buffer . the tissues were processed for routine haematoxylin and eosin staining to assess the general architecture and osteonecrosis of the femoral and humeral heads . we defined histopathological osteonecrosis as the diffuse presence of empty lacunae or pyknotic osteocyte nuclei in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis ( figure 1 ) ( ichiseki et al . histopathological appearance of proximal femoral epiphysis of weight - bearing ( wb ) rat with haematoxylin and eosin staining . all experiments adhered to the guidelines of the ministry of sports , culture , science and technology of japan and followed protocols approved by the animal ethics committee of the sapporo medical university ( # 09 - 008 ) . femoral head osteonecrosis was observed in seven of 11 ( 63.6% ) wb rats and six of 11 ( 54.5% ) nwb rats , with no significant intergroup difference observed ( p = 0.66 ) . figure 2 shows the histopathological appearance of the femoral head after haematoxylin and eosin staining in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , empty lacunae were seen at the necrotic bone trabeculae , and the number of hematopoietic cells was decreased in the medullary space at most areas of the femoral head ( figure 2a , b ) . in the nwb group , empty lacunae in addition , the number of haematopoietic cells was again decreased , and necrotic cell debris was accumulated in the medullary space at most areas of the femoral head ( figure 2c , d ) , as observed in the wb group . on the other hand , no humeral head osteonecrosis was observed in any of the wb or nwb rats . typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . both wb and nwb group shows diffuse presence of empty lacunae in the bone trabeculae , accompanied by surrounding bone marrow cell necrosis at most areas of femoral head . figure 3 shows the histopathological appearance of the humeral head in typical specimens from the wb ( a , b ) and nwb ( c , d ) groups . in the wb group , normal trabeculae , as well as haematopoietic and fat cells , were observed ( figure 3a , b ) . normal trabeculae were also observed in the nwb group rats . in comparison with the wb group , a large number of haematopoietic cells , but few fat cells were observed in the nwb group rats ( figure 3c , d ) . typical specimens of weight - bearing ( wb ) ( a , b ) and non - weight - bearing ( nwb ) ( c , d ) rats . osteonecrosis of the femoral head often develops after corticosteroid therapy for inflammatory diseases , such as autoimmune diseases ( fukushima et al . , it has been reported that mega - dose corticosteroid therapy for trauma , such as spinal cord injury , does not induce onfh ( wing et al . therefore , we developed a non - traumatic onfh animal model in which onfh was encouraged by the administration of lps ( a ligand of toll - like receptor 4 , and implicated in inducing inflammatory status ) and methylpredonisolone treatment in the rats . in this animal model , however , rats were allowed to walk and move independently in individual cages , with the hip joints supporting the loading from the lower body . we could not exclude the possible influence of weight bearing on the hip joints on the induction of onfh . therefore , we employed a hindlimb unloading procedure , in which the rat tail was fitted on to a suspension system with the rats in a head - down tilted position to prevent weight bearing on the hindlimbs . in this system , we observed that nwb group rats developed onfh at a comparable rate as did wb group rats . taken together , the results of the present study confirm that weight bearing does not contribute to the development of non - traumatic onfh in rats . to clarify the pathogenesis of onfh , several quadruped animal models of osteonecrosis , including both traumatic and non - traumatic forms , have been reported ( yamamoto et al . 1995 ; norman et al . however , the quadruped animals in these models fail to progress to end stage mechanical collapse similar to that in bipedal animals , such as humans ( conzemius et al . xu et al . ( 2010 ) reported that this is caused by a difference in weight bearing through the hip joint between quadruped and bipedal animals . however , our results showed that weight bearing did not contribute to the initial development of onfh in nwb rats . therefore , it is speculated that the rat model may help us to elucidate the pathogenesis of onfh and to devise a prevention strategy for the condition , but may not be useful for the observation of the mechanism of femoral head collapse . in conclusion , the present study revealed that weight bearing is not related to the development of non - traumatic onfh in the rat model , although the aetiology , pathogenesis and prognosis of non - traumatic onfh remain unclear .

the hip joint is one of the major structures in the human body and the resultant force acting through the hip joint is 300% of body weight . therefore , weight bearing , as a cause of ischaemia , may contribute to the development of non - traumatic osteonecrosis of the femoral head ( onfh ) . however , it remains unclear whether weight bearing is related to the development of non - traumatic onfh . therefore the aim of this study was to clarify the role of weight bearing in the development of non - traumatic onfh . non - weight - bearing ( nwb ) rats were tail suspended to prevent any weight coming to bear on the hindlimbs from day 1 to the time of sacrifice . the weight - bearing ( wb ) group rats were also housed individually , although without tail suspension . all rats were injected with lipopolysaccharide and methylprednisolone to promote the development of non - traumatic onfh . all animals were sacrificed three weeks after the final methylprednisolone injection . histopathological analysis was performed . osteonecrosis of the femoral head was observed not only in the nwb but also in the wb rats ; however , no osteonecrosis of the humeral head was observed in either group . we confirmed that non - traumatic onfh developed in nwb rats , suggesting that weight bearing does not contribute to the development of non - traumatic onfh in rats .

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with the global debates around health policy reforms , there have been increasing calls for nurses to be actively engaged in national policy processes [ 13 ] . first , nurses comprise the majority of the global health care workforce , working closely with patients and their families in a variety of settings . thus , nurses ' experiences and insights may help guide improvements in the quality of health service delivery and inform health systems strengthening . second , many health sector policies have an impact on nurses ' work environments and consequently their professional practice . thus , nurses ' input on health sector policies could help ensure that supportive work environments for clinical practice are taken into account when policies are reformed . given nurses ' pivotal role in health care delivery , their engagement in policy changes in the system has been described as both a moral and professional obligation as well as a responsibility to the people they serve [ 2 , 4 ] . the engagement of nurses in national policy processes is especially important in lower income settings where nurses comprise an even larger proportion of the health sector workforce and where the largest burden of illness exists . historically , nurses have had limited involvement in national policy and political decisions that affect health service delivery [ 58 ] . for instance , a quantitative study of national nurse leaders and hospital based nurses in thailand revealed that majority of nurses were not involved in policy formulation or modification stage . literature further reveals several reasons for nurses underengagement in policy including lack of awareness of policy related issues and processes , inadequate knowledege and skills on how to contribute to policies , and lack of opportunities to contribute to policy decisions [ 911 ] . nurses ' limited participation in policy processes has also been attributed to gender differences in the health care system [ 12 , 13 ] . nursing is predominately viewed as a woman 's occupation involving caring for others , while men are typically seen in positions of power and decision - making [ 1416 ] . thus , men are more likely than women to be involved in public policy and to influence policy decisions . in kenya , nurses ' underengament has also been attributed , in part , to an inadequate policy focus in undergraduate and graduate nursing programs . studies provide several strategies that can be applied to enhance nurses ' engagement in policy processes . several studies have emphasized that nurses can engage in policy processes directly by assuming formal leadership positions in the health care system , and indirectly through lobbying and advocacy with policy - makers via professional organizations [ 12 , 17 ] . the nurse leaders can identify issues strategically , work with decision - makers , and understand who holds the power in the health care organizations and who controls the resources for health care in order to influence them . another strategy is to build nurses ' understanding of and competencies in policy and politics by integrating pertinent courses in nursing training and fellowship programs [ 1720 ] . examples of programs developed to enhance nurses ' competency in leadership and policy influence exist mainly in developed countries [ 21 , 22 ] . for instance in the usa a program was developed to train nurse leaders who would influence policy change . the training helped the nurse leaders to understand how policy reforms affect health care , the policy formulation process , and the challenges affecting health care including access , quality , and care as well as the role of media . several authors have emphasized the need for nurses to utilize research as credible evidence to inform policy decisions . however , the studies have found that nurses have not been adequately supported to generate evidence to inform health policies [ 2326 ] . furthermore , nurses from low- and middle - income countries ( lmics ) have inadequate training and mentoring in research as well as scarce resources for conducting research . thus , there is a need to strengthen lmic nurses ' research abilities to enhance their ability to understand , generate , and utilize research knowledge that is useful for policy change . very few emperical studies have examined extent of nurses ' involment policy developemnt process in the african context . a survey of nurse leaders involvement in health policy process in east africa found that nurse leaders ' participation in all stages of policy development was limited and inconsistent . another study looked at nurses involvement in hiv / aids policy in four lmics including kenya and uganda . the study revealed nurses ' lack of involvement in policy processes , poor communication of policies from the national level , and a lack of resources for nurses to implement the policies . published studies are mainly descriptive reviews and have not used a critical theory lens to analyze nurses ' contributions to policy processes . furthermore , studies have rarely compared the perspectives of nurses and other decision - makers regarding nurses ' engagement in policy development and implementation processes . this study applied a critical theory perspective to examine how nurses had been involved in national health policy processes in kenya , eliciting the perspectives of nurses and decision - makers working at various levels of the health care system . feminist theories are related to critical theory but focus on gender domination and discrimination within patriarchal societies [ 28 , 29 ] . from feminist perspectives , analysis examines formal power structures that create or maintain gender inequality , thereby systematically elevating men above women [ 31 , 32 ] . in this study , critical theory helped interrogate historical hierarchies and structures as well as gender - related issues affecting nurses ' participation in policy processes . the overall study applied a two - phased mixed method ( qualitative / quantitative ) design to examine the influence of policies on nurses ' work as well as how nurses were involved in policy processes in the kenyan health care system . this paper focuses on the qualitative results , describing how nurses were involved in national policy processes in the kenyan public health sector . the perspectives of nonnursing decision - makers , national level nurse leaders , and frontline nurses and managers were sought . the specific objectives were ( 1 ) to analyze the extent of nurses ' involvement in the national policy development processes in the health sector ; ( 2 ) to analyse the reasons for nurses ' inadequate involvement in the national policy development in the health sector ; and ( 3 ) to describe mechanisms though which nurses ' involvement in policy development processes can be improved . the study was carried out in the kenyan public health care system , since this sector has primary responsibility for policy development and provides about 55% of health care services . the health delivery system is organized in a pyramidal structure with six levels of care . the community is the first level , followed by dispensaries and medical clinics , health centers and nursing homes , district level hospitals , and provincial level and national level hospitals . decision - making structures exist at national , provincial , and district levels , all of which include nurse representation . although these nurses work at all levels of the health care system , the majority are employed at the district levels . the majority of nurses entered the health care service with diploma or certificate level preparation until the early 1990s when the country started training degree level nurses . kenya 's nursing data base for 2011 indicates that among those employed in the public sector , the majority were certificate nurses ( 53% ) , followed by diploma nurses ( 46% ) , with a small minority of degree level nurses ( 1% ) . decision - makers at national , provincial , and district levels were purposively sampled based on their positions involving policy development or implementation . national level nonnursing and nursing decision - makers employed by the ministry of health were eligible for selection . two provinces ( nyanza and central ) were purposively selected because of their varied geographical locations and differences in health indicators . nyanza province had poor health indicators and few health service resources , while the central province had better health indicators and more health service resources . one district was then purposefully selected from each of these provinces and the two main district public hospitals were the sites for sampling district - level nurses and managers . nurses and nurse managers in these district hospitals were selected based on a sufficiently long work history in the system and 2 years or more in the facility which gave them potential familiarity with many of the policy changes affecting nursing and health system reforms in kenya . open - ended interview guides tailored to participant groups were used with all key informant interviewees . the groups included national level nursing and nonnursing decision - makers , decision - makers at provincial and district levels , and frontline nurses and managers at the hospital level . participants were asked questions about national level health policy - making processes ; extent to which nurses engaged in policy - making and implementation processes ; and ways to improve nurses ' involvement in policy processes . data collection was stopped when saturation was reached . additionally , the researcher kept field notes during the visits . audio - taped data were transcribed verbatim and they were analyzed simultaneously with data collection . transcripts from each participant were read repeatedly before and after coding to gain a sense of the whole interview . interview data were clustered in three groups including nonnursing decision - makers , nurse leaders and frontline nurses , and nurse mangers ; then all the data sets and field notes were triangulated as the analysis proceeded . data was initially coded and divided into contents including nurses ' engagement in policy , reasons for under engagement , and ways to improve nurse ' engagement . matrices were then used to compare categorized responses of national level nonnursing decision - makers , national nurse leaders , and frontline nurses and managers [ 36 , 37 ] . further analysis and interpretation of findings was then done using critical and feminist lens to identify hierarchies and gender related factors that influenced nurses ' engagement in policy development . credibility was enhanced throughout the study process by maintaining accuracy in data collection , analyzing , and reporting . critical reflexivity involving cyclical and continuous comparisons of data collected were used throughout data collection and analysis . these included journaling and writing reflections on processes and decisions [ 36 , 37 ] . transferability was enhanced through the purposive recruitment of participants at different health care system levels . triangulation of data from different types of respondents was done making it possible to compare and confirm similar perceptions across different groups . ethical approval was obtained from university of ottawa health sciences and science research ethics board in canada and great lakes university of kenya ethics review committee . administrative approval was also obtained from the ministry of health in kenya , and appropriate authorities in provincial health offices , district offices , and the two district hospitals where study participants were recruited . a total of 32 interviews were conducted ( table 1 ) . at the national level , five interviews were conducted with nonnursing decision - makers at the ministry of health , one interview with the world health organization country representative and three interviews with national nurse leaders . each of the three national nurse leaders had risen up the ranks from the district level to the national level and had worked for more than 20 years in public service . at the provincial level , two nonnursing decision - makers and two nurse leaders were interviewed in nyanza and one nonnursing decision - maker and two nurse leaders interviewed in central province . at the district level , one nurse manager in each district office and most respondents were of the opinion that nurses were not adequately involved in the health sector policy development processes at the national level . they mentioned that only three nurse leaders from the offices of the chief nurse , the nursing council and the , national nurses association had been invited to participate in national policy committees as part of their jobs . the nurse leaders ' input on these committees was described as revolving around representing nurses ' issues at selected policy tables , communicating policies to nurses , and regulating nursing education and practice . the chief nurse was charged with the highest responsibility of representing all kenyan nurses at policy meetings . two of the nonnursing decision - makers felt that kenyan nurses were adequately involved in national policy - making processes through the nurse leaders ' representation at the national level . in contrast with this view , all nursing participants felt that this representation was inadequate given the large number and diversity of nurses in the country.generally nurses have not been fully involved in policy formulation . many a time nurses are involved after policies have been formulated , and maybe just a few nurses [ national nurses leaders are involved in the policy formulation stage ] ; the province is just informed later that this is happening , but really there has been no good input from nurses . ( national nurse leader ) generally nurses have not been fully involved in policy formulation . many a time nurses are involved after policies have been formulated , and maybe just a few nurses [ national nurses leaders are involved in the policy formulation stage ] ; the province is just informed later that this is happening , but really there has been no good input from nurses . ( national nurse leader ) furthermore , the contributions of these three nursing leaders to the national policy formulation processes were considered inadequate by both the nurse leaders and nonnursing decision - makers but for different reasons . while nonnursing decision - makers emphasized the limitation of nurse leaders ' singular focus on nursing issues , the nurse leaders described the problem of inadequate representation of nurses on a wider set of national policy committees . they described representation on these committees as being primarily heads of departments , the majority of whom were doctors . the public health act , which allows doctors to be the heads of ministerial departments , as well as provincial and district health offices , affords doctors this role.normally the heads of the various departments sit down ; many of them are headed by doctors because of the public health act , and others would be the technical people . ( national nurse leader ) normally the heads of the various departments sit down ; many of them are headed by doctors because of the public health act , and others would be the technical people . ( national nurse leader ) the findings also revealed nurse leaders ' uneven participation in policy meetings . according to the nurse leaders , nurses only attended those policy meetings requiring reports on nurse situations , while doctors were active in every policy meeting regardless of the policy content . furthermore , nurse leaders noted that many of the final policy decisions had been made without nurse leaders being present . frontline nurses and nurse managers at district levels described their primary policy role as being implementers of national policies . even though policy - making was considered a national level activity , nurses at the frontline were concerned that they were not involved . they added that their contribution could be important since nurses better understood the care environment.nurses should be consulted before policies are started because we are the majority , but they decide for us ( ) . nurses have everything that they need , education , brains , but they lack chance to air views . ( provincial nurse leader ) nurses should be consulted before policies are started because we are the majority , but they decide for us ( ) . nurses have everything that they need , education , brains , but they lack chance to air views . ( provincial nurse leader ) a common view , among frontline nurses , was that they were excluded from policy decisions even in the workplace.we are not involved , yet we are the ones who really are on the ground , we are the backbone of the hospitals . we are like a mother in the kitchen , we are the ones who know how much we need , so we should be really involved in every step , because you can not just tell me now cook and even i was not there in the shopping but you want to see what i am cooking ; we should be involved from the word go . ( frontline nurse ) we are not involved , yet we are the ones who really are on the ground , we are the backbone of the hospitals . the other people come to write papers and go away ( ) . we are like a mother in the kitchen , we are the ones who know how much we need , so we should be really involved in every step , because you can not just tell me now cook and even i was not there in the shopping but you want to see what i am cooking ; we should be involved from the word go . ( frontline nurse ) due to their limited involvement , frontline nurses and managers felt that their priority work - related issues such as staff shortages , motivation , and resource inadequacies were not being addressed . yet they felt that policies were just pushed on them , so they sat back and watched or did only what they could , as one nurse manager commented : lack of nurses ' involvement in formulation is the cause of poor policy implementation . they are not positive towards it , but since they have to comply , they do it , but not in the right way . when you are involved from the beginning , you understand and own it , and during implementation , you have inward drive to accomplish what you planned ( ) . we feel like we are pushed at the corner , so we remain there and watch things as they are done , or do things because they must be done , not that we feel motivated to do them . they are not positive towards it , but since they have to comply , they do it , but not in the right way . when you are involved from the beginning , you understand and own it , and during implementation , you have inward drive to accomplish what you planned ( ) . we feel like we are pushed at the corner , so we remain there and watch things as they are done , or do things because they must be done , not that we feel motivated to do them . the participants gave several reasons as to why there was inadequate nurses ' participation in national health policy processes . one reason was that policy - making primarily followed a top down approach , emanating from the national level . respondents felt that this approach denied nurses opportunities for direct involvement in national policy decisions because most nurses are employed at lower levels of the health care system . as one nurse manager said for me policy - making is a thing for those at the top , whether they are doing the right thing or not , to them it does not matter . another frontline nurse commented that policies are made from above , they are not made according to how you want things to be done , they are just brought and you are supposed to follow them . other reasons for nurses ' lack of engagement in policy formulation mentioned by nonnursing decision - makers were their lack of knowledge about the policy - making process and lack of skills to engage in this process . nurse leaders concurred that nurses did not generally understand the broader issues outside nursing that were necessary to influence policies . they largely blamed this on the omission of any curriculum related to policy issues in basic nursing training . others felt that nurses ' low participation was due to the general design of the health care system , which excluded nurses from policy - making structures.it is by design of health system that nurses are not brought at the policy tables [ the ] majority of nurses lack knowledge and skills ( ) to support that policy . therefore nurses should be made to add value when an opportunity comes for them to sit at the policy - making tables , not to be a passenger but to be a participant . ( national nurse leader ) it is by design of health system that nurses are not brought at the policy tables [ the ] majority of nurses lack knowledge and skills ( ) to support that policy . therefore nurses should be made to add value when an opportunity comes for them to sit at the policy - making tables , not to be a passenger but to be a participant . ( national nurse leader ) nurses were also perceived as being isolated from other professionals . nonnursing decision - makers blamed the nursing profession for its tradition of being a closed professional group , concerning themselves only with professional issues and not broader issues affecting the health care system . a few of the decision - makers felt that nurses had failed to market themselves as important providers in the health care system . in addition , they felt that nursing had a hierarchical system that hindered junior nurses from participation . one national nonnursing decision - maker was particularly strident in his / her remarks : nurses are allergic to change . nursing profession discourages nurses from thinking because it 's the matron who decides , ( ) . they have put a self - imposed mind block by relying on another person to make decisions ( ) . they go through a very hierarchical system unlike other professions . while a nurse will still report to a nursing officer , she can not make decisions on her own , it is the biggest weakness , there is no freedom , and they maintain the status quo . nursing profession discourages nurses from thinking because it 's the matron who decides , ( ) . they have put a self - imposed mind block by relying on another person to make decisions ( ) . they go through a very hierarchical system unlike other professions . while a nurse will still report to a nursing officer , she can not make decisions on her own , it is the biggest weakness , there is no freedom , and they maintain the status quo . ( nonnursing national decision - maker ) another reason expressed for nurses ' nonengagement was the inability of nurses to bring forward research evidence to inform policy formulation . nurse leaders commented that nurses had inadequate skills for generating and utilizing research evidence to influence policy decisions at national levels . frontline nurses complained that a further impediment was the failure of nurses to take united action to influence policy . they had different professional associations ( the kenya registered nurses ' association and kenya professional nurses association ) , which served different nursing groups and tended to divide nurses and their leaders . participants gave several suggestions regarding how nurses ' participation in the policy process could be enhanced . they mentioned actions to be taken by decision - makers to enhance nurses ' participation in policy - making . these included creating more opportunities for nurses to contribute to policy processes , increasing nurses ' representation on policy - related structures , and reviewing the public health law to allow nurses to compete for leadership positions in the system . in addition , frontline nurses felt that national policy - makers should consult frontline nurses when developing national policies . it was their view that this would enhance bottom - up input on policy - making . as one frontline nurse said : i believe there is a way of channeling suggestions until they reach the national level , maybe through the nurses in charge of hospitals to the provincial then to the national level through writing and meetings . i believe there is a way of channeling suggestions until they reach the national level , maybe through the nurses in charge of hospitals to the provincial then to the national level through writing and meetings . as their representatives , the frontline nurses felt that nurse leaders should come to the ground more often and get views from frontline nurses to include in policies : nurse leaders are at the top , when there are issues to be discussed , they can come down , involve us in discussions and gather information , so that when they make these policies they know it is something that has been discussed , it has been understood and it is needed , because occasionally policies have been made [ and ] when they come down here they do not even work . ( frontline nurse manager ) nurse leaders are at the top , when there are issues to be discussed , they can come down , involve us in discussions and gather information , so that when they make these policies they know it is something that has been discussed , it has been understood and it is needed , because occasionally policies have been made [ and ] when they come down here they do not even work . ( frontline nurse manager ) some nursing participants mentioned that the opinion of frontline nurses could be sought through research and regular meetings and then brought to policy - making processes . in particular , they identified the need to empower nurses through training to enable them to better understand and participate in policy formulation . while the experiences of nurses were thought to be important assets for this input , a broader knowledge of the health care system , of policy - making , and of decision - making processes , were described as essential prerequisites for constructive contributions.the bottom - line is that you must be knowledgeable , you may be a real nursing guru , ( ) your contribution should not only be based on care , of course care is very critical , but you must have general knowledge like how the government operates ( ) . majority of nurses do not know anything apart from nursing , you are supposed to have general knowledge , like international monetary funds policies , how does it affect us ? ( nurse leader 2 ) the bottom - line is that you must be knowledgeable , you may be a real nursing guru , ( ) your contribution should not only be based on care , of course care is very critical , but you must have general knowledge like how the government operates ( ) . majority of nurses do not know anything apart from nursing , you are supposed to have general knowledge , like international monetary funds policies , how does it affect us ? ( nurse leader 2 ) other areas identified for skill - building included political skills needed to engage the government to address policy gaps that affected nurses ' work as one nurse leader said that nurses in this country lack political skills , they should have political skills in order to dialogue with politicians and other leaders in this country . in addition the participants mentioned that nurses need data management and research skills to bring evidence to policy . finally , they mentioned actions to be taken by nurses themselves to enhance their ability to participate in policy - making . nonnursing decision - makers strongly suggested that nurses should take the lead to change their situation . they mentioned that nurses should market themselves , progress in their careers , diversify , venture outside the nursing profession , and broaden their curriculum in order to understand broader policy related issues.nurses build empires around themselves ( ) . nurses must be free to venture into other areas and be competent in those areas and not tied to nursing . more often , you find doctors in areas , which are not related to medicine and they fit well . they do not have to link strongly to their profession , they are more independent . ( national nonnurse decision - maker ) nurses build empires around themselves ( ) . nurses must be free to venture into other areas and be competent in those areas and not tied to nursing . more often , you find doctors in areas , which are not related to medicine and they fit well . they do not have to link strongly to their profession , they are more independent . ( national nonnurse decision - maker ) congruent with this decision - maker 's opinion , some of the nurse leaders interviewed added that more nurses should position themselves to take up leadership roles in the system . they indicated that nurses should diversify their knowledge , be united to build a strong voice , collaborate and network within and beyond the country , and be assertive about what they wanted . they added that the nursing council and the professional association should be strengthened to enhance their respective roles in influencing national policies that affect nurses ' work . frontline nurses added that nurse leaders should be more proactive in policy activities so as to influence policy - makers . some frontline nurses felt that this type of advocacy would be an incentive for front line nurses to participate in decision - making in the health care sector . from a critical perspective , two major issues influencing nurses ' involvement in policy processes emerged . these issues were hierarchies and structures influencing decision - making in the health sector and nurse and nursing professional issues . three intersecting hierarchies hindered nurses ' active engagement in policy processes in kenya : health systems hierarchies , interprofessional hierarchies , and intraprofessional hierarchies . the kenyan health systems decision - making structure is organized following political administrative levels including central headquarters and provincial and district levels . the political structures mandate the central administration as the policy - making and service planning level . the kenyan health policy framework of 19942010 emphasized that policy formulation was the role of the central office . this top - down policy - making process limited nurses ' opportunities to engage directly in policy formulation in the health care system . this policy framework delineates a policy implementation rather than a policy formulation role for nurses . given the top - down nature of policy - making processes in the health sector , particularly with the many stakeholders and foreign donors involved , the findings speak to the importance of finding ways to give nurses some voice in decision - making at all levels . the interprofessional hierarchy described within the health sector identified medical doctors as being the dominant policy decision - makers at various levels of the health care system . social constructs of racialized status and gender imbue health care hierarchies and are grounded in the legacy of kenya 's colonial period that ended in 1963 . white colonialists developed health care institutions that favored a male- and white - dominated medical profession over a female- and african - dominated nursing profession . the education system prepared physicians with first degrees while nurses acquired certificates and later diplomas . although baccalaureate degree nursing education in kenya began in the late 1990s , only one percent of nurses in the public health care sector have baccalaureate degrees . resultantly , medical doctors have higher status and are granted more decision - making authority in the system as opposed to nurses whose education and status were , and are considered low , further precluding nurses from higher decision - making positions . the interprofessional hierarchy is further sustained by the legal system , specifically the public health act , which led to the systematic exclusion of nurses from senior leadership positions at various levels of the health care system . according to the act , physicians were to serve as the heads of various departments , administrative directors of districts and hospitals , and managers of various programs , while senior nurses could only be placed in positions to represent nurses , thus limiting their participation in policy and other health care decisions . this legislation thus legitimized the gendered professional hierarchy and further limited the types of issues that nurse leaders could bring to the policy table . nurse leaders ' participation at policy tables was considered nursing centric by both nursing and nonnursing leaders . while the majority of decision - makers blamed nurse leaders for nursing centric participation , this kind of participation was also shaped by the system , which mandated nurse leaders to report only on nursing issues . this is consistent with a global study of nurse leaders focusing on chief nursing officers , which revealed that , even though these nursing leaders had varied responsibilities associated with their positions , they were only asked to provide nursing - related advice . the study revealed how an intraprofessional hierarchy within the nursing profession played out in the policy arena . nurse leaders at the national level were recognized as the sole professional representatives of other nurses in the country who would provide input on national policy decisions . the intraprofessional hierarchy influenced channels of policy communication to frontline nurses who received new policy directives from their supervisors at every level of the system . this hierarchical pattern of decision - making within nursing may have been exacerbated by the fact that there are only a few nurses in key national level positions , limiting the scope of their policy influence at the national level and precluding them from engaging nurses more proactively at the district level or on the front lines in policy - oriented discussion . in addition , nurses at lower levels felt oppressed by senior nurses who made most of the decisions . from the findings , the nonnursing decision - makers had more to say about nurses ' oppression than nurses themselves . nonnursing decision - makers indicated that nursing was a profession where the oppression of lower level nurses was a problem . however , they were generally pessimistic , indicating that nurses were not yet ready to make a change . the oppression of lower cadre nurses by higher cadre nurses in decision - making has been documented in other studies [ 4042 ] . in the absence of feedback from the grassroots on policy implementation , as identified by participants in this study , the voices of nurses at lower levels might be suppressed further . given the fact that kenya has a decentralized health care system , more participatory approaches could be introduced to enhance nurses ' potential engagement in policy - making , thus improving bottom up planning and decision - making . this would increase the way in which those who have less power in a hierarchy get voice . both nursing and nonnursing participants attributed nurses ' underengagement in policy to issues related to the nurses and the nursing profession . respondents consistently indicated that nurses had inadequate knowledge and skills required to contribute to policy processes . they described this deficiency in nurses ' knowledge as being attributable to a failure of the nursing profession to incorporate policy issues and policy process into nurses ' training curricula , thus maintaining the status quo for nurses ' role in decision - making . studies in other contexts have revealed how lack of policy content in nursing training contributes to nurses ' lack of understanding of policy . given the need to strengthen nursing leadership to influence policies , other programs have emerged to train nurse leaders in ways to influence policy . in the african context , many nurse leaders have been enrolled and trained in a leadership training program offered by international council of nurses . this participation may help strengthen nurses ' competency in engaging in national health care decision - making . study findings also revealed that nurses had inadequate advocacy , research , and political skills required to influence policies . these issues are similar to what has been identified in studies from higher income countries [ 12 , 17 , 23 ] . in these studies , the profession is seen to have been slow in incorporating these issues in nursing education . nonnursing decision - makers in this kenyan study felt that the profession had reinforced its own weaker professional status by failing to enhance nurses ' abilities and competencies to influence decisions at strategic levels in the system . their comments reflected an openness to incorporate nurses ' views if nurses were competent in national policy processes and more knowledgeable about general health care system issues . the findings also revealed that nurses have worked in isolation from other professionals in the system . with the emphasis on professional transformation towards equality and diversity [ 44 , 45 ] this would require nursing champions to take up leadership in working with other health cadres to influence policy change . while leadership competencies have been recognized as key attributes to shaping and influencing policy [ 46 , 47 ] , in this study nursing leadership was viewed as internal to the nursing profession , with concerns about the focus on nurses ' work issues and not general health care issues . nursing champions should not focus on the profession alone ; they must be educated in the broader health systems issues and become patient advocates for health care as they engage with other professionals in leading policy change . kenya has many district level hospitals but data from frontline nurses was mainly collected from two districts given the limitation in funding . in conclusion nurses in kenya have not been adequately involved in national policy development . applying critical theory perspective in the analysis has revealed important health system structures and hierarchy as well as nursing professional factors hindering nurses ' participation in policies making processes . these factors must be addressed if nurses ' input in policy - making is going to be significantly altered . to enhance nurses ' participation in policy processes , the policy - makers should address hierarchies , domination , and gender issues inherent in the health care system and establish mechanisms through which the views of nurses could get to national level decision - makers when policies are being formulated . this can be achieved by enacting legislation in kenya that would allow nurses with competencies in leadership and policy to hold senior positions in the health care system just like doctors . with governance opportunities created through decentralization of power , there is a need for all health professionals to have equal opportunities to be in leadership and management positions that are embedded within the decision - making structures in the health care system . to enhance bottom - up policy - making , national level decision - makers should solicit the views of nurses at the frontline with regard to policies that might affect their work and quality of care . there are well - described processes for this purpose [ 49 , 50 ] but they can not be implemented in isolation of process changes at the district and national levels , which build mechanisms for continuous quality improvement . leadership and advocacy skills should be developed within nursing to enhance nurses ' abilities to carry out policy advocacy at all levels . icn has developed programs aimed at building the capacity for such leadership in societies around the world . these programs include the leadership in negotiation program , the global nursing leadership institute , and the leadership for change program . nurses in this region can benefit from these programs . to enhance their role in advocacy , nurses should unite to build a strong voice to advocate for improvements in national policies that affect their work and quality of care . working in collaboration with other health professionals , nurses can influence decisions to strengthen the health care system , resource allocation , and primary health care as well as patient care . in addition , nursing regulators and educators should be clear that policy involvement is part of nurses ' scope of practice . nurses , particularly at the frontline , should be given orientation on existing national policies in the sector and how these policies are affecting their work environment and patient outcomes . this would be an important step towards better communication of policy impact , creating a much needed feedback chain between those implementing policy and those formulating policy . as members of nursing associations , frontline nurses should encourage and support their nurse leaders to speak for nurses at the national policy tables . in their workplaces , decision - makers should allow other nurses to actively engage in organizational policy decisions during policy implementation . the nursing profession should consider and implement strategies to strengthen nurses ' capacity to contribute to policy decisions that would enhance service delivery . in order to effectively do this , nurses in kenya will need to understand that the policy cycle is complex and influencing policy decisions depends on having a sufficient understanding of the broader issues in the health care system , as well as knowledge and competencies in policy formulation , implementation , and evaluation . to enhance nurses ' awareness , literacy , and competency in policy , nursing educators should introduce a course on policy and policy processes , as well as policy influencing in the nursing training curriculum . finally , the nursing profession should carry forward the notion of evidence - based policy - making . they should develop skills in research and conduct both health systems and clinical research to inform policy - making decisions . research courses should be linked to policy courses and training curricula should include current and locally relevant examples of how evidence can be used to shape policies . this would include strategies and tools for communicating research to policy - makers , like writing policy briefs and position statements based on research evidence . sharing of policy relevant research would contribute to policy change and resource allocation to improve nurses ' work . this is one of the first studies examining the perspectives of both nurses and nonnurses at different system levels regarding nurses ' involvement in national policy - making processes . with the continuing health care policy reforms , further examination of issues of hierarchy , gender , and power inherent in the health care system the question is how do we unravel the deeply embedded hierarchies and power processes that sustain nurses ' status quo in policy change process ? of particular importance is how the hierarchies affect the role and performance of nurses at lower levels and how can nurses influence national leaders to enhance participation in health policy change processes . furthermore , this study did not carry out an in - depth examination of the competencies held by nurse leaders that would be useful for policy change ; thus future research should examine these aspects .

the aim of this study was to critically examine how nurses have been involved in national policy processes in the kenyan health sector . the paper reports qualitative results from a larger mixed method study . national nonnursing decision - makers and nurse leaders , and provincial managers as well as frontline nurse managers from two kenyan districts were purposefully selected for interviews . interviews dealt with nurses ' involvement in national policy processes , factors hindering nurses ' engagement in policy processes , and ways to enhance nurses ' involvement in policy processes . critical theory and feminist perspectives guided the study process . content analysis of data was conducted . findings revealed that nurses ' involvement in policy processes in kenya was limited . only a few nurse leaders were involved in national policy committees as a result of their positions in the sector . critical analysis of the findings revealed that hierarchies and structural factors as well as nursing professional issues were the primary barriers constraining nurses ' involvement in policy processes . thus , there is need to address these factors both by nurses themselves and by nonnursing decision makers , in order to enhance nurses engagement in policy making and further the contribution to quality of services to the communities .

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uti is a common problem in infants and young children ( 1 , 2 ) ; however , its frequency , symptoms and causative organisms varies in accordance with sex , age , and gender ( 3 ) . unlike other age groups neonates present with non - specific signs and symptoms ( 4 ) warranting a complete sepsis workup followed by a 7 to 14 day - course of antibiotic therapy ( 2 ) , typically parenteral ampicillin plus an aminoglycoside ( 5 ) or a third - generation cephalosporin ( 6 , 7 ) . recent reports on emerging patterns of resistance toward empirical antibiotics ( 812 ) , especially ampicillin ( 1317 ) , have made the early choice of antibiotics a more challenging decision ( 8) . the criticalness of issue lies in the fact that those diagnosed earlier and treated accordingly , are less likely to bear long - term consequences imposing an increased burden on health care systems ( 18 ) . clinical manifestations of uti in neonates are mostly non - specific and of a systemic nature ( 15 ) like fever , irritability , lethargy , vomiting , growth failure , abnormal urination- namely oliguria , polyuria or malodorous urine , and jaundice ( 14 , 1921 ) . the non - specific presentations often make uti hard to diagnosis ; however , its reported prevalence ranges from 4 - 25% ( 22 ) in in very low weight infants ( birth weight 1000 g ) to 0.1 - 1% in term infants ( 23 ) . additionally , uti in newborns could lead to urosepsis or neonatal sepsis that is why it is often managed as a case of sepsis ( 24 ) . most utis in infants are caused by gram - negative bacilli ( 23 , 2527 ) . e. coli has been the most prevalent organism causing uti in all ages including neonatal period ( 1 , 21 , 2830 ) accounting for as many as 80% of isolates in many studies ( 25 ) . other enterobacteriaceae causing uti in neonates include klebsiella , enterobacter , citrobacter , proteus , providencia , morganella , serratia , and salmonella ( 6 , 26 , 27 ) . gram - positive bacteria like staphylococcus and enterococcus have less been retrieved from neonates with uti ( 2 , 26 ) . this is not possible unless a continuous surveillance is done and let therapeutic choices to be updated accordantly . these motivated researchers to retrospectively review all uti cases managed in an inpatient setting of neonatal division and aim to this end . the primary intention of this study was to see the frequency of uti causative organisms in neonates admitted to neonatal division of bahrami children s hospital over an eight year period . the study accomplished at the neonatal division of bahrami children s hospital - tehran , iran , the largest tertiary pediatric center in the eastern end of tehran . all cases over an eight - year period ( june 2004 to june 2012 ) constituted the population of current study the source of extracted data were the hospital records of the patients regarding age ( day ) , sex , date and method of urine sample collection , urine culture results , and antibiogram reports . the latter routinely was based on the report of laboratory unit of bahrami children s hospital which reports antibiogram by semi - quantitative approachbased on diffusion technique , and the available laboratory standards . current study designed as a case series using simple non - random sampling to recruit all cases with diagnosis of uti admitted to neonatal division of bahrami children hospital tehran , iran in 2004-to-2012 period . in this study , uti was defined ( 6 ) as : i ) a pure growth of single bacteria from a urine sample obtained by suprapubic aspirate , ii ) 10 cfu / ml from a urine sample obtained by catheterization . cases with more than one episode of uti were separately analyzed if they occurred more than 14 days apart . only the result of the first positive urine culture was extracted . in the event of two separate initial positive cultures , result of suprapubic aspirate cases diagnosed on the basis of a urine bag sample were not included in the study . those with incomplete records , including absence of two confirmatory progress notes regarding urine sample collection method were excluded from the study . to increase accountability of results , only those cases included which had a physician progress note and nursing staff note on the way urine is collected . mixed growth urine cultures denoting growth of two or more organisms in one sample were excluded , as they are likely to represent contaminated samples . 17 ) software spreadsheets . ultimately , quantitative data displayed as minimum , maximum , means , standard deviation , and qualitative data as the relative frequency . all research ethics and regulations adopted by the iranian medical commission of scientific research council were considered in every steps of this study and researchers will adhere to it . academic honesty and trustworthiness , impartiality and avoiding certain trends have been respected ; however , the study was not posed with any other ethical prohibition and was approved by the research ethics committee of the tehran university of medical sciences . the study accomplished at the neonatal division of bahrami children s hospital - tehran , iran , the largest tertiary pediatric center in the eastern end of tehran . all cases over an eight - year period ( june 2004 to june 2012 ) constituted the population of current study the source of extracted data were the hospital records of the patients regarding age ( day ) , sex , date and method of urine sample collection , urine culture results , and antibiogram reports . the latter routinely was based on the report of laboratory unit of bahrami children s hospital which reports antibiogram by semi - quantitative approachbased on diffusion technique , and the available laboratory standards . current study designed as a case series using simple non - random sampling to recruit all cases with diagnosis of uti admitted to neonatal division of bahrami children hospital tehran , iran in 2004-to-2012 period . in this study , uti was defined ( 6 ) as : i ) a pure growth of single bacteria from a urine sample obtained by suprapubic aspirate , ii ) 10 cfu / ml from a urine sample obtained by catheterization . cases with more than one episode of uti were separately analyzed if they occurred more than 14 days apart . only the result of the first positive urine culture was extracted . in the event of two separate initial positive cultures , result of suprapubic aspirate cases diagnosed on the basis of a urine bag sample were not included in the study . those with incomplete records , including absence of two confirmatory progress notes regarding urine sample collection method were excluded from the study . to increase accountability of results , only those cases included which had a physician progress note and nursing staff note on the way urine is collected . mixed growth urine cultures denoting growth of two or more organisms in one sample were excluded , as they are likely to represent contaminated samples . 17 ) software spreadsheets . ultimately , quantitative data displayed as minimum , maximum , means , standard deviation , and qualitative data as the relative frequency . all research ethics and regulations adopted by the iranian medical commission of scientific research council were considered in every steps of this study and researchers will adhere to it . academic honesty and trustworthiness , impartiality and avoiding certain trends have been respected ; however , the study was not posed with any other ethical prohibition and was approved by the research ethics committee of the tehran university of medical sciences . a total of 73 cases ( mean age , 14.14 7.68 days ; mean birth weight of 3055.85 623.00 grams ; and mean admission weight of 3180.55 610.25 grams ) fulfilled the inclusion criteria and recruited in the study . boys constituted 69.9% ( 51 neonates ) of the study population , bringing a boys - to - girls ratio of 2.32 . four different bacteria were isolated from retrospectively assessed cases , namely e. coli , enterobacter , klebsiella , and s. epidermidis no cases of proteus , pseudomonas , serratia , s. aureus , enterococcus were found over the study period . e. coli was the most prevalent organism of the study with a relative frequency of 64.4% ( 47 cases ) followed by enterobacter with a relative frequency of 19.2% ( 14 case ) . however , frequency of isolated organisms as well as their relative frequency in study population is represented in table 1 distribution of organisms isolated from the urine in the study in this study , e. coli found to be mostly resistant toward ampicillin ( 93.6% ( 44 cases ) ) , cefixime 85.7% and cephalexin 77.3% respectively . the most sensitivity of e. coli toward the antibiotics commonly used as first line neonatal empirical therapy were toward cefotaxime ( 63.6% ) ; however , its most susceptibility was to norfloxacin ( 100% ) and ciprofloxacin ( 100% ) respectively in 14 and 12 patients . the general overview of e. coli susceptibility pattern toward all applied discs is summarized in table 2 . distribution of study escherichia coli antibiotic susceptibility pattern enterobacter susceptibility pattern was representative of a high resistance to amikacin ( 100% ) and ampicillin ( 92.8 ) respectively in 4 and 13 cases , and high sensitivity to ceftazidime ( 100% ) and norfloxacin ( 100% ) respectively in 5 and 6 cases ; nonetheless , its most susceptibility toward commonly used neonatal empirical therapy antibiotics was in face of ceftizoxime ( 71.4% ) . notably less frequent isolated uropathogens of this study , klebsiella and s. epidermidis , were found to be completely ( 100% ) resistant to ampicillin . distribution of study enterobacter antibiotic susceptibility pattern distribution of study s. epidermdiis antibiotic susceptibility pattern distribution of study klebsiella antibiotic susceptibility pattern current study showed e. coli to be the most prevalent ( 64.4% ) organism causinguti in neonates admitted to neonatal division of bahrami children s hospital over an eight year . besides gram negative bacteria comprised the leading cause ( 95.8% ) of uti in the study population . this finding is in agreement with most of past studies ( e.g. , 1 , 21 , 28 - 30 ) over the subject . it is already known that uti incidence in boys exceeds that of girls in first few months of life ; however , this sex - related dominancy would change toward girls by end of first year and thereafter ( 2 , 6 ) . based on epidemiological data on neonatal uti , this boys - to - girls incidence ratio estimated as 2 to 1 ( 6 ) . current study 69.9% frequency of uti among boys comes to a boys - to - girls ratio of 2.32 , which is compatible with foreseeable higher incidence of in boys . on an overview of previous national studies , e. coli has been reported as the leading uropathogen in neonates with uti with various relative frequencies : 80.5% in qazvin ( 14 ) , 76.5% in tehran ( 20 ) , 76.3% in tehran ( 33 ) , 50% in kashan ( 34 ) , and 50% in uromia ( 35 ) . uunlike previous national based studies , proteus ( 14 ) , pseudomonas ( 14 , 35 ) , serratia ( 36 ) , s. aureus ( 20 ) were not retrieved in this study . in compare to other age groups , continuous assessment of uropathogen epidemiology in neonatal period is of profound significance , as uti is hard to diagnose at this age group and could potentially lead to systemic infection leaving potential life threatening and long term sequel . moreover this would enable clinicians to choose an empirical therapy regimen that is most clinically and epidemiologically ideal till further microbiologic results are available . furthermore it is an accepted practice that neonatal divisions take into account the prevailing antibiotic sensitivities of local bacterial isolates before opting an empirical regimen ( 32 ) . having said all these , to our knowledge , this is an area which has least been underattention of neonatal divisions across the country . most of previous studies on neonatal uti were focused on the relationship of neonatal uti and some non - specific signs and symptoms such as neonatal jaundice . recent observed resistance patterns towards antibiotics commonly being used in empirical therapy of neonates with uti and or sepsis , namely ampicillin along with aminoglycoside or a third - generation cephalosporin ( 1317 ) , have raised concern over the efficacy of current treatment protocols and even the need for changes in present regimens ( 912 ) . resistant toward ampicillin has been reported by many studies ( 1317 ) . however , there are few studies assessing susceptibility pattern of neonatal uti organisms toward a wide spectrum of antibiotics ( 8 , 31 , 32 ) . on the view point of susceptibility to neonatal empirical therapy antibiotics at this study , e. coli was resistant to ampicillin in 93.6% , ceftizoxime in 78.9% gentamycin in 59.5% and amikacin in 56.7% cases . less frequently isolated organisms also showed different but consistent susceptibility patterns : enterobacter resistance was : 92.9% to ampicillin , 69.2 - 100% to aminoglycosides ( gentamycin and amikacin ) , and 0 - 28.6% to third - generation cephalosporins ; klebsiella and s. epidermidis were found to be sensitive to gentamycin 50% and 100% respectively . comparing our results with few past national - based studies is representative of some shift in sensitivity patterns of uropathogens isolated from neonates with uti . movahedian et al . ( 34 ) reported e. coli resistant ratio to be 78.9% , 64.7% , 5.25% , and 5.25% toward cephalexin , ampicillin , amikacin , and gentamycin respectively in 2007 . our study found a dramatic almost ten - fold resistance to amikacin and gentamycin and 144% increase -in resistance rate to ampicillin ; however , the resistance to cephalexin has a little decreased . it is not clear whether this changein susceptibility pattern of e. coli toward ampicillin , amikacin , and gentamycin is secondary to overusage of these antibiotics , as one of accepted empirical regimen , or it is due to an original different in susceptibility patterns in kashan and tehran cities , where the studies accomplished . on the same comparative view to movahedian et al study , klebsiella susceptibility toward ampicillin , cephalexin , and gentamicin has not changed but klebsiella exhibits an almost ten - fold resistance rate to amikacin . the strengths of our study include : i ) the large sample size relative to low prevalence of urinary tract infection in term neonates , 0.1% to 1% ( 23 ) . it needs to be mentioned that in bahrami children hospital , pre - term neonates with diagnosis of sepsis most often would be admitted to nicu than neonatal division where our study accomplished . ii ) the long period of the study which enabled researcher to recruit larger number of cases . iii ) the accountability of the urine samples included in the study ( suprapubic aspirate or catheterization ) . all included cases were diagnosed upon a suprapubic aspirate or catheterizations which are the most accepted means ( 6 , 37 ) of collecting urine sample in acute settings in need of instituting an empirical antibiotic . besides , all cases with incomplete double - notes on the method of urine collection ( physician note and nursing staff note ) were excluded from the study population to : i ) adhere to study inclusion criteria , ii ) decreasing chance of the recruiting contaminated samples in the study i ) inevitably miss some cases due to their incomplete records , ii ) report the susceptibility results on the basis of number of individual antibiotic discs applied over total cases , iii ) not having the chance of assessing isolated uropathogens toward a wide spectrum of antibiotics . nonetheless , ampicillin was the sole antibiotic which had been applied to almost all studied cases . the strengths of our study include : i ) the large sample size relative to low prevalence of urinary tract infection in term neonates , 0.1% to 1% ( 23 ) . it needs to be mentioned that in bahrami children hospital , pre - term neonates with diagnosis of sepsis most often would be admitted to nicu than neonatal division where our study accomplished . ii ) the long period of the study which enabled researcher to recruit larger number of cases . iii ) the accountability of the urine samples included in the study ( suprapubic aspirate or catheterization ) . all included cases were diagnosed upon a suprapubic aspirate or catheterizations which are the most accepted means ( 6 , 37 ) of collecting urine sample in acute settings in need of instituting an empirical antibiotic . besides , all cases with incomplete double - notes on the method of urine collection ( physician note and nursing staff note ) were excluded from the study population to : i ) adhere to study inclusion criteria , ii ) decreasing chance of the recruiting contaminated samples in the study . limitations of our study include its retrospective nature which made us to : i ) inevitably miss some cases due to their incomplete records , ii ) report the susceptibility results on the basis of number of individual antibiotic discs applied over total cases , iii ) not having the chance of assessing isolated uropathogens toward a wide spectrum of antibiotics . nonetheless , ampicillin was the sole antibiotic which had been applied to almost all studied cases . according to previous studies ( 31 , 32 ) , the guiding key to choose the empirical antibiotic with the most efficacy , and the least side effect is understanding of causative organisms susceptibility pattern . otherwise , it is less likely to manage as efficacious as possible . the susceptibility patterns not only are different over various centres but also vary over the time . the most prevalent organisms implicated in this study were significantly resistant to ampicillin - a frequent combining to empirical antibiotic regimen . having reviewed neonatal uropathogens over the past 8 years , authors believes a third generation cephalosporins plus an aminoglycoside would be a better choice to ampicillin plus an aminoglycoside as the first line of empiric therapy of neonatal uti . lack of extensive and multi - central studies over neonatal uti in iran warrants prospective future studies on this subject , and authors believes the results of this study could stress the necessity to conduct such projects and motivate further researcher on assessing the subject . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

abstractbackgroundsusceptibility pattern of organisms causing urinary tract infection ( uti ) in neonate would potentially improve the clinical management by enabling clinicians to choose most reasonable first line empirical antibiotics . this study aimed to this end by studying isolated organisms from neonates with uti in an inpatient setting.methodscurrent retrospective study has recruited all cases of neonatal uti diagnosed through a suprapubic / catheterized sample , admitted to neonatal division of bahrami children s hospital , tehran , iran , from june 2004 to june 2012.resultsescherichia coli was the dominant ( 64.4% ) bacteria among a total of 73 cases ( 69.9% boys and 30.1% girls ; aged 14.14 7.68 days ; birth weight of 3055.85 623.00 g ) and enterobacter ( 19.2% ) , klebsiella ( 12.3% ) , and staphylococcus epidermdisis ( 4.1% ) were less frequent isolated bacteria . e. coli was mostly resistant to ampicillin ( 93.6% ) , cefixime ( 85.7% ) and cephalexin ( 77.3% ) , and sensitive to cefotaxime ( 63.6% ) . enterobacter found to be most resistant to amikacin ( 100% ) , ampicillin ( 92.85% ) , and most sensitive to ceftizoxime ( 71.4%).conclusiona high ratio ( > 92.85% ) of resistance toward ampicillin was observed among common neonatal uti bacterial agents . having this finding along with previous reports of emerging resistance of neonatal uropathogensto ampicillin could be a notion that a combination of a third generation cephalosporin and an aminoglycoside would be a more reasonable first choice than ampicillin plus an aminoglycoside .

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congenital adrenal hyperplasia ( cah ) describes a group of inherited autosomal recessive disorders that cause a deficiency in an adrenal enzyme resulting in altered cortisol and aldosterone secretion . the most frequent cah variant , accounting for 95% of all affected patients , is 21-hydroxylase deficiency ( 21-ohd ) and is caused by inactivating mutations in the 21-hydroxylase gene ( cyp21a2 ) . the deficiency of 21-hydroxylase causes a decrease in negative feedback to the hypothalamic - pituitary - adrenal ( hpa ) axis , and subsequent hyperplasia of the adrenal gland . cortisol and aldosterone precursors are subsequently diverted to androgens , causing increased virilization and a chronic hyperandrogenic state . there are different clinical forms of cah associated with 21-ohd : classical cah , the most severe form comprises both salt - wasting ( sw ) and simple virilizing ( sv ) forms , and the non - classical ( nc ) form which may be asymptomatic or associated with symptoms of postnatal or even adult onset hyperandrogenism . several studies have suggested high concordance rates between genotype and phenotype in patients with the classic form of cah . treatment of cah consists of replacement of glucocorticoids and , when necessary , mineralocorticoids to prevent adrenal crises and to suppress the abnormal secretion of androgens and steroid precursors from the adrenal cortex . conventional glucocorticoid replacement can not , however , mimic the natural circadian rhythms of cortisol release . even in adequately treated patients , there are reports of clinical evidence of both over - suppression with glucocorticoid including iatrogenic cushing 's syndrome , obesity , and decreasing growth velocity and under - suppression including virilization , menstrual disorders and infertility , growth suppression , and development of adrenal tumors . cah constitutes an interesting natural model for studying interactions between hormones and the brain . until now , only a few studies described brain magnetic resonance imaging ( mri ) abnormalities in cah patients . hormonal imbalances during early development and exposure to excess exogenous glucocorticoids are the most probable explanations for these mri findings.[711 ] in the present study , we investigated the frequency of white matter changes and temporal lobe structures dysgenesis in patients with cah due to 21-ohd . we studied 26 patients with cah due to 21-ohd ( 11 males and 15 females , mean age sd = 27.4 8.2 years , range = 16.5 - 48 years ) , who were followed at the department of endocrinology in mahdia and sfax , in tunisia . this sample includes all patients with cah , aged more than 16 years and regularly followed at these two clinics since 1982 . the diagnosis of cah was based on clinical and biochemical criteria ( i.e. , elevated levels of 17-hydroxyprogesterone ( 17-ohp ) and androstenedione , acth stimulation test ) . ten patients ( 6 m , 4 f ) had the sw form of cah . they had been diagnosed in their first year of life and had been treated , from the time of diagnosis , with glucocorticoids and mineralocorticoids . eight patients ( 5 m , 3 f , age at diagnosis : between birth and 6 years ) were diagnosed as classical sv patients . in the other eight patients ( women only in this group ) , the nc form was diagnosed between 15 and 44 years of age . twenty - one patients were started on a regimen of hydrocortisone ( hc ) , given 2 or 3 times daily , whereas the remaining five patients ( three with the nc form and two with the classic sv form ) were treated with dexamethasone ( once daily ) . salt wasters were treated with 27.9 9.6 mg / m per day of hc the first 2 years of life , and the doses were decreased to 17.6 6.6 mg / m per day during childhood . daily doses of hc in patients with classical and nc forms were respectively , 17.3 4.6 mg / m and 16.04 3.4 mg / m during adulthood . for dexamethasone , clinical and brain magnetic resonance imaging findings in 26 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency the adequacy of therapy was monitored periodically on the basis of clinical and laboratory data , in accordance with current guidelines . patients were classified as under adequate hormonal control if 50% or more of the total serum androgen levels were within normal limits for age or if 50% or more of the baseline serum 17-ohp concentrations were 2.0 - 10 ng / ml ( 6 - 30 the mean duration of the follow - up period was 18.5 9.3 ( range 3 - 41.5 ) years . seven patients ( five with the sw form and two with the sv form ) had experienced a sw adrenal crisis in the neonatal period . one female patient ( patient she was started on l - thyroxine 10 g / kg per day at the age of 6 months , but she was irregular in follow - up and was not compliant with treatment . 4 ) with the sw form developed hypertension at 8 years of age and responded well to nifedipine . detailed investigations ( renal doppler ultrasound , adrenal ct scan , urinary metanephrine , 11-deoxycortisol , and aldosterone concentrations ) failed to detect any cause for secondary hypertension , and a diagnosis of essential hypertension was made . all our patients or their parents gave their informed consent to participate in the study and underwent neurological examination and brain mri . the imaging protocol included axial and sagittal spin - echo ( se ) t1-weighted [ repetition time ( tr ) 550 ms , echo time ( te ) 15 ms , flip angle 90 ] , axial and coronal enhanced t1-weighted ( tr 550 ms , te 15 ms , flip angle 90 , axial fast se ( fse ) t2-weighted ( tr 5000 ms , te 94 ms , flip angle 90 ) , axial proton - density ( pd ) t2-weighted ( tr 2500 ms , te 18 ms , flip angle 90 ) , and coronal fluid - attenuated inversion recovery [ flair ; tr 7500 ms , te 94 ms , inversion time ( ti ) 2200 , flip angle 90 ] sections . white matter changes were defined as areas of increased signal intensity on intermediate and t2-weighted sequences . thin coronal mri slices , perpendicular to the axis of the hippocampus were used for determining hippocampal dysplasia ( hippocampus with distorted anatomy , small volume , disturbed internal architecture , and increased signal on t2 and flair images ) . amygdala was regarded as dysplastic if it was smaller than the contralateral side , if increased signal on t2 and flair sequences was observed , or both . further investigations , including viral serologies , amino acid profile , organic acid profile , lactate , ceruloplasmin , lumbar puncture and arylsulfatase a activity , were performed in a16.5-year - old patient ( patient no . 4 ) with the classical sw form of cah and who presented with hypertension , obesity , mental decline , tremor , cerebellar syndrome , white matter hyperintensities , and agenesis of the corpus callosum . we studied 26 patients with cah due to 21-ohd ( 11 males and 15 females , mean age sd = 27.4 8.2 years , range = 16.5 - 48 years ) , who were followed at the department of endocrinology in mahdia and sfax , in tunisia . this sample includes all patients with cah , aged more than 16 years and regularly followed at these two clinics since 1982 . the diagnosis of cah was based on clinical and biochemical criteria ( i.e. , elevated levels of 17-hydroxyprogesterone ( 17-ohp ) and androstenedione , acth stimulation test ) . ten patients ( 6 m , 4 f ) had the sw form of cah . they had been diagnosed in their first year of life and had been treated , from the time of diagnosis , with glucocorticoids and mineralocorticoids . eight patients ( 5 m , 3 f , age at diagnosis : between birth and 6 years ) were diagnosed as classical sv patients . in the other eight patients ( women only in this group ) , the nc form was diagnosed between 15 and 44 years of age . twenty - one patients were started on a regimen of hydrocortisone ( hc ) , given 2 or 3 times daily , whereas the remaining five patients ( three with the nc form and two with the classic sv form ) were treated with dexamethasone ( once daily ) . salt wasters were treated with 27.9 9.6 mg / m per day of hc the first 2 years of life , and the doses were decreased to 17.6 6.6 mg / m per day during childhood . daily doses of hc in patients with classical and nc forms were respectively , 17.3 4.6 mg / m and 16.04 3.4 mg / m during adulthood . for dexamethasone , clinical and brain magnetic resonance imaging findings in 26 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency the adequacy of therapy was monitored periodically on the basis of clinical and laboratory data , in accordance with current guidelines . patients were classified as under adequate hormonal control if 50% or more of the total serum androgen levels were within normal limits for age or if 50% or more of the baseline serum 17-ohp concentrations were 2.0 - 10 ng / ml ( 6 - 30 the mean duration of the follow - up period was 18.5 9.3 ( range 3 - 41.5 ) years . seven patients ( five with the sw form and two with the sv form ) had experienced a sw adrenal crisis in the neonatal period . one female patient ( patient she was started on l - thyroxine 10 g / kg per day at the age of 6 months , but she was irregular in follow - up and was not compliant with treatment . 4 ) with the sw form developed hypertension at 8 years of age and responded well to nifedipine . detailed investigations ( renal doppler ultrasound , adrenal ct scan , urinary metanephrine , 11-deoxycortisol , and aldosterone concentrations ) failed to detect any cause for secondary hypertension , and a diagnosis of essential hypertension was made . all our patients or their parents gave their informed consent to participate in the study and underwent neurological examination and brain mri . all patients underwent neurological and mri examinations . the mri examination was performed with a 1 t. siemens magnetom impact system . the imaging protocol included axial and sagittal spin - echo ( se ) t1-weighted [ repetition time ( tr ) 550 ms , echo time ( te ) 15 ms , flip angle 90 ] , axial and coronal enhanced t1-weighted ( tr 550 ms , te 15 ms , flip angle 90 , axial fast se ( fse ) t2-weighted ( tr 5000 ms , te 94 ms , flip angle 90 ) , axial proton - density ( pd ) t2-weighted ( tr 2500 ms , te 18 ms , flip angle 90 ) , and coronal fluid - attenuated inversion recovery [ flair ; tr 7500 ms , te 94 ms , inversion time ( ti ) 2200 , flip angle 90 ] sections . white matter changes were defined as areas of increased signal intensity on intermediate and t2-weighted sequences . thin coronal mri slices , perpendicular to the axis of the hippocampus were used for determining hippocampal dysplasia ( hippocampus with distorted anatomy , small volume , disturbed internal architecture , and increased signal on t2 and flair images ) . amygdala was regarded as dysplastic if it was smaller than the contralateral side , if increased signal on t2 and flair sequences was observed , or both . further investigations , including viral serologies , amino acid profile , organic acid profile , lactate , ceruloplasmin , lumbar puncture and arylsulfatase a activity , were performed in a16.5-year - old patient ( patient no . 4 ) with the classical sw form of cah and who presented with hypertension , obesity , mental decline , tremor , cerebellar syndrome , white matter hyperintensities , and agenesis of the corpus callosum . all these investigations revealed negative or normal results . on neurological examination , mental retardation was evident in three patients ( patient nos . 4 , 6 and 23 ) . on intellectual quotient test , it was moderate in patient nos . 4 and 6 ( both had developed salt losing crisis during the neonatal period ) and profound in patient no . 23 other neurological signs included postural and action tremor in two patients ( patient nos . 4 and 23 ) , 1 ) , and static and kinetic cerebellar syndrome in one patient ( patient no . 4 ) . at the time of inclusion in the study , mean morning baseline 17-ohp in serum for the whole cohort was 22.51 ng / ml ( range , 1.1 - 77 ) ; mean morning androstenedione levels were 5.25 ng / ml ( range , 1.6 - 24 ) and mean morning plasma acth levels were 118.31 pg / ml ( range , 6.5 - 537 ) . during the treatment period , poor hormonal control , with elevation of 50% or more of 4-androstenedione and 17-ohp serum measurements , was present in 16 patients ( 61.5% ) . these were chronically non - compliant patients , mainly because of ignorance and lack of understanding . the remaining patient ( patient no . eleven patients ( five males and six females , 42.3% ) showed mri abnormalities : one patient was over - treated ( patient no . 8) , two patients had good hormonal control ( patient nos . 13 and 15 ) and eight patients were poorly controlled due to non - compliance with their medication . eight patients ( seven with the classic form and one with the nc form ) evidenced white matter hyperintensities on t2-weighted and flair images . these hyperintensities were located in periventricular regions in seven patients [ figure 1a ] and in cerebellar white matter in the remaining patient [ patient no . 11 ) with the classical sv form showed moderate right temporal lobe atrophy [ figure 3 ] . indeed 21 , hippocampus was globular in shape and its internal structures were not identified [ figure 5 ] . other mri findings included partially empty sella in one patient ( patient no . 25 ) and cortico - subcortical atrophy associated with complete agenesis of the corpus callosum in another patient [ patient no . a ) t2-weighted axial mri sequence showing bilateral periventricular white matter hyperintensities and cortico - subcortical atrophy in a 16.5-year - old patient affected by swcah ( patient no . 4 ) t1-weighted sagittal mri sequence showing complete agenesis of the corpus callosum in a 16.50 year old patient affected by swcah ( patient no . 4 ) axial flair and t2 mri showing bilateral cerebellar white matter hyperintensities in a 21-year - old man affected by sw cah ( patient no . 1 ) axial t1-weighted image showing moderate atrophy in the right anterior temporal lobe in a 47.5-year - old man affected by simple virilizingcah ( patient no . 11 ) t2-weighted mri , coronal section , showing right hippocampal atrophy in a 36-year - old woman affected by non - classic cah ( patient no . 23 ) coronal t2-weighted mri in a 30.5-year - old woman affected by non - classic cah . the right hippocampus is moderately round - shaped and its internal structures are not identified ( patient no . on neurological examination , mental retardation was evident in three patients ( patient nos . 4 , 6 and 23 ) . on intellectual quotient test , it was moderate in patient nos . 4 and 6 ( both had developed salt losing crisis during the neonatal period ) and profound in patient no . 23 other neurological signs included postural and action tremor in two patients ( patient nos . 4 and 23 ) , 1 ) , and static and kinetic cerebellar syndrome in one patient ( patient no . at the time of inclusion in the study , mean morning baseline 17-ohp in serum for the whole cohort was 22.51 ng / ml ( range , 1.1 - 77 ) ; mean morning androstenedione levels were 5.25 ng / ml ( range , 1.6 - 24 ) and mean morning plasma acth levels were 118.31 pg / ml ( range , 6.5 - 537 ) . during the treatment period , nine patients ( 34.6% ) showed well - controlled cah . poor hormonal control , with elevation of 50% or more of 4-androstenedione and 17-ohp serum measurements , was present in 16 patients ( 61.5% ) . these were chronically non - compliant patients , mainly because of ignorance and lack of understanding . the remaining patient ( patient no . eleven patients ( five males and six females , 42.3% ) showed mri abnormalities : one patient was over - treated ( patient no . . 13 and 15 ) and eight patients were poorly controlled due to non - compliance with their medication . eight patients ( seven with the classic form and one with the nc form ) evidenced white matter hyperintensities on t2-weighted and flair images . these hyperintensities were located in periventricular regions in seven patients [ figure 1a ] and in cerebellar white matter in the remaining patient [ patient no . 11 ) with the classical sv form showed moderate right temporal lobe atrophy [ figure 3 ] . indeed 21 , hippocampus was globular in shape and its internal structures were not identified [ figure 5 ] . 25 ) and cortico - subcortical atrophy associated with complete agenesis of the corpus callosum in another patient [ patient no . a ) t2-weighted axial mri sequence showing bilateral periventricular white matter hyperintensities and cortico - subcortical atrophy in a 16.5-year - old patient affected by swcah ( patient no . 4 ) t1-weighted sagittal mri sequence showing complete agenesis of the corpus callosum in a 16.50 year old patient affected by swcah ( patient no . 4 ) axial flair and t2 mri showing bilateral cerebellar white matter hyperintensities in a 21-year - old man affected by sw cah ( patient no . 1 ) axial t1-weighted image showing moderate atrophy in the right anterior temporal lobe in a 47.5-year - old man affected by simple virilizingcah ( patient no . 11 ) t2-weighted mri , coronal section , showing right hippocampal atrophy in a 36-year - old woman affected by non - classic cah ( patient no . 23 ) coronal t2-weighted mri in a 30.5-year - old woman affected by non - classic cah . the right hippocampus is moderately round - shaped and its internal structures are not identified ( patient no . our study provides evidence of an increased frequency of white matter abnormalities and temporal lobe structures dysgenesis in cah patients . in the past , only a few studies described brain mri abnormalities in cah.[711 ] table 2 summarizes the major results . overall , these studies documented abnormalities affecting white matter signal , temporal lobe and amygdala structure and function . major previous reports of brain magnetic resonance imaging abnormalities related to congenital adrenal hyperplasia white matter hyperintensities , or leukoencephalopathy , related to cah are an interesting finding in this population . in a previous italian study , disease phenotype does not seem to affect the frequency of such lesions , which may even be present in heterozygous carriers of cyp21 mutations . brain white matter impairment in cah involve typically periventricular regions or , less frequently , cerebellar area as seen in one of our patients . these signal changes are non - specific and without side prevalence,[79 ] but they differ from those due to ischemic causes , as the latter involve namely subcortical and deep structures . white matter hyperintensities in cah are most often subclinical or , less frequently , associated with unremarkable neurological signs . in the present study , the data in the literature concerning the clinical significance of white matter lesions are controversial . it has been suggested that a threshold area of white matter lesions must be present before cognitive deficits are installed . the mechanisms that could explain mri lesions in cah remain poorly understood . hormonal imbalance related to a deficiency of cortisol and aldosterone and an overproduction of 17-oh - progesterone and androgen would cause a destabilization of the myelin molecule leading to its degeneration . in addition , exogenous glucocorticoids may alter the process of neuronal maturation and myelination by inhibiting the differentiation of oligodendrocyte precursors . thus both , overdose and discontinuous therapy have been suggested as factors favoring the emergence of brain mri abnormalities . in our patients , chronic hyperandrogenism , a result of non - compliance with treatment , may be the main mechanism involved in the genesis of brain mri abnormalities . aldosterone is provided with a regulating effect on the arterial structure by stimulating fibroblast proliferation and collagen synthesis via direct activation of local receptors . in this regard , aldosterone deficiency could induce changes of cerebral arterial structure , and , as consequence , a subclinical cerebral ischemia . this ischemia could also be indirectly promoted by glucocorticoids that alter glucose and lipid metabolism and reduce platelet aggregability . in our study , similarly , nass , et al . reported some cah patients with white matter lesions , with localizations that were typical of demyelinating diseases ( located in the corpus callosum and cerebellum ) . have described a patient whose condition was diagnosed at birth as the sw subtype of cah in whom relapsing - remitting multiple sclerosis developed . the fact that a susceptibility locus for multiple sclerosis is in the hla region that comprises the cyp21 genes suggests a possible genetic link between multiple sclerosis and cah . the organizational effects refer to permanent changes in brain structure , organization or function in utero or during critical periods of development . the activational effects tend to be transient and refer to the acute effects of circulating steroids during adulthood . two structures are known to have many androgen and glucocorticoid receptors and are particularly susceptible to the effects of excess glucocorticoids : the hippocampus , a central neural substrate of declarative memory , and the amygdala , a key to emotional coding . the hippocampus also plays an important role in the fine - tuning of the hpa axis by participating in its glucocorticoid negative feedback regulation . alterations in amygdala function have been implicated in the pathophysiological characteristics of anxiety and depressive disorders in both adults and children . the effect of hypercortisolism on the amygdala has not been systematically studied ; however , patients with classic cah , who have prenatal glucocorticoid deficiency with possible postnatal iatrogenic glucocorticoid excess , have smaller amygdala volume than healthy age - and sex - matched controls . cah female patients have also enhanced response to negative facial emotions ( anger and fear ) compared with neutral expressions . animal studies and human studies of adult patients with chronic hypercortisolemia have shown that prolonged exposure to glucocorticoid excess could result in extensive hippocampus damage and permanent memory impairment . the mechanisms leading to brain atrophy include impaired neurogenesis , reduction in neurotrophic factors and reduction in cerebral glucose metabolism . interestingly , it has been suggested that at least some of the deleterious effects of prolonged hypercortisolemia on cognitive functioning and hippocampal volume are reversible . our present study showed temporal lobe atrophy in one patient and hippocampal dysgenesis in two patients . this study is the first one demonstrating hippocampal abnormalities on mr imaging in patients with cah , and it showed , also for the first time , brain mri abnormalities in patients with the nc form of cah . studies of cognitive function in cah patients have reported discrepant findings . it was suggested that patients with the most severe form of cah and those who have experienced sw adrenal crises with abnormal electrolytes as neonates are at risk for cognitive impairement . in the present study , the remaining one had the nc form , but also had an uncontrolled congenital hypothyroidism which may also contribute to his altered mental status . in conclusion , brain mri abnormalities are an interesting finding in cah patients and could affect white matter signal and temporal lobe structures . these abnormalities could be the consequence of hormonal imbalance during brain development and corticosteroid treatment . more extensive studies are required to define better the relationships between brain involvement and different cah phenotypes and treatment regimens .

background : congenital adrenal hyperplasia ( cah ) is an inherited recessive disorder of adrenal steroidogenesis . the enzymes most commonly affected are 21-hydroxylase . past reports suggested brain magnetic resonance imaging ( mri ) abnormalities in cah patients , affecting white matter signal , temporal lobe and amygdala structure and function.aims:in the present study , we aimed to investigate the frequency of white matter changes and temporal lobes structures dysgenesis in a population of patients having cah due to 21-hydroxylase deficiency.materials and methods : neurological examination and brain mri were performed in 26 patients.results:neurological examination revealed mental retardation in three patients , tremor in two patients , tendon reflexes asymmetry in one patient , and cerebellar syndrome in one patient . eleven patients ( 42.3% ) showed mri abnormalities : eight of them had white matter hyperintensities , one patient had moderate atrophy in the right temporal , and hippocampal dysgenesis was found in the remaining two patients.conclusions:brain mri abnormalities in cah patients include white matter hyperintensities and temporal lobe structures dysgenesis . the mechanisms involved seem related to hormonal imbalances during brain development and exposure to excess exogenous glucocorticoids . clinical implications of such lesions remain unclear . more extensive studies are required to define better the relationships between brain involvement and different cah phenotypes and treatment regimens .

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delivery of the human genome draft sequence by publicly funded and corporate projects promises to precipitate significant biomedical advances this century . to rise to this challenge , biologists must become adept at navigating the vast expanses of genomic dna data that may seem , at first glance , to be devoid of features . yet lying beneath this facade of uniformity are rich veins of knowledge awaiting exploitation . surveying and signposting this apparently bland genomic landscape should guide investigators towards experiments that address specific hypotheses about gene function . ? different communities of biologists speak in dialects that are not always mutually comprehensible , particularly with respect to the umbrella term ' function ' . where one investigator might be interested in designing active - site inhibitors using high - resolution protein structural data , another might require information on gene pathways , and another might be focused on relating genotype to phenotype . if the genome is to offer up its secrets to all scientific communities , its surveyors need to adopt universal vocabularies . broadly speaking , there are two common ways of annotating ( assigning names and functions to ) genes . websites such as locuslink , genecards , eugenes and ensembl ( see table 1 ) integrate information from diverse sources relevant to individual human genes . thus one may browse sequence information , for example concerning single - nucleotide polymorphisms ( snps ) , alongside both descriptions of molecular and cellular function and information relevant to human disease . such annotation is essential , yet it is currently restricted to the minority of human genes - those that have been characterized experimentally . a key to the databases mentioned in this article the second type of annotation relies not on empirical observations but rather on predicted evolutionary relationships . all genes that are thought to have arisen from a common ancestor are defined as homologs : where additional copies have arisen by gene duplication within a single genome they are defined as paralogs , whereas corresponding genes in different species are orthologs . sometimes homologous gene products have strong sequence similarities , such that an inference of homology is straightforward ; one such example is the drosophila melanogaster gene branchless , which encodes a homolog of human fibroblast growth factor ( fgf ) . on other occasions , protein homologs have subtle or indiscernible sequence similarities that try the patience and expertise of genome wayfarers . for example , human fgf and interleukin-1 have highly similar tertiary structures despite insignificantly similar sequences but , as a result of their similar growth - factor - type functions , they are in fact very likely to be homologs . the importance of annotating the genomic landscape on the basis of homology is that protein homologs invariably have similar tertiary structures and frequently also have similar functions . the surveying and way - marking of each new gene , therefore , need n't always be an arduous process of discovering structure and function from scratch , since clues can be inferred from what has already been experimentally gleaned from its homologs . by applying the concept of homology , the problem of broadly predicting the functions of all genes is brought within the realms of possibility . there is a pitfall to be avoided when annotating genes by homology : homology is defined on the basis of evolution , rather than function . on one hand , homologs may be related only by evolution and not by similarities in molecular mechanism ; relatives of enzymes that now lack catalytic sites are just such examples . on the other hand , examples abound of divergent homologs , or even non - homologs , whose functions overlap . consequently , homology assignment indicates only an approximate direction for future empirical determination of function , perhaps analogous to laying down a compass bearing rather than an exact map reference . the first completely sequenced genomes , such as that of haemophilus influenzae , were annotated following searches of all available sequence databases with each predicted gene product . this approach assumes that matters are straightforward : although homologous genes may not always be similar in function , genes are either homologous or they are not . a major problem in gene annotation arises , however , when one encounters sequence - similar , homologous portions of genes embedded in otherwise sequence - dissimilar , non - homologous contexts . the presence of these domains demonstrates that evolution has constantly fused and divided genes , using a repertoire of pre - existing components ( figure 1 ) . domain architectures of the human signalling proteinsvav , grb2 , nck1 and crk , as predicted using smart . although these proteins share two common domains ( namely src - homology 2 and 3 , or sh2 and sh3 , domains ) they are not all homologous to one another . the complex architectures of these proteinsimply that the co - occurrence of these two domain types has arisen on more than one occasion , and thus that the domain combinations seen in the four proteins are not likely to have arisen from a common ancestor with whom they share a common architecture . domains are homologous portions of sequences that are encoded in different gene contexts and have survived the evolutionary tests of time without fragmentation . in three dimensions , domains are observed to be compact units of structure , often with a hydrophobic interior and a hydrophilic exterior , and they are not divisible into smaller units . consequently , domains represent the finite vocabulary of protein evolution : if domains are words , then multidomain proteins are complete sentences . just as there is a dictionary or lexicon for every language , there is one - or in this case several - for the vocabulary of domains . pfam is the widest - ranging lexicon of domain families , predicting at least one domain for more than two - thirds of all entries in the swiss - prot protein database . smart is a more concise collection , focusing on those domains that are widespread and difficult to detect . prosite also has a dictionary of domain profiles , as does celera ( called panther ) and the institute for genomic research , tigr . each of these resources detects domains using numerical representations of multiple sequence alignments , either hidden markov models ( hmms ) or generalized profiles ( gps ) . although the constructions of hmms and gps are very different , formally they are equivalent . homology assignments are guided by comparisons of hmms or gps with protein sequence databases , and by implementation of an upper threshold value for e , the number of unrelated sequences expected purely by chance that are aligned with a particular score , or higher , in the search . this procedure has been shown on many occasions to identify subtle , yet informative , sequence similarities among distant homologs . taken together , pfam , smart and the other domain resources carry considerable redundancy , although each has its own merits . an additional resource , interpro , has been derived in part to avoid the onerous task of querying each of these domain lexicons separately for each protein sequence of interest . interpro ( release 3.0 ) combines the domain and motif sets of prosite , pfam , prodom , prints and smart in a hierarchical manner and is thereby able to provide annotation for 74% of all swiss - prot ( protein database ) and trembl ( translated dna sequence database ) entries . pfam , smart and interpro were recently chosen by the public and private consortia to annotate their human genome draft sequences . to be more precise , annotations were applied to the proteome , the current predicted set of all expressed proteins encoded by the draft . the proportion of the proteome that could be annotated , even minimally , using these resources is low , at approximately 40 - 60% . the resources were used to annotate the proteome according to lists of component domains rather than protein or gene names . thus , protein sequences were identified not , for example , as the product of the lbc oncogene , but rather as containing rhogef ( dbl - homology ) and pleckstrin homology ( ph ) domains ( figure 2 ) . this is protein annotation viewed through the evolutionary lens , rather than faithful extracts from the current body of experimental knowledge . by implication , proteome annotation by domain content implies that a gene product 's function is a synthesis of the generalized functions of its component parts ( see figure 2 ) . although such descriptions of molecular function are approximate and pale in comparison to accounts of experimentally derived characteristics , they provide the best predictions that can be mustered for the uncharacterized majority of human genes . domain architectures of the lbc oncogene product , and the experimentally uncharacterized protein kiaa1415 , as predicted using smart . the predicted domains in kiaa1415 suggest that it facilitates guanine nucleotide exchange on rho - type small gtpases ( it has rho guanine - nucleotide exchange factor , or rhogef , and pleckstrin homology or ph domains ) , it is localized to the plasma membrane ( ph domain ) and it may interact with the carboxyl termini of transmembrane receptors or channels ( it has pdz domains ) . the generalized functions of dep domains are not currently known . it should be noted that prediction of this protein 's domain architecture does not allow prediction of either its cellular function or the phenotypic effect of its deficiency . it is important to emphasize that annotation of individual proteins or complete proteomes using domains is achieved automatically rather than by manual curation . this is relevant , because a newly sequenced genome 's proteome is generally in a high state of flux , with additions and deletions resulting from sequence updates , enhanced understanding of gene structure and identification of previously overlooked genes . the animal genome sequencing projects already underway will proceed in the same manner as the publicly funded human project , through numerous draft stages and on towards completion . providing up - to - date , and necessarily automatic one might suggest that gene annotation by use of domains is a relatively short - term measure that will be made redundant by results from high - throughput studies of non - vertebrate model organisms . it can be argued that detailed predictions of the functions of most human genes can be inferred from studies of their orthologs , as these are the most likely members of a family to have similarities in molecular and cellular roles . in contrast to previous expectations , the human genome draft publications found that the great majority of human genes have no orthologs in each of three important model organisms whose genome sequences are known , namely drosophila ( fruitfly ) , caenorhabditis elegans ( nematode worm ) and saccharomyces cerevisiae ( baker 's yeast ) . thus , the contribution of domain identification to gene - function prediction will remain until such a time as reliable results from high - throughput studies on a more closely related organism , such as the mouse , are available . deconvolution of human proteins into their constituent domains has also played a major role in understanding the evolution of chordates . three significant differences were detected between the repertoires of human domain families and those of the nematode worm , fruitfly , the mustard cress arabidopsis thaliana and yeast . first , only a small proportion ( 7% ) of human domain families are absent from the other proteomes . second , numerous domain families were greatly expanded in terms of the number of members in humans , whereas others were considerably reduced . this finding demonstrated that domain ' invention ' in the chordate lineage has made only a minor contribution to proteome diversity , whereas expansions and contractions of domain families , and domain additions and deletions , have all added greatly to proteome innovation . such studies demonstrate the power of comparative proteomics in relating gene content to the evolution of eukaryotic organisms . but as with the argument that the complexity of organisms is only loosely coupled to total gene number , the question of whether the number of representatives of a domain family in a proteome is directly related to that family 's contribution to cellular and organismal function remains open . it is hoped that future proteome comparisons will progress beyond the simple enumeration of homologous genes and domains towards an understanding of the biology that underlies the variability of domain family sizes . although it provides information about molecular structure , function and evolution , by and large it is unable to predict functional aspects , such as cellular or organismal role , protein - binding partners or post - translational modifications . fortunately , other views that address these aspects have been incorporated with domain predictions into web - based resources such as locuslink , genecards , eugenes and ensembl . each of these sites represents a confluence of diverse information sources that are mapped to specific regions of genomic sequence . whilst navigating these sites it should be borne in mind that they often fail explicitly to distinguish between annotations that are experimentally derived and those that are predicted by homology - based methods . nevertheless , these sites are a significant boon to biologists since they provide views of genomes from multiple vantage points , from protein tertiary structure through to snps and on to human disease . as viewed now , the human genome appears to be a relatively featureless landscape , punctuated by islands of annotations for well - characterized and biomedically important genes . as biological sciences progress into a more knowledge - rich , as well as data - rich , era , the cartography of this genome will become more complex with numerous different functional characteristics being assigned to a growing fraction of human genes . it will be increasingly important to restrict descriptions of function to a common and broad vocabulary that is compatible with computational approaches . fortunately , a cross - community approach to dealing with this issue is already underway . the gene ontology project ( go ) has created an initial hierarchy of defined terms that encompasses many of the flavours of ' function ' commonly described in biology . go has begun to permeate throughout genomics and it will do so more rapidly as its scope and attention to detail improves . two further domain - centric approaches look set to guide the efficient navigation of genomes : orthology prediction and the partitioning of a domain family into subfamilies with distinct functions . orthology is a valuable concept from which to infer functional information between species , and orthologs from the genomes of 30 bacteria , archaea and the yeast saccharomyces cerevisiae can be predicted directly using the cogs database . pairs of orthologs from animal genomes are also available on the web ( for example from the jackson laboratory ) . no resource is yet available that accurately predict sets of orthologs for several multicellular eukaryotes , however , such as the fruitfly , worm , arabidopsis , mouse and human . a more difficult problem is the partitioning of a homologous domain family into multiple subfamilies representing multiple functions . homologous proteins with divergent sequences frequently have distinct functions that are characterized by contrasting patterns of conserved amino acids . one productive approach to the analysis and prediction of functional subtypes identified key sites in multiple protein sequence alignments that specify the different functional subtypes . this method has performed well in defining functional subtypes with prediction accuracies of up to 96% . a key element in ensuring a central function will be provided by sophisticated web forums that accumulate and automatically present integrated functional data . the best example so far is the distributed annotation system , or das , which seeks to amalgamate annotations donated by experimentalists worldwide . even these schemes , however , will face a major challenge in integrating functional information from the huge datasets that arise , for example , from microarray and proteomics experiments . the human genomic landscape , relatively featureless now , will soon be teeming with evidence , pointers , and clues . ultimately , the success of the genome projects will be measured not in the completion of sequences , but in how access to integrated data opens up new avenues of research and therapy .

the evolutionary history of eukaryotic proteins involves rapid sequence divergence , addition and deletion of domains , and fusion and fission of genes . although the protein repertoires of distantly related species differ greatly , their domain repertoires do not . to account for the great diversity of domain contexts and an unexpected paucity of ortholog conservation , we must categorize the coding regions of completely sequenced genomes into domain families , as well as protein families .

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over the last 25 years , heart rate monitors have been widely used in sports and sports science . since the development of handy heart rate monitors ( hrms ) with the size of a watch and appropriate flexible chest belts with electrodes to transmit signals wirelessly to the watch , measurement of heart rate ( hr ) has become an easily accessible and valuable tool for training and coaching purposes and conducting laboratory or field studies . validity and reliability have been shown to be high for these tools compared to ecg measurements for both hr and heart rate variability ( hrv ) [ 16 ] . owed to its widespread use in physical activity not only for individuals but also for team sports , recent technical development focused on real - time monitoring instead of post - exercise evaluation of recorded data . currently , several different systems are available on the market , of which four were tested in the present study : polar team pro , acentas team monitoring system , suunto pro team pack and activio sport system . all systems allow direct and real - time monitoring of several individuals at the same time . data are transmitted wirelessly to a receiver which is connected with a standard laptop with relevant software . however , a direct comparison of these systems and validity of recorded data are missing in literature . therefore the main purpose of this study was to compare all systems in a field test against each other . operating distance and validity against a commercially available ecg were evaluated as well as the installation and use of software and hardware of the above - mentioned systems . a total of 12 active people ( 11 men and 1 woman ; age : 26.4 3.2 yr ; height : 177.9 8.7 cm ; mass : 78 7 kg ) participated in the study . ten men of an amateur soccer team conducted an online field test during a real football match . one male subject participated for validity of hr monitoring systems against ecg whereas the female subject was only involved in operating distance measurement . because of the low case number of the last two tests , data can only be considered as a pilot study for ecg validation and distance measurement . the following four commercially available systems were used for all tests : polar team pro ( polar electro oy , kempele , finland ) , acentas team monitoring system ( acentas gmbh , hrgertshausen , germany ) , suunto pro team pack ( suunto oy , vantaa , finland ) , and activio sport system ( activio ab , stockholm , sweden ) . a quality rating system similar to rating systems used in trade journals which is specialised on the testing of mobile and electronic communication tools . for this purpose , the authors decide to subdivide the rating system into three main categories : measurement , software , and hardware . at this , the core area of the study focused on the adequate online measurement of the heart rate . therefore , the rating system was set in favour of the measurement which was represented by 200 scores of maximal 500 . the latter 300 were equally divided on quality of the hardware and the usability of the software . subcategories were weighted subjectively by the authors in consideration of heart rate monitoring of team sports including the monitoring of 10 or more subjects in parallel . in addition , the following subcategories were rated once by an independent observer who was not familiar with any of the monitor systems to get a first impression : instruction manual , packing , chest belts ( quality ) , and initial software handling . the remaining categories were judged once by an experienced person . with exception of wearing comfort and failing quota in field , all subcategories were judged and quantified by using a ten - stepped visual analogue scale which is known for pain assessment . visual analogue scaling was weighted by multiplying itself with factor f : ( f = maximal score of subcategory/10 ) . parameters leading the judgement of the subcategories are described in tables 2 and 4 . wearing comfort of the chest belts was evaluated via a short anonymous questionnaire which all 12 participants ( see section 2.1 ) had to complete directly after wearing . the questionnaire contained five questions about the handling and individual fitting of the chest belts . the questions one and two are positively formulated whereas questions three to five are negative ( see table 3 ) . all questions had to be answered by an even scaled rating system including six categories . maximum score for the whole questionnaire was 25 ; hence , each question was scored with maximal 5 points . the main field test was measurement of failure ratio during the half time of a real soccer match . all players apart from goal keeper were equipped with chest belts of the relevant system and measured during first half (= 45 min ) of a league soccer game during four home matches in summer 2009 . temperature range was within 9c , and weather conditions were similar ( no precipitation ) for all measurements . the receiver of the relevant system was always positioned three meters in projection of midline of the soccer ground . in order to improve transmission distance , receiver could not be excluded a priori , a mean failure ratio was determined only with transmitters sending valid data . each deviation / loss of one percent of whole duration measurement of 45 min (= 2700 s ) was subtracted with 10 scores , and a maximal score of 100 could be obtained . if , for example , a total loss time of all ten transmitters of a system was 270 s , a total score of 90 would be credited to this system . a failure ratio of 10 percent of whole duration measurement would be credited with a zero score . distance measurement was conducted on a uphill street with a constant slope and in far distance to high - voltage line or radio tower to reduce possible interference . receiver antenna was placed on highest elevation of the hill one meter above ground , and unit was connected to a laptop ( toshiba satellite series ; toshiba inc , ny , usa ) . the participants wearing the chest belts were equipped with a gps device ( nokia n79 , nokia group , espoo , finland ) to measure distance . by moving backwards additionally , the participants were instructed to move arms to keep hr variable with time . attempts were only started when both valid signals of gps and hr were available and stopped when hr signal was lost . tests were conducted twice , and mean value of both attempts was calculated and taken as maximal range . maximal range distance was credited with 70 scores whereas the system with the absolute maximal covered range was taken as reference system and credited maximum score . percentage deviation of distance of other systems was analogously deducted as percentage of scores . if , for example , reference system had 100 m range , it was credited 70 scores . if a second system had only 50 m range , it would be credited 50% of maximum score , hence 35 . comparison against ecg was conducted in a laboratory by using ecg tool vicardio ecp-6 standard ( energy - lab technologies , hamburg , germany ) . a maximum score of 30 could be obtained at this test . a deviation of 1 beat per minute ( bpm ) was deducted with two scores . only complete accordance of both values was credited with maximum score . a plus ( + ) is a positive feature ; a minus ( ) is a negative one . a plus / minus ( ) indicates aspects which are both positive and negative . in context of the supplied hardware acentas and polar lead the field of monitor systems , both reaching 100 and 106 scores in sum for the hardware . the leading position of polar depends on the storage capacity of the chest belt increasing data safety but also decreasing wearing performance which is indicated by the chest - belt questionnaire presented in table 3 . in case of wearing comfort , it has to be noted that both chest belts of activio and acentas were supplied by the identical manufacture . both polar and acentas delivered high - quality receivers . results of software evaluation are summarized in table 4 . again , similar to hardware evaluation , a plus ( + ) is a positive feature , and a minus ( ) is a negative one . if all systems were similar or identical , there is no separate classification . a plus / minus ( ) indicates aspects which are both positive and negative . here , activio offered the harmonious concept which led to the highest ranking . the installation procedure was easiest with the acentas software , but acentas did not focus on extensive statistical analysis . in this case , polar as well as activia provide a more developed software tool . as data of ecg comparison were only obtained with a single subject , they have to be regarded with caution . suunto 's deviation was 80 to 85 bpm , activio 's deviation was 81 to 85 bpm , and polar 's deviation was 82 to 85 bpm , respectively . same caution has to be taken as only one subject 's data were included in analysis . nevertheless , acentas gave reference with 349 m , followed by activio with 141 m and suunto with 107.5 m. it has to be noted that activio 's system was supposed to reach 300 m. last but not least , polar reached 102 m which corresponded to the manufacturer information for this system . nevertheless , all other systems had a wider range of data acquisition , and hence score distribution was credited as follows : acentas 70 , activio 28 , suunto 21 , and polar 20 . regarding failure quota , average downtime was 148 s ( 0,6% ) for acentas , 286 s ( 1,0% ) for polar , and 552 s ( 2,0% ) for activio . single belts were recognised from time to time , but no consistent data acquisition could be accomplished . similarly , five out of ten polar chest belts had signal loss and could not be reactivated whereas activio managed 10 out of 10 signals , and for the acentas system 9 out of 10 were used for data evaluation as one person was injured during match play . the purpose of the present study was to compare four commercially available online heart rate monitoring systems and especially their use in everyday situation in team sports . the system of polar could score especially with a clear design , useable features of the software , and a convincing product quality . only polar uses wlan for data transmission and also adds a mobile terminal in pocket format which can easily be used outdoor and with bad weather conditions . but the high energy consumption of wlan transmission would terminate the battery capacities and additionally the duration of measurement . the system of activio has a functional and useful software and the instruction manual was very detailed and easily comprehensible . acentas could score with a huge operating distance , a valid heart rate signal compared with ecg , a low failure quota , and an easy handling . in contrast , the system of suunto had a total breakdown during competition measurements and could not be reactivated . prospectively , it would be a great challenge to realise online monitoring for swimmers , possibly leading to new opportunities for the training regime . in this context the system of polar and suunto had the advantage that data are also recorded on the chest belt memory during water immersion and could be evaluated after exercise . meanwhile , some of the tested heart rate monitoring systems are updated and upgraded with new functions , respectively . for example , acentas included an offline memory in the chest belts and added a wrist monitor for direct heart rate analysis from up to twenty athletes by the coach . in summary , acentas system reached the best mark of all systems , but every system has its advantages and disadvantages depending on the using purpose , location , and weather . so this present comparative study can not recommend a single system but rather shows strength and weakness of all systems and additionally can be used for further system improvements . the investigators were responsible for the study design , data collection , data analysis , data interpretation , and submission of the paper for publication , independently of all funding sources . the authors declare that they have no financial conflict of interests to disclose with any of the companies named in this paper .

recent technical development focused on real - time heart rate monitoring instead of postexercise evaluation of recorded data . there are several systems on the market that allow direct and real - time monitoring of several individuals at the same time . the present study compared the systems of polar , acentas , activio , and suunto in a field test with twelve subjects regarding failure quota , operating distance , and ecg validity . moreover , the installation and use of software and hardware were evaluated with a quality rating system . chest belts were evaluated with a questionnaire , too . overall the system of acentas reached the best mark of all systems , but detailed results showed that every system has its advantages and disadvantages depending on using purpose , location , and weather . so this evaluation can not recommend a single system but rather shows strength and weakness of all systems and additionally can be used for further system improvements .

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in the 1960s , mcculloch and till proposed three functional characteristics that came to form the definition of stem cells . their findings supported the hypothesis that a specific population of cells was responsible for propagation and sustained growth in a hierarchical manner , a theory first proposed in the middle of the 19 century by virchow . first , stem cells have the ability to make identical copies of themselves ( self - renewal ) . assessment of a cell 's ability to proliferate extensively and self - renew requires the induction of these behaviors . thus , the cell must be isolated and the behaviors induced in vitro for the cell to be classified as a stem cell . to more efficiently isolate candidate stem cells , much work has turned to the use of cell surface proteins unique to stem cells , which permit isolation by flow cytometry , magnetic bead isolation , fluorescent protein tagging , and immunostaining . these markers also enable stem cells to be tracked and their functions to be characterized . advances in stem cell biology have provided insight into the role of stem cells in the lung . due to the relative quiescence of the lung epithelia , with a proliferative fraction of just 0.06% to 1.3% in the bronchiolar and tracheal epithelia , identification of putative stem cells has been more difficult than in tissues with higher rates of turnover , such as the epithelia of the gut or skin . despite these challenges , populations of stem cells have been identified throughout the lung by way of their role in the response to injury of the airway . in each case , the functional tenants of stem cell behavior namely , self - renewal , proliferation , and multipotency have been observed . a growing body of evidence now indicates that the earliest stages of lung malignancies may be related to a deregulation of this stem cell - based repair mechanism . the epithelial lining of the adult airway progresses from proximal to distal in a stereotyped fashion . proximal airways consist of pseudostratified epithelium , middle airways consist of a simple columnar epithelium , and the most distal airways consist of a cuboidal epithelium . multiple studies indicate that cells capable of stem - like behavior in response to airway injury are present throughout the airway . in proximal airways , studies of airway repair in the trachea identified a population of tracheal basal cells that are pluripotent , retain bromodeoxyuridine , and express keratin-5 ( k5 ) . a second study using a naphthalene inhalation injury model identified an additional population of tracheal basal cells , this time expressing the keratin-14 ( k14 ) marker . these k14 cells were able to proliferate to give rise to cells of multiple phenotypes in response to airway injury . it appears that in the trachea , basal cell populations expressing either k5 or k14 are capable of the functional aspects of stem cell niches . squamous cell carcinoma frequently arises from the more proximal tracheobronchial airways , yielding centrally located tumors that often exhibit elevated levels of k5 basal cells . as such , it appears that cancers with histology consistent with squamous cell carcinoma are more likely to arise from k5 stem cells . although there is no targeted therapy available against k5 cells to date , this stem cell niche of squamous cell carcinoma may provide a worthwhile direction for targeted research , providing a unique biomarker that can be used to target the cell of origin in these tumors . in the middle airways , a rat model of nitrogen dioxide ( no2)/ozone ( o3 ) inhalation injury revealed that mature rat clara cells could proliferate into phenotypically diverse progeny . this ability to move from quiescent and fully differentiated epithelial cells to actively proliferating multipotent cells led evans et al . to deem these clara cells facultative progenitor cells , as they are capable of but not limited to behavior indicative of a stem cell . in the setting of injury to a clara cell - depleted airway , as is the case with the naphthalene model of injury , clara - cell secretory protein ( ccsp)-positive cells located within neuroepithelial bodies ( neb ) have been shown to proliferate to repair the airway . these neb ccsp cells are able to independently repopulate the middle airways with phenotypically diverse progeny after naphthalene injury , characteristic of true bronchial airway stem cells . small cell lung carcinomas , frequently arising in the mid - level bronchioles , have been shown to contain fractions of cells expressing cell surface markers associated with mid - airway stem cell subpopulations . in particular , the cell surface markers cd44 and multidrug resistance 1 ( mdr1 ) have previously been identified in a series of small cell lung carcinoma cell lines . cells expressing cd44 have also very recently been implicated in lung cancer progression and metastasis by the epithelial - to - mesenchymal transition ( emt ) . in their 2011 study , tellez et al . found that bronchial epithelial cells exposed to carcinogens gained stem - like properties and that emt led to a progressively motile and invasive phenotype . again , while no targeted therapies are presently available to target cells expressing cd44 and mdr1 , the potential exists for improved delivery by using agents directed at this cell or origin in conjunction with the current standard of care . for the most distal airway , there is no consensus on the cell surface marker phenotype of representative lung stem cells . first identified pluripotent , neb - independent , ccsp cells in the bronchoalveolar duct junction ( badj ) that could , in response to naphthalene injury , yield diverse populations of cells . cells expressing surface markers surfactant protein c ( sp - c ) , stem cell antigen-1 ( sca-1 ) , and cd34but not expressing cd45 or pecam were shown to act as pluripotent and to have the ability to proliferate and self - renew in culture . however , others have argued that these ccsp / sp - c cells are insufficient as the distal lung stem cell population because they are unable to differentiate into type ii alveolar cells . instead promoted id2 as a more suitable cell surface marker for distal lung epithelial stem cells . thus , while there is consensus that stem cells form a niche population in the badj , the cell marker phenotype remains under debate . carcinomas of the distal airway most frequently lung adenocarcinoma and bronchioloalveolar carcinoma have been associated with stem cells from the badj region . the hierarchical or cancer stem cell hypothesis has proven to be an important theory with which to understand lung tumorigenesis , and may have important implications for future management of lung tumors . in this model , relatively rare , multipotent cells gain unlimited proliferative potential that may then drive tumor growth . the multipotent nature of these cells leads to heterogeneity within the tumor by aberrant differentiation and epigenetic changes in the progeny . it follows , then , that a stem - like cell , with the ability to rapidly proliferate into phenotypically diverse progeny , could act as this cancer stem cell . stem cells are also known to express high levels of anti - apoptotic and drug - resistant proteins , which may explain why chemotherapy is extremely effective in shrinking the size of primary tumors but frequently ineffective against localized recurrence and metastatic disease . proponents of the theory of lung cancer stem cells propose that although chemotherapy is able to effectively kill non - stem cell cancer cell types in these settings , chemoresistant lung cancer stem cells to continue to proliferate unchecked . this understanding has sparked new energy in the development of targeted therapies against specific cell types within the heterogeneous tumor . as introduced above , common tumors from each representative airway zone have been associated with stem cell niches based on shared cellular expression of surface markers . in addition , recent studies have successfully identified cells within lung cancer specimens that exhibit stem - like properties . identified tumor cell subpopulations that exhibited several key functional properties multidrug resistance , increased telomerase expression , and self - renewal that are characteristic of stem cells . in addition , cd133 cells isolated from freshly resected human lung cancer specimens can grow indefinitely in culture , a characteristic usually only seen in transformed cell lines . cd133 cells are also able to proliferate into phenotypically diverse tumors , both in culture and when injected into immunodeficient mice . of high clinical interest are recent observations that patients with lung tumors enriched with these cd133 lung cancer stem cells tend to have worse prognosis than those with fewer cd133 lung cancer stem cells . in addition , these cd133 subpopulations exhibit increased resistance to cisplatin - based therapies , suggesting that the presence of cd133 cell populations may warrant changes to chemotherapeutic regimens in these patients . that lung cancer stem cells impact prognosis raises the possibility , then , that major therapeutic advances in lung cancer treatment , through the specific targeting of lung cancer stem cells or the pathways that confer stem cell - like properties to lung cancer cells , are possible in the near future . in addition , measurement of the lung cancer stem cell fraction in a tumor could prove a useful modality for better differentiating between transient tumor debulking effects of chemotherapy and longer lasting therapies . the study of the lung epithelial response to airway injury has shown compelling evidence for the role of stem cells as a component of airway repair . while there is still controversy in the field , there is also a growing body of evidence to suggest that these same lung stem cells may play a key role in the initiation and proliferation of human lung cancers . two primary pieces of evidence reviewed here support the hypothesis that lung stem cells may play a role in lung tumorigenesis : 1 ) cells sharing the functional properties of stem cells are present in many tumors , and 2 ) many tumor sites of origin match known stem cell niches . because characterization of putative stem cells requires manipulation of the cells themselves to induce a specific phenotype , it remains exceptionally difficult to discriminate between potential or facultative and true stem cells . on account of the inducible abilities that form the critical features of stem cells , it remains possible that these cancer stem cells may exist on a continuum of inducible features that is , if genetic and epigenetic changes can confer stem cell characteristics upon a given tumor cell , then any tumor cell may in fact have the potential to drive tumor growth and resistance . this spectrum of qualities may yield a mixed model , as proposed by rahman et al . , in which stem - like cells may have variable potential to drive tumor growth , depending on the conditions presented to the cell . several key questions remain unanswered with respect to the role of stem cells in lung tumorigenesis . it remains to be conclusively determined whether healthy adult stem cells undergo oncogenic transformation leading to the initiation of tumorigenesis , or whether healthy airway cells undergo oncogenic transformation leading to their de - differentiation and subsequent adoption of stem cell - like properties . in addition , the relationship between the tumor microenvironment and the lung cancer stem cell remains poorly characterized . however , the sum of the available evidence to date , in conjunction with ever - improving modalities for the identification and isolation of putative stem cell populations , should provide a wealth of future directions that hold great promise in improving the management of patients with lung tumors .

lung cancer is the leading cause of cancer - related deaths worldwide . recently , advancements in our ability to identify and study stem cell populations in the lung have helped researchers to elucidate the central role that cells with stem cell - like properties may have in lung tumorigenesis . much of this research has focused on the use of the airway repair model to study response to injury . in this review , we discuss the primary evidence of the role that cancer stem cells play in lung cancer development . the implications of a stem cell origin of lung cancer are reviewed , and the importance of ongoing research to identify novel therapeutic and prognostic targets is reiterated .

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tubularized incised plate ( tip ) repair is recommended as the ideal repair for distal penile hypospadias ; it has shown excellent results in the management of primary , redo , distal , and proximal hypospadias . a number of groups have recently been engaged in studying the plate characters and its impact on the success of tip . while some authors have shown the importance of the plate characters in the success of tip repair , others have shown that the technique could be used for all cases of hypospadias without concern for the plate configurations . these previous studies have inspired us to undertake the current study to examine the role of the plate character in the success of tip repair among the study population in yemen . here , we present a study evaluating the urethral plate characters , such as shape and the width in the success of tip urethroplasty for the management of primary anterior penile hypospadias in 100 children . between january 2010 and december 2013 , a total of 100 children with primary anterior penile hypospadias were undergone tip repair . all cases were undergone in mayo hospital , hodiedah , yemen . we used snodgrass technique with modifications for our current study . all children were examined under general anesthesia ; the type of the plate is defined and reported by a single surgeon . the plates were measured at the widest area ( in mm ) at the time of surgery . we divided the study population according to the width of urethral plate > 8 mm ( group - a ) , while patients with urethral plate < 8 mm ( group - b ) . following the classification made by nguyen et al . , the plates were categorized as flat , cleft , and deeply grooved [ figures 13 ] . in addition , examination of the penis was also carried out to evaluate the presence or absence of chordee . first a u - shaped incision was made around the plate associated with circumferential incision for complete degloving of the penis . an artificial erection was achieved by saline injection , and the intrinsic ventral curvature was corrected with a dorsal plication if needed . one deep midline incision in the urethral plate was made from the tip of the penis to the urethral meatus as described by snodgrass . the plate was tubularized around 6 or 8 french ( fr ) nelaton catheter using polyglycolic acid 6/0 subcuticular running suture . glanuloplasty was performed in a standard manner with interrupted 6 - 0 monofilament polyglyconate synthetic absorbable sutures . postoperative urethral feeding tube or silicone foley catheter 8 fr was used for urinary drainage for average 1 week . intraoperative single dose antibiotic was used ( ampicillin / gentamycin weight - adjusted dose in all cases ) . postoperative management included ampicillin / gentamycin weight - adjusted dose in all cases and oxybutynin for relieving bladder spasms . nonsteroidal antiinflammatory drugs , oral or suppository , were used in weight - adjusted doses . success of surgery and postoperative complications were assessed in patients categorized on the basis of the shape of the plate or its width . postoperatively , patients were followed - up for evaluating meatal stenosis , fistula formation , and partial or complete glandular dehiscence at the 1 , 3 and 6 months . patients were followed - up for urethral calibration by urethral sound 8 fr at the third and 6 months of follow - up . the flow rate was selectively indicated for patients with voiding difficulty and/or a narrow meatal orifice or weak stream . a meatal stenosis was diagnosed when the flow rate was < 5 ml / s . success was defined as the presence of an anatomically positioned vertical slit - like meatus , a normal urinary stream , and a satisfactory cosmetic appearance resembling a circumcised penis . data were statistically analysed using epi info program ( centers for disease control and prevention , atlanta , georgia , usa ) . fischer exact test for 2 2 tables was performed when expected cell count in > 25% of cases was < 5 . first a u - shaped incision was made around the plate associated with circumferential incision for complete degloving of the penis . an artificial erection was achieved by saline injection , and the intrinsic ventral curvature was corrected with a dorsal plication if needed . one deep midline incision in the urethral plate was made from the tip of the penis to the urethral meatus as described by snodgrass . the plate was tubularized around 6 or 8 french ( fr ) nelaton catheter using polyglycolic acid 6/0 subcuticular running suture . glanuloplasty was performed in a standard manner with interrupted 6 - 0 monofilament polyglyconate synthetic absorbable sutures . postoperative urethral feeding tube or silicone foley catheter 8 fr was used for urinary drainage for average 1 week . intraoperative single dose antibiotic was used ( ampicillin / gentamycin weight - adjusted dose in all cases ) . postoperative management included ampicillin / gentamycin weight - adjusted dose in all cases and oxybutynin for relieving bladder spasms . nonsteroidal antiinflammatory drugs , oral or suppository , were used in weight - adjusted doses . success of surgery and postoperative complications were assessed in patients categorized on the basis of the shape of the plate or its width . postoperatively , patients were followed - up for evaluating meatal stenosis , fistula formation , and partial or complete glandular dehiscence at the 1 , 3 and 6 months . patients were followed - up for urethral calibration by urethral sound 8 fr at the third and 6 months of follow - up . the flow rate was selectively indicated for patients with voiding difficulty and/or a narrow meatal orifice or weak stream . a meatal stenosis was diagnosed when the flow rate was < 5 ml / s . success was defined as the presence of an anatomically positioned vertical slit - like meatus , a normal urinary stream , and a satisfactory cosmetic appearance resembling a circumcised penis . data were statistically analysed using epi info program ( centers for disease control and prevention , atlanta , georgia , usa ) . fischer exact test for 2 2 tables was performed when expected cell count in > 25% of cases was < 5 . mean age of the patients underwent surgery was 4.3 years , ranging between 9 months and 12 years . mean operative time was 115 min ( ranging 70 - 140 min ) and the average period of hospital stay was 4.6 days ( ranging 1 - 15 days ) . urethral stenting for urine diversion was used for an average 7.7 days ( ranging 3 - 14 days ) , and the patients were followed - up for an average period of 6.4 months ( ranging 2 - 24 months ) . we found mild insignificant chordee in 15 cases , improved after complete degloving of the penile skin without any additional procedures . five infants had subcoronal hypospadias along with unilateral undescended testes ; these infants underwent orchiopexy at the same time along with hypospadias surgery . location of the meatus was reported as coronal in 46 , subcoronal in 50 and anterior penile in 4 patients . we divided the study population according to the width of urethral plate as 74 cases ( 74% ) with urethral plate > 8 mm ( group - a ) , whereas 26 cases ( 26% ) with urethral plate < 8 mm ( group - b ) . table 1 shows that group - b patients with urethral plate < 8 mm had a statistically significant higher fistula rate compared with the group - a patients with urethral plate > 8 mm ( p = 0.004 ) . similarly , group - b patients also had significantly higher incidences of failed 8 fr calibration compared with group - a ( p = 0.01 ) [ table 1 ] . complications correlated to urethral plate width we also divided our study population ( 100 patients ) according to the character of urethral plate as flat ( 26 patients ) , cleft ( 52 patients ) , and deeply grooved ( 22 patients ) and monitored for postoperative complications . table 2 shows that higher fistula rate as well as failed 8 fr calibrations is associated with patients having flat urethral plate compared with the patients with either cleft or deeply grooved urethral plates , although not statistically significant . irrespective of urethral plate character , overall 8% patients developed fistula , 12% patients had failed 8 fr calibration , while partial dehiscence occurred in 1 case ( 1% ) . in addition , redo surgery was also indicated in 1 patient and fistulectomy in 8 patients . the aim of hypospadias surgery is the creation of a straight penis with slit - like meatus at the tip of a glans with enough skin coverage . tip urethroplasty is the most common procedure used for hypospadias repair since its introduction in 1994 by snodgrass . tip repair is versatile and can be used for creation for neourethra with satisfactory slit - like meatus . the urethral plate shape and width are generally evaluated when the tip repair is opted for the management of hypospadias . in this study , we also categorised our study population based on both urethral plate width and shape . based on the plate width , patients were divided into group - a with width > 8 mm and group - b < 8 mm . we found significantly higher incidences of fistula in group - b patients when compared to the group - a patients ( p = 0.004 ) . ideal urethral plate allows the creation of the neourethra > 10 fr in children after a midline incision to widen the urethral plate . however , a narrow plate < 8 mm is inadequate for the creation of neourethra , which might be the cause of the statistically significant higher fistula rate in group - b patients with urethral plate width < 8 mm . our findings are in accordance with the earlier reports by both holland and smith and nguyen et al . where statistically significant higher fistula was found in patients with urethral plate < 8 mm width however , nguyen et al . reported 1 case ( 3.3% ) over 30 cases with narrow urethral plate versus one case ( 0.7% ) over 129 cases with urethral plate > 8 mm ; therefore , the result was statistically insignificant . the higher fistula rate with narrow urethral plate occurs because of the fact that midline incision of the urethral plate is not enough to create a neourethra with too much width around 8 fr catheter ; as a consequence , the urethral plate makes the anastomoses under tension . therefore , tubularisation with tension on suture line increases the incidence of fistula formation in cases with narrow urethral plate . failure in 8 fr calibration in association with fistula formation could be resolved with urethral dilation . in our study , two of the children who had fistula resolved with urethral dilation . modification to midline incision of the plate by extending it beyond to the tip of glans prevents meatal stenosis as described by khairallah and bader - eldin ; however , it only decreases fistula rate and meatal stenosis to < 1% . we also divided our study population based on the urethral plate character as flat , cleft , and deeply grooved . a higher fistula rate was noted in patients with flat urethral plate compared to the patients with cleft or deeply grooved ; however , the result is not statistically significant ( p = 0.4 ) . this is also in agreement with the previous reports , where no significant relation was found between urethral plate depth and incidence of fistula . although higher failed 8 fr calibration rate was observed in patients with flat urethral plate compared with the other two groups cleft and deeply grooved , the result is statistically insignificant ( p = 0.09 ) [ table 2 ] . similar to earlier reports , our study also provide evidences of excellent outcome in hypospadias management through tip repair with overall 8% patients developing fistula , 12% patients with failed 8 fr calibration , while partial dehiscence occurred in 1 case ( 1% ) ; redo surgery was indicated in only 1 patient . in the current study , we found patients with flat and narrow urethral plate developed more complications . in this regard , tavakkoli tabassi and mohammadi rana advocated the use of buccal mucosal graft ( bmg ) with tip repair to augment the narrow urethral plate ( 6 - 8 mm ) and they concluded that the use of mucosal graft decreased fistula rate formation and meatal stenosis . ye et al . also combined tip with bmg in 53 patients and found better outcome . therefore , urethral plate augmentation is recommended when the urethral plate is narrow and inelastic requiring augmentation with free graft . based on our findings , we conclude that the tip urethroplasty has good cosmetic and functional results and offer a modality for hypospadias repair that has the following advantages : preservation and utilisation of urethral plate without the need for additional flaps , single suture line of the neourethra , creation of glandular and penile urethra , short operative time and low complication rate . this technique can be used for primary anterior penile hypospadias with satisfactory results . using this technique with plates flat plates may need augmentation by bmg as advised by some authors to improve the results in this group of patients .

background : today , tubularized incised plate ( tip ) urethroplasty is the most commonly performed operation for distal and mid - penile hypospadias . reports from different centers worldwide confirm its nearly universal applicability and low complications rate.aim:evaluation of the urethral plate characters and its effect on the outcome of tip urethroplasty.materials and methods : between 2010 and 2013 , 100 children with primary distal penile hypospadias underwent tip urethroplasty . urethral plates were categorized as flat , cleft , and deeply grooved . postoperatively , patients were followed - up for evaluation of meatal stenosis , fistula formation , and glandular dehiscence at 1st , 3rd and 6th months . patients were followed - up for urethral calibration by urethral sound 8 fr at 3rd and 6 months follow - up . data were statistically analysed using epi info program to correlate between the width , plate shape , and complications.results:mean age at surgery was 4.3 years . patients were followed - up for an average period of 6.4 months . pre - operative location of the meatus was reported as coronal in 46 , subcoronal in 50 and anterior penile in 4 cases . urethral plate characters were flat in 26 cases , cleft in 52 , and deeply grooved in 22 . urethral plate width was > 8 mm in 74 cases and < 8 mm in 26 . patients with urethral plate < 8 mm had a statistically significant higher fistula rate ( p = 0.004 ) and failed 8 fr calibrations in 26.9% ( p = 0.01 ) compared with the patients with urethral plate > 8 mm . in addition , we also founds higher fistula rate and failed 8 fr calibrations in flat urethral plate.conclusions:an adequate urethral plate width ( > 8 mm ) is essential for successful tip repair . lower success rates with flat plates may need buccal mucosal augmentation to improve the results .

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a biodegradao de materiais que ocorre na cavidade bucal pode interferir na resistncia de unio entre o braquete e o esmalte , causando baixos valores de resistncia de unio in vivo , em comparao aos valores in vitro . o objetivo do presente estudo foi desenvolver um prottipo para mensurar , in vivo , a fora de descolagem de braquetes e avaliar , in vitro , a resistncia de unio obtida com o uso do prottipo . um alicate ( 3 m unitek ) foi modificado por meio da adio de um extensmetro diretamente conectado garra do alicate . um circuito eletrnico realizou a leitura do extensmetro , e um software instalado em um computador registrou os valores de fora de descolagem do braquete em kgf . os braquetes ortodnticos foram colados com material adesivo na face vestibular de 30 dentes incisivos bovinos . no grupo 1 ( n = 15 ) , a descolagem foi realizada com o prottipo , enquanto no grupo 2 ( n = 15 ) foi realizado teste de resistncia de unio trao , utilizando - se uma mquina universal de ensaios . de acordo com o teste de t de student ( = 0,05 ) , os grupos 1 ( 2,96 mpa ) e 2 ( 3,08 mpa ) no foram estatisticamente diferentes o prottipo mostrou - se confivel para se obter , in vivo , os valores de resistncia de unio dos braquetes ortodnticos . a stable interface between the bonding material and the bracket , as well as between the bonding material and the enamel , is important to withstand the loads generated by an activated arch . using universal testing machines , direct bracket bonding to dental enamel and the forces required to debond the brackets have been studied , in vitro , by means of shear or tensile strength . regardless of the precision obtained in the measurement of the debonding forces , the universal testing machines are expensive , large and complex to handle . due to the proportions , it may be impossible to use it for in vivo studies . furthermore , the complex interaction processes that exist in the oral cavity can not be completely reproduced by in vitro studies . the oral cavity is a challenging environment for dental materials , especially regarding the adhesion process . the contact of adhesive systems and composite resins with the oral medium is associated with an ageing pattern , which may alter the composition and the mechanical properties of the polymers . material biodegradation may interfere in the bonding between the bracket and the enamel , causing lower bond strength values in vivo , in comparison with in vitro values . because in vitro bond strength can not be correlated with clinical failure indexes , and facing the necessity of creating a method to test the effects of the oral medium on the composite resins used in orthodontics , different devices have been developed to measure the bracket debonding force in vivo . usually , strain gauges are bonded to the handle or to the arm of the pliers . the force obtained is a measure of the deformation that occurs in the handle or arm of the pliers during the bracket debonding procedure , and the results can be influenced by the way pliers are holded . no study has developed a device using strain gauges connected directly to the part of the pliers where the bracket is attached for the debonding procedure . thus , the aim of the present study was to develop a prototype to measure the bracket debonding force in vivo , using a strain gauge connected directly to the extremity of the pliers where the bracket debonding occurs . the bond strength obtained with the prototype was also compared in vitro by using the tensile bond strength test in a universal testing machine . a polypropylene plier ( debracketing instrument - 444 - 761 , instrument kit - 3m / unitek , monrovia , ca , usa ) was modified to develop the prototype ( fig 1a ) , which consists of three basic parts : figure 1a ) original plier . b ) exploded image of the intermediate part of the prototype ( the arrow indicates the localization of the strain gauges ) . 1 ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . figure 1c shows a photograph of the prototype.2 ) measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . 3 ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) was applied to the claw , and the measurements were recorded in kgf . a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . figure 2a ) prototype after electronic instrumentation ; b ) measurement and control circuit ; c ) usb communication cable . figure 3representative diagram of the parts of the prototype . to measure the accuracy and precision of the prototype , the following weights were used : 1 , 2 , 3 , 4 , 5 , and 6 kg . each weight was connected to the claw of the prototype by a stainless steel wire , and the measurement was done by means of displacing the active arm and lifting the weight . the measurements were undertaken by two examiners , who did not standardize the position of the hand on the pliers . twenty repetitions were performed for each weight . the numerical value recorded by the software installed on the computer was noted , and the arithmetic mean was calculated . the mean temperature in the laboratory during the experiments was 23 2c and the humidity was 70% . the exposed enamel surface was lightly abraded with # 600 grain silicon carbide abrasive paper in a polisher to obtain a flat enamel surface . the maxillary central incisor brackets ( eurodonto , curitiba , pr , brazil ) were bonded to the enamel using the following procedure : a ) prophylaxis with pumice stone and water ; b ) etching with phosphoric acid at 37% for 15 s , followed by washing and drying ; c ) application of scotchbond multi - purpose adhesive ( bond ) ( 3 m , st . paul , mn , usa ) on the enamel ; d ) application of z100 composite resin ( 3 m , st . paul , mn , usa ) at the base of the bracket and positioning on the tooth with light manual pressure , followed by removal of the excess with a periodontal scaler ; e ) light curing for 40 s ( 10 seconds on each side ) with the optilight plus appliance ( gnatus , ribeiro preto , brazil ) . the specimens were stored in 100% relative humidity at 37c for 24 hours . after storage , specimens were divided into two equal groups : group 1 - bracket debonding with the prototype : each specimen was placed on a bench lathe . a single operator held the prototype , which was placed on the bracket with the supports aligned in an occlusal - gingival direction in contact with the enamel . with the claw hitched onto one of the bracket wings , the prototype was activated by moving the active arm until bracket debonding occurred . the value of the force necessary to remove the bracket was recorded by the software in kgf , transformed into newtons and divided by the bracket area ( 14.12 mm ) , to obtain resistance values in mpa . group 2 - bracket debonding by tensile testing in a universal testing machine : the specimen was fixed in a metal sleeve on the bottom part of the emic - dl 2000 universal testing machine ( so jos dos pinhais , paran , brazil ) . for tensile testing , a claw made of stainless steel wire , 0.40 mm in diameter and 5 cm in length , was connected to one of the bracket wings . at the top part of the claw , a 2 mm thick and 20 mm long wire segment was welded , which was hitched onto the top part of the universal testing machine . , tokyo , japan ) at 10x magnification to assess the adhesive remnant index ( ari ) : score 0 = no composite resin left on the tooth ; score 1 = less than half of the composite resin left on the tooth ; score 2 = more than half of the composite resin left on the tooth ; score 3 = all composite resin left on the tooth , with a distinct impression of the bracket mesh . student s t test ( = 0.05 ) for paired samples was used for comparison between the examiners . student s t test ( = 0.05 ) was performed to compare the groups . a polypropylene plier ( debracketing instrument - 444 - 761 , instrument kit - 3m / unitek , monrovia , ca , usa ) was modified to develop the prototype ( fig 1a ) , which consists of three basic parts : figure 1a ) original plier . b ) exploded image of the intermediate part of the prototype ( the arrow indicates the localization of the strain gauges ) . 1 ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . figure 1c shows a photograph of the prototype.2 ) measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . 3 ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . ) modified plier : the pliers have undergone changes from its original design . to install the strain gauge , the original pliers were cut to receive an intermediate part positioned between the active arm of the pliers and the claw ( fig 1b ) . one strain gauge ( 45% ni ; 55% cu ) with 10 mm of a sheet type grid and with temperature compensation for steel ( kyowa , chofu , tokyo , japan ) was directly connected to the claw . measurement and control circuit : corresponds to the electronic circuit that performs the reading of the strain gauge mounted on the intermediate part of the prototype and changes the obtained value into an 8-bit binary number . the value 0 ( zero ) represents a strength of 0 kgf , and the value 255 ( 8 bits ) is the maximum measured force of 6.8 kgf . prior adjustments are necessary for the required performance of the circuit ( wheatstone bridge , amplifier gain and the ad converter range ) . ) pc : a specific software was developed to record the values ( in kgf ) of the bracket debonding force . the software was programmed in c ( programming language ) and was designed to enable appropriate use in a research environment . the software has a calibration system , and calibration was performed with weights ranging from 0.5 kg to 6 kg . a known load ( kg ) was applied to the claw , and the measurements were recorded in kgf . a simple linear regression analysis was applied to adjust the data ( r = 0.99 ) . figure 2a ) prototype after electronic instrumentation ; b ) measurement and control circuit ; c ) usb communication cable . to measure the accuracy and precision of the prototype , the following weights were used : 1 , 2 , 3 , 4 , 5 , and 6 kg . each weight was connected to the claw of the prototype by a stainless steel wire , and the measurement was done by means of displacing the active arm and lifting the weight . the measurements were undertaken by two examiners , who did not standardize the position of the hand on the pliers . twenty repetitions were performed for each weight . the numerical value recorded by the software installed on the computer was noted , and the arithmetic mean was calculated . the mean temperature in the laboratory during the experiments was 23 2c and the humidity was 70% . the exposed enamel surface was lightly abraded with # 600 grain silicon carbide abrasive paper in a polisher to obtain a flat enamel surface . the maxillary central incisor brackets ( eurodonto , curitiba , pr , brazil ) were bonded to the enamel using the following procedure : a ) prophylaxis with pumice stone and water ; b ) etching with phosphoric acid at 37% for 15 s , followed by washing and drying ; c ) application of scotchbond multi - purpose adhesive ( bond ) ( 3 m , st . paul , mn , usa ) on the enamel ; d ) application of z100 composite resin ( 3 m , st . paul , mn , usa ) at the base of the bracket and positioning on the tooth with light manual pressure , followed by removal of the excess with a periodontal scaler ; e ) light curing for 40 s ( 10 seconds on each side ) with the optilight plus appliance ( gnatus , ribeiro preto , brazil ) . after storage , specimens were divided into two equal groups : group 1 - bracket debonding with the prototype : each specimen was placed on a bench lathe . a single operator held the prototype , which was placed on the bracket with the supports aligned in an occlusal - gingival direction in contact with the enamel . with the claw hitched onto one of the bracket wings , the prototype was activated by moving the active arm until bracket debonding occurred . the value of the force necessary to remove the bracket was recorded by the software in kgf , transformed into newtons and divided by the bracket area ( 14.12 mm ) , to obtain resistance values in mpa . group 2 - bracket debonding by tensile testing in a universal testing machine : the specimen was fixed in a metal sleeve on the bottom part of the emic - dl 2000 universal testing machine ( so jos dos pinhais , paran , brazil ) . for tensile testing , a claw made of stainless steel wire , 0.40 mm in diameter and 5 cm in length , was connected to one of the bracket wings . at the top part of the claw , a 2 mm thick and 20 mm long wire segment was welded , which was hitched onto the top part of the universal testing machine . tokyo , japan ) at 10x magnification to assess the adhesive remnant index ( ari ) : score 0 = no composite resin left on the tooth ; score 1 = less than half of the composite resin left on the tooth ; score 2 = more than half of the composite resin left on the tooth ; score 3 = all composite resin left on the tooth , with a distinct impression of the bracket mesh . student s t test ( = 0.05 ) for paired samples was used for comparison between the examiners . student s t test ( = 0.05 ) was performed to compare the groups . table 1 compares the mean values obtained by the two examiners , with each weight , during the gauging procedure of the prototype . according to student s t test for paired samples , there were no significant differences ( p < 0.05 ) between the examiners at any weight . table 1comparison between the examiners in each weight.comparisonnmean ( kgf)standard deviationp valuea - 1 kg 201.040.080.30b - 1 kg 201.070.08a - 2 kg 202.030.070.20b - 2 kg 202.060.09a - 3 kg 202.950.060.08b - 3 kg203.010.09a - 4 kg 204.080.080.44b - 4 kg 204.040.09a - 5 kg205.010.090.16b - 5 kg 205.050.07a - 6 kg 206.080.080.32b - 6 kg 206.050.09*p > 0.05 indicates no significant difference between the examiners a - examiner a.b - examiner b. * p > 0.05 indicates no significant difference between the examiners according to student s t test , the mean bond strength obtained in group 1 ( 2.96 mpa ) and in group 2 ( 3.08 mpa ) were statistically similar ( table 2 ) . table 2comparison of mean bond strength ( mpa ) obtained in each group.groupnmeansdpgroup 1152.960.32 > 0.05group 2153.080.40group 1 : prototype.group 2 : tensile test in a universal testing machine . adhesive remnant index score 3 was predominant in the two groups ( table 3 ) . score 0score 1score 2score 3group 1 - 1 ( 7%)1 ( 7%)13 ( 86%)group 2- -2 ( 13%)13 ( 86%)group 1 : prototype.group 2 : tensile test in a universal testing machine . the prototype of the present study was an evolution of a device which had two strain gauges bonded to the anterior and posterior 2/3 surfaces of the active arm of the pliers . because the strain gauges were placed in the active arm of the pliers , they suffered from the influence of the operator s hand . the present prototype has been developed in order to control this problem by placing the strain gauge near the claw , and not in the active arm of the pliers . strain gauges are defined as localized mechanical deformation sensors , since all and any phenomenon responsible for mechanical deformation can be analyzed . therefore , strain gauges are transducers that convert mechanical displacement into a range of electrical resistance . strain gauges are very sensitive and temperature alterations are able to influence the measurements . to compensate for any possible temperature alterations , the following procedures were adopted : a ) use of an electrical resistance strain gauge with temperature compensation for steel that self - compensates eventual temperature variations , and ; b ) control of the laboratory mean temperature during the experiments . in the present study , the strain gauges were bonded in the intermediate part positioned between the active arm of the plier and the claw . one strain gauge was directly connected to the claw . when the plier was used to debond the brackets , the strain gauge metal grid resistance generated a tension ( mv ) . the small amount of tension generated was multiplied by an amplifier circuit in the electronic circuit , which performed the reading of the strain gauge , into binary numbers that were converted into kgf . the mode of holding the prototype was not standardized between the two examiners , and there was no statistically significant difference in the values obtained between the examiners at any weight . this means that the prototype was not influenced by the way that it was held - as in another study - , confirming that the new prototype is superior to the previous one . after gauging the prototype , in vitro orthodontic bracket debonding the bracket debonding performed with a universal testing machine is not dependent on operator variation , the values obtained with this method were compared to the values obtained with the prototype to evaluate its reliability . the tensile test on the universal testing machine ( group 2 ) tried to reproduce the same conditions as debonding with the prototype . for this purpose , a 0.40 mm in diameter stainless steel wire claw was made reproducing the same thickness as the prototype claw ( group 1 ) . along with that , the claw made for the test machine was also connected on a single wing of the bracket at the testing time , reproducing the same position used by the prototype claw at the debonding moment . although it is named tensile test , it is known that not only tensile forces occur at the debonding moment of the bracket , but that there is also an interaction of tensile , shear and compressive forces . debonding of the bracket was performed under a constant crosshead speed of 0.5 mm / min in the universal testing machine . this load speed is generally used in spite of not corresponding to clinical conditions due to the fact that in vivo debonding occurs at a higher speed . therefore , when the prototype was applied , a speed higher than 0.5 mm / min was probably used for bracket removal . despite the fact that differences existed between the methodologies used for debonding , the bond strength did not differ statistically between the prototype ( 2.96 mpa ) and the tensile test on the universal testing machine ( 3.08 mpa ) . this suggests that the prototype might be a useful tool for measuring bond strength in vivo . orthodontic bracket bonding to teeth requires that the bond system applied must provide sufficient bond strength to withstand the forces that occur during the orthodontic mechanics and chewing . for clinical situations bond strength values above 10 mpa are potentially dangerous since they may cause enamel fractures during bracket debonding . however , it is difficult to establish a numerical value because these values depend on several factors , such as the condition of the substrate , the material involved , the devices chosen for the test , cure time , and the location where force is applied to the bracket . due to the difficulty of comparing and extrapolating the results obtained from in vitro studies to the clinical situation , it remains unknown what minimum bond strength values are required for safe clinical performance of orthodontic bonding procedures . thus , it would be useful to determine the bracket bond strength values in vivo , by using a device to measure the bracket debonding force directly in the oral cavity . the ari scores showed that the fracture pattern was predominantly score 3 in both groups . the composite resin remained completely bonded to the enamel after bracket debonding , which is advantageous since score 3 has the least probability of harming the enamel . therefore , debonding with the prototype was shown to be a safe method for preserving the enamel surface .

abstractintroduction : material biodegradation that occurs in the mouth may interfere in the bonding strength between the bracket and the enamel , causing lower bond strength values in vivo , in comparison with in vitro studies . objective : to develop a prototype to measure bracket debonding force in vivo and to evaluate , in vitro , the bond strength obtained with the prototype.methods : a original plier ( 3 m unitek ) was modified by adding one strain gauge directly connected to its claw . an electronic circuit performed the reading of the strain gauge , and the software installed in a computer recorded the values of the bracket debonding force , in kgf . orthodontic brackets were bonded to the facial surface of 30 bovine incisors with adhesive materials . in group 1 ( n = 15 ) , debonding was carried out with the prototype , while tensile bond strength testing was performed in group 2 ( n = 15 ) . a universal testing machine was used for the second group . the adhesive remnant index ( ari ) was recorded.results : according to student s t test ( = 0.05 ) , group 1 ( 2.96 mpa ) and group 2 ( 3.08 mpa ) were not significantly different . ari score of 3 was predominant in the two groups.conclusion : the prototype proved to be reliable for obtaining in vivo bond strength values for orthodontic brackets .

pubmed

trichomonas vaginalis infection is one of the most common curable sexually transmitted infections . who estimates that 173 million of new cases occur annually . trichomonal infection has been associated with adverse reproductive health outcomes including pelvic inflammatory disease , low infant birth weight and premature delivery [ 36 ] . furthermore , trichomoniasis has been shown to increase transmission and acquisition of other sexually transmitted diseases , including hiv infection [ 1 , 8 , 9 ] . the frequency of trichomonal infection is not monitored in most countries nor do control programs exist . peruvian sti guidelines do not include trichomonas diagnostic testing ; the syndromic management of suspected vaginal trichomoniasis is the current standard for clinical care . trichomonal infection is most likely more common among women and men than is generally understood , and long standing , asymptomatic infections may lead to continued community level transmission . as trichomonas prevalence depends on many factors such as age , sexual activity , and the number of sex partners , a better understanding of the epidemiology of this infection in women is needed to foster disease control programs in populations at risk for reproductive health complications . this study was a cross - sectional analysis of the behavioral and biological data from the baseline assessment of a cohort subsequently enrolled in the nimh collaborative hiv / std prevention trial collected from 2003 to 2005 in peru . we enrolled participants from low - income neighborhoods in three coastal peruvian cities : lima , trujillo , and chiclayo . we chose neighborhoods based on data from the peruvian national institute of statistics and informatics . socially marginalized women were characterized through informal interviews and participant observation , and based on ethnographic information collected prior to the study baseliness were shown to have high levels of sexual risk behaviors . we defined socially marginalized women as those women living in low - income neighborhoods who contradicted peruvian social norms by spending time with and having sex with poor unemployed men . most of these women were also unemployed , and although the majority reported a stable sex partner , most of them also reported having multiple sex partners , further defying the social norms of peru . eligible women were recruited by outreach workers from venues such as volleyball fields , hair salons , parks , bars , and street corners . the number of venues selected per city was approximately proportional to the city 's overall population . eligible participants were restricted to women between 18 and 40 years of age who reported having had sex in the past six months , intended to stay in the neighborhood for at least two years , and who frequented the target venue at least twice a week . participants were invited to a temporary project office in their neighborhood , where we privately administered a 30-minute structured survey in spanish , wherein trained interviewers read questions to participants and entered their answers into a portable computer . the survey instrument collected sociodemographic information as well as detailed sexual risk behavior with the last five sex partners , number of sex partners , sti - related symptoms , alcohol and drug , use and hiv status . upon completing the survey , participants underwent pretest counseling for stis including hiv infection , conducted by a trained counselor . trained laboratory technicians then explained to each participant how to self - collect a vaginal specimen using a dacron swab . a second dacron swab was stored for chlamydia trachomatis and neisseria gonorrhoeae pcr testing ( ct / ng amplicor pcr , roche diagnostics , nj , usa ) , and a blood sample was also collected for hiv-1 ( genetic systems bio - rad laboratories , hercules , calif , usa ) , herpes virus simplex type 2 antibodies ( herpeselect ; focus technologies , cypress , calif , usa ) , and syphilis testing ( rprnosticon , shield diagnostics , dundee , uk and serodia - tppa , fujirebio diagnostics inc , tokyo , japan ) . participants returning for results underwent posttest counseling to ensure their understanding of the meaning of both positive and negative results and for treatment in case of a positive result . treatment for trichomoniasis consisted of 2 g , single dose metronidazole by mouth administered by trained health personnel . all participants with positive results were encouraged to share test results with recent sex partners . reimbursement for travel costs and time spent during the assessment was given to each participant at the rate of 15 peruvian soles ( approximately $ 5 ) at their first visit and an additional 10 peruvian soles ( approximately $ 3 ) at their posttest visit . all vaginal swab specimens were immediately inoculated and transported at ambient temperature using the in pouch tv culture media ( biomed diagnostics , white city , ore , usa ) to the cayetano heredia university sexual health laboratory in lima , chiclayo , or trujillo . cultures were transported to the laboratory within 68 hours after sample collection and incubated at 37c once they arrived at the laboratory . to monitor the performance of test and as part of the quality control of the study , known positive samples were inoculated in the field , transported , and cultured under the same conditions . all samples , including the controls , were observed microscopically at 400x power for the presence of motile , oval flagellated protozoan daily for 5 days . the observation of motile trichomonads was reported as positive , and specimens showing no organisms after 5 days of incubation were considered negative for t. vaginalis . laboratory work was performed in accordance with the college of american pathology quality assurance and quality control program . as odds ratios overestimate the estimated risk when the prevalence is high , we calculated prevalence ratios to avoid that bias . to model the prevalence ratios we used poisson family generalized linear models and adjusted the standard errors to account for the clustered nature of sample . multivariate models were created taking into account all variables that were significant at p - value < .1 in bivariate analysis . the variables added to the multivariate model were selected based on likelihood ratio tests comparing nested to saturated models and included only if the likelihood ratio test had a p - value < .05 . the study was approved by the committee on human research of the university of california , san francisco ; university of california , los angeles ; naval medical research center detachment and cayetano heredia university of peru . we collected data only from participants that gave written informed consent to participate in the study . between 2003 and 2005 , we recruited and assessed 308 women from 20 low - income communities in coastal peru . the overall prevalence of t . women with t. vaginalis infection were more likely to be older ; the highest prevalence was found in the oldest age group , 3640 years ( figure 1 ) . those with less education were 3 times more likely to have trichomonal infection compared to those who completed high school , and being single the prevalence of trichomonal infection among women with vaginal discharge and painful urination in the prior six months was lower than the prevalence among those who did not report vaginal discharge during the same period , although the difference was not statistically significant . women with hsv-2 infection and syphilis infection were twice as likely to have trichomonal infection . in the multivariate model , because only the number of partners in the past 6 months significantly improved the model , no other variables were entered into the model . vaginalis infection among socially - marginalized young women from low - income communities in coastal peru . t. vaginalis infection occurred more often among older women ( see figure 1 ) , women who had not completed high school , single women , women who reported unprotected sex with a nonsteady partner , and women who reported multiple sex partners . no association was found between any vaginal symptoms and t. vaginalis . while most epidemiological studies on trichomonal infection have been focused on pregnant women , adolescents , and female sex workers accessing care , there are few studies at the population - level and in particular , socially - marginalized women [ 5 , 1519 ] . our findings are important because such hard to reach populations are often under - represented because they are not seeking health care and represent a high - risk target population and potential core population that maintain endemic trichomonal infections at the population level . in latin america trichomonal prevalence has varied from 2.9% to 16.5% , where the highest prevalence was found in population samples in the jungle and the highlands of per , and the lower prevalences were found in clinical settings such as family planning clinics , pharmacies , and obstetrics services . in general , tested populations in those studies were most often symptomatic women [ 15 , 20 , 21 ] . the association between higher prevalence of trichomonal infection and older age is unusual for nonviral sexually transmitted infections . the increased prevalence of trichomonal infection in older women suggests long - term prevalent infection that does not spontaneously resolve and would be missed by screening programs focused on younger age women . the prevalence of trichomonal infection was not different between women with or without presence of recent painful urination or vaginal discharge . that finding suggests that syndromic management of t. vaginalis may result in both over and under treatment . the asymptomatic nature of trichomonal infection also could contribute to a long duration of infection and continued spread of infection in the population [ 1 , 7 , 23 ] . our data , and others [ 4 , 21 , 24 ] , attest to the asymptomatic nature of t. vaginalis and the need for screening and treatment programs to control the spread of trichomonal infection . results from other peruvian studies as well as the results in the present study indicate that there is no direct relationship between t. vaginalis infection and symptoms making syndromic treatment unlikely to impact the population burden of infection [ 20 , 21 ] . first , the sample size was small resulting in limited power to assess multivariate models between demographic and behavioral characteristics and t. vaginalis . furthermore , the results presented here may not be generalizable to other female populations . while response bias is always a concern , we believe that the use of standardized interviews by trained staff helped to minimize social desirability and other information biases . importantly as all epidemiologic information was collected prior to testing , there was no reason to suspect differential bias associated by trichomonal infection . selection bias was also a concern , but because most women were asymptomatic we do not believe that those with potential trichomonal infection were more likely to participate compared with those without infection . since t. vaginalis is a common sexually transmitted pathogen , the screening and treatment of sex partners of infected women must be also prioritized as a public health measure to prevent reinfection and reduce infection prevalence and complications in untreated males [ 23 , 25 ] . as the majority of the assessed population in this study was in a fertile age range , our findings support the need for improved disease control activities to reduce adverse trichomoniasis - associated reproductive health outcomes such as ectopic pregnancy , low infant birth weight , preterm labor , and non - hpv associated cervical neoplasia [ 14 , 26 , 27 ] . as t. vaginalis is widely prevalent and one of the easiest infections to treat with an inexpensive antimicrobial , further studies are needed to determine if screening and treatment of trichomoniasis will improve the reproductive health and birth outcomes of women .

objective . the epidemiology of trichomonas vaginalis infection among sexually active socially - marginalized women in three urban , coastal peruvian cities was examined in order to quantify the prevalence of trichomonas infection and identify associated risk factors . methods . we conducted a cross - sectional , venue - based study of women from socially - marginalized populations in three coastal peruvian cities . results . among the 319 women enrolled , the overall prevalence of trichomonal infection was 9.1% ( 95% ci , 5.9%12.3% ) . the mean age was 26.3 years , and 35.5% reported having had unprotected intercourse with nonprimary partners and 19.8% reported two or more sex partners in the last three months . trichomonal infection was associated with increased number of sex partners ( pr 2.5 , 95% ci 1.44.6 ) and unprotected sex with nonprimary partner in the last three months ( pr 2.3 , 95% ci 1.14.9 ) . conclusions . a moderately high prevalence of trichomonal infection was found among women in our study . trichomonal infection was associated with unprotected sex and multiple sex partners . efforts to control the continued spread of trichomonal infection are warranted .

pubmed

lennox - gastaut syndrome ( lgs ) , one of the catastrophic epilepsies of childhood , is classified by the international league against epilepsy as a symptomatic generalized epilepsy syndrome . originally described in 1966 , this tonic seizures ( especially during sleep ) , atonic ( astatic or drop attacks ) seizures , and atypical absence seizures are most commonly observed ; some patients also develop myoclonic , generalized tonic - clonic , or partial seizures . the second feature required by the definition is an interictal awake electroencephalogram ( eeg ) pattern consisting of slow spike wave discharges ( less than 3 hz ) , usually with a generalized distribution . another characteristic eeg feature is paroxysms of low voltage fast activity at about 10 hz during sleep . the third component of the definition of lgs is cognitive impairment involving moderate to severe mental retardation and behavioral disorders including aggression and autistic features . over 75% of children with lgs these include numerous congenital or acquired etiologies , such as cortical maldevelopment , perinatal hypoxia - ischemia , cns infection , or neurometabolic disorders . about 20% of children with lgs have prior infantile spasms ( west syndrome ) and evolve into lgs with age . the typical age of onset of lgs is between 2 and 5 years ; boys are affected about 5 times more often than girls.5 the prognosis of lgs is poor , with regard to both seizures and cognitive outcome . risk factors for a poor cognitive prognosis include symptomatic etiology , history of nonconvulsive status epilepticus , prior infantile spasms , and early age of seizure onset.6 due to the encephalopathic nature and multiple seizure types , lgs is notoriously difficult to treat.7 many drugs reduce seizures initially , only to lose effectiveness over time . children often end up on polypharmacy with numerous anticonvulsants , which adds to the cumulative side effects and drug drug interactions.3 furthermore , the seizures themselves are thought to contribute to the cognitive impairment and behavioral comorbidities . difficulties in diagnosing lgs are discussed in detail in a recent review.4 sometimes the classic clinical and eeg features are not present at the onset of the syndrome . due to the heterogeneous causes , for example , it can be difficult to differentiate between spasms and tonic seizures and to identify and quantify atypical absence seizures accurately . systematic difficulties complicate performance of drug trials in lgs , including the very frequent occurrence ( often nearly uncountable ) of atypical absence seizures , the inaccuracy of parental reports of seizure semiology and frequency , the wide range of etiologies , and the evolution of seizure types over time . a few randomized , double blind , placebo - controlled trials of single agents have been performed in lgs.5,7 felbamate,8 lamotrigine,9 and topiramate10 reduced the occurrence of atonic and tonic - clonic seizures in children with lgs . all of these studies entailed addition of the study drug to other medications , and the studies varied considerably in their experimental design and patient selection criteria . no head - to - head trial comparing more than one drug has been published . even with the new generation of anticonvulsants , valproic acid is considered the most useful initial medication of choice for drop attacks , atypical absences and myoclonic seizures in lgs.5,11,12 although there are no controlled studies , valproic acid is reportedly effective against multiple lgs seizure types including atypical absence , myoclonic , and other situations in which slow spike - wave discharges are found on eeg . caution must be exercised in using valproic acid in children under the age of 2 years , especially if they are receiving several other anticonvulsants , because of the risk of hepatotoxicity . in that age group , it has been recommended to try topiramate or lamotrigine first.11 felbamate could also be considered as an alternative to valproic acid , because felbamate lacks the sedative side effect seen with other anticonvulsants ( eg , topiramate , benzodiazepines ) , which exacerbates seizure occurrence . owing to the risk of aplastic anemia and hepatoxicity with felbamate use , this medication must be used with caution and appropriate patient monitoring of blood levels , liver function , and hematologic indices . a recent study showed that clobazam significantly reduced both drop and non - drop seizures in a dose - dependent manner in patients with lgs.13 clobazam reportedly has less sedative effects than other benzodiazepines , making it an attractive potential adjunctive treatment for lgs . an animal model of lgs does not exist , hampering progress in design of therapeutics . it is not surprising that there is no experimental model , since lgs comprises so many distinct seizure types and lacks a consistent underlying etiology . treatment of rats with a cholesterol synthesis blocker produces atypical absence seizures with slow spike waves , providing an opportunity to study the mechanism of this specific seizure type , which appears to involve gabab receptors.14 these observations have not yet been exploited therapeutically . some general treatment considerations include recommendations to use as few anticonvulsants concurrently as possible to avoid side effects from polytherapy , avoid excessive drowsiness which exacerbates several of the seizure types in this syndrome , and consider the cognitive and psychological comorbidities which result from both lgs and its treatment . clearly , a multidisciplinary approach is required to address the medical and psychosocial aspects of lgs . at present , authorities recommend valproic acid as the first line medication , followed by one or two of the second - line agents ( lamotrigine , rufinamide , topiramate , clobazam , felbamate , levetiracetam).5,12 if those therapies fail to achieve treatment goals , zonisamide , the ketogenic diet , vagus nerve stimulation , or corpus callosotomy can be tried ( the latter targeting drop attacks ) . of note , carbamazepine and gabapentin can exacerbate some of the seizure types in lgs.11,15 the role of newer anticonvulsants such as vigabatrin and zonisamide remain undetermined in lgs . rufinamide [ 1-(2,6-difluoro - phenyl)methyl-1h-1,2,3-triazole-4-carboxamide ] is a triazole derivative granted orphan drug status for the adjunctive treatment of lgs in the united states in 2004 . , rufinamide was approved by the united states food and drug administration for treatment of lgs in children 4 years of age and older . it is also approved for adjunctive treatment of partial seizures in adults and adolescents.16 rufinamide is structurally unrelated to other anticonvulsants . its mechanism of action reportedly involves decreased firing of high frequency sodium - dependent action potentials and prolongation of sodium channel inactivation.17 enhancing sodium channel inactivation would prevent a neuron from generating subsequent bursts of high frequency action potentials . however , data implicating an effect of rufinamide on sodium channels is presently published only in abstract form and , given the preclinical profile , other mechanisms of action are likely to operate.18 rufinamide has a wide spectrum of anticonvulsant effectiveness in animals including the maximal electroshock model ( for generalized tonic - clonic and partial seizures ) and the subcutaneous pentylenetetrazol model ( for clonic seizures).19 in support of the hypothesized action of rufinamide on sodium channels , seizure protection in those in vivo models is also afforded by other anticonvulsants that block sodium channel function . rufinamide also blocks seizures induced by subcutaneous strychnine , bicuculline , and picrotoxin.20 rufinamide has an excellent safety profile in animals and lacks obvious cognitive side effects and behavioral toxicity at clinically relevant doses . on the rotorod test of motor coordination , rufinamide had a higher safety index than phenytoin , phenobarbital , and ethosuximide.19 the drug also appears to be relatively devoid of cognitive side effects in humans . in a multicenter study of 189 adolescent and adult patients with partial seizures , 12 weeks of treatment with rufinamide failed to cause deficits ( compared to placebo ) in several measures of cognitive function , including psychomotor speed and alertness , processing speed , and working memory.21 taken orally , rufinamide is well absorbed ( 85% after an oral dose).22 the absorption rate is slow and the extent of absorption decreases as the dose is increased . after a single 400 mg oral dose in healthy adults , the time to maximum plasma concentration ranges from 1.5 to 10 hours with an average of about 6 hours , with a mean maximum plasma concentration ( cmax ) of 3.03 g / ml.23 rufinamide has low protein binding ( 34% ) and food does not affect the time to maximum plasma concentration or peak plasma concentration.22 the plasma half life is 6 to 10 hours and is unaffected by renal disease . rufinamide is eliminated primarily via metabolism , the principal metabolite being a carboxylic acid derivative . this metabolite primarily appears in the urine , and only about 2% of rufinamide occurs in the urine unchanged . the low plasma binding rate of rufinamide suggests that drug drug interactions are likely to be minimal . however , in conditions like lgs , in which polypharmacy is usual , careful elucidation of drug drug interactions is necessary . in all of the clinical trials with rufinamide , , there were no significant effects of rufinamide on the plasma concentrations of valproic acid , lamotrigine , or topiramate.24 there is no study of the effects of rufinamide on felbamate concentration . at average steady state concentrations of rufinamide , this comparison includes population pharmacokinetic analyses of carbamazepine , lamotrigine , phenobarbital , phenytoin , and valproic acid . however , phenytoin clearance was decreased at average steady state levels of rufinamide up to 21% , suggesting that phenytoin levels should be closely monitored in patients on concurrent rufinamide . the clearance of rufinamide is not significantly affected by several other anticonvulsants including carbamazepine , phenytoin , primidone , and phenobarbital . however , rufinamide clearance was decreased by valproic acid with elevated rufinamide levels of up to 70%.20 therefore , valproic acid therapy should be introduced cautiously and at relatively low doses in children already on rufinamide . early sponsored studies , including open label extensions , provided data that rufinamide had a beneficial effect as add - on therapy for partial seizures in adolescents and adults . for lgs , a single double - blind randomized placebo controlled trial has been published.24 this study involved 138 patients between the ages of 4 and 30 years who had lgs diagnoses for a median of 7.5 years . study subjects were randomized to either oral rufinamide ( titrated up to 45 mg / kg / day over 14 days ) ( n = 74 ) or placebo ( n = 64 ) in addition to their other antiepileptic drugs . this study involved a 28-day baseline followed by an 84-day parallel group treatment ( 14 days titration , 70 days maintenance ) . primary end points were the percent change in seizure frequency and the parent / guardian ratings of seizure severity . the investigators found a significant decrease in total seizures compared with placebo ( 32.7% vs 11.7% : p = 0.0015 ) , and tonic - atonic seizures compared with placebo ( 42.5% vs + 1.4% : p < 0.0001 ) . the patients on rufinamide also had a greater improvement in seizure severity and a higher 50% responder rate compared with placebo for both total seizures and tonic - atonic seizures . adverse effects in this study were modest , with 24% of treated patients experiencing sedation and 21% experiencing vomiting . eight percent of the rufinamide group withdrew because of adverse side effects . cognitive or psychiatric adverse events were less common among rufinamide - treated patients than in the placebo group . it was concluded that rufinamide is well tolerated and efficacious for seizures in lgs . a 3-year open - label followup study , published in abstract form only , reports continued rufinamide effectiveness.25 that study included 124 patients treated for a median of 432 days at a dose of 10 to 45 mg / kg / day . compared to the placebo group , there was a decrease in total seizures at all time points assessed up to 3 years , ranging from 42.6% to 79.3% . furthermore , rufinamide was well tolerated over the long term , with only 12 patients discontinuing the drug because of adverse side effects ( most commonly vomiting , pyrexia , and somnolence ) . a recently published study from europe , using observational retrospective data from multiple centers , examined the effectiveness of rufinamide in children and adults with refractory epilepsy , including lgs.26 in the subgroup with lgs , 17 of 31 patients ( 55% ) had a response rate with greater than 50% reduction in countable seizures . investigators found fatigue , vomiting , anorexia in 10% to 20% of patients but no serious adverse effects . again , this study concludes that rufinamide is effective and well tolerated in patients with refractory epilepsy , including lgs . although promising as a novel anticonvulsant in lgs , several caveats arise regarding rufinamide . in part , issues relate to the study populations . as discussed above , patients with lgs are notoriously difficult to treat , are often treated with multiple anticonvulsant medications , and seizures are of several different types and difficult to monitor and count . therefore , the question arises as to what place rufinamide will hold in overall treatment algorithm for lgs . however , a larger number of patients must be treated with this medication to establish its role in the treatment armamentarium . the role of this new medication in relationship to prior drugs with some reported effectiveness in lgs needs to be further defined . taken orally , rufinamide is well absorbed ( 85% after an oral dose).22 the absorption rate is slow and the extent of absorption decreases as the dose is increased . after a single 400 mg oral dose in healthy adults , the time to maximum plasma concentration ranges from 1.5 to 10 hours with an average of about 6 hours , with a mean maximum plasma concentration ( cmax ) of 3.03 g / ml.23 rufinamide has low protein binding ( 34% ) and food does not affect the time to maximum plasma concentration or peak plasma concentration.22 the plasma half life is 6 to 10 hours and is unaffected by renal disease . rufinamide is eliminated primarily via metabolism , the principal metabolite being a carboxylic acid derivative . this metabolite primarily appears in the urine , and only about 2% of rufinamide occurs in the urine unchanged . the low plasma binding rate of rufinamide suggests that drug drug interactions are likely to be minimal . however , in conditions like lgs , in which polypharmacy is usual , careful elucidation of drug drug interactions is necessary . in all of the clinical trials with rufinamide , , there were no significant effects of rufinamide on the plasma concentrations of valproic acid , lamotrigine , or topiramate.24 there is no study of the effects of rufinamide on felbamate concentration . at average steady state concentrations of rufinamide , this comparison includes population pharmacokinetic analyses of carbamazepine , lamotrigine , phenobarbital , phenytoin , and valproic acid . however , phenytoin clearance was decreased at average steady state levels of rufinamide up to 21% , suggesting that phenytoin levels should be closely monitored in patients on concurrent rufinamide . the clearance of rufinamide is not significantly affected by several other anticonvulsants including carbamazepine , phenytoin , primidone , and phenobarbital . however , rufinamide clearance was decreased by valproic acid with elevated rufinamide levels of up to 70%.20 therefore , valproic acid therapy should be introduced cautiously and at relatively low doses in children already on rufinamide . results from randomized controlled clinical trials of rufinamide are just appearing . early sponsored studies , including open label extensions , provided data that rufinamide had a beneficial effect as add - on therapy for partial seizures in adolescents and adults . for lgs , a single double - blind randomized placebo controlled trial has been published.24 this study involved 138 patients between the ages of 4 and 30 years who had lgs diagnoses for a median of 7.5 years . study subjects were randomized to either oral rufinamide ( titrated up to 45 mg / kg / day over 14 days ) ( n = 74 ) or placebo ( n = 64 ) in addition to their other antiepileptic drugs . this study involved a 28-day baseline followed by an 84-day parallel group treatment ( 14 days titration , 70 days maintenance ) . primary end points were the percent change in seizure frequency and the parent / guardian ratings of seizure severity . the investigators found a significant decrease in total seizures compared with placebo ( 32.7% vs 11.7% : p = 0.0015 ) , and tonic - atonic seizures compared with placebo ( 42.5% vs + 1.4% : p < 0.0001 ) . the patients on rufinamide also had a greater improvement in seizure severity and a higher 50% responder rate compared with placebo for both total seizures and tonic - atonic seizures . adverse effects in this study were modest , with 24% of treated patients experiencing sedation and 21% experiencing vomiting . cognitive or psychiatric adverse events were less common among rufinamide - treated patients than in the placebo group . it was concluded that rufinamide is well tolerated and efficacious for seizures in lgs . a 3-year open - label followup study , published in abstract form only , reports continued rufinamide effectiveness.25 that study included 124 patients treated for a median of 432 days at a dose of 10 to 45 mg / kg / day . compared to the placebo group , there was a decrease in total seizures at all time points assessed up to 3 years , ranging from 42.6% to 79.3% . furthermore , rufinamide was well tolerated over the long term , with only 12 patients discontinuing the drug because of adverse side effects ( most commonly vomiting , pyrexia , and somnolence ) . a recently published study from europe , using observational retrospective data from multiple centers , examined the effectiveness of rufinamide in children and adults with refractory epilepsy , including lgs.26 in the subgroup with lgs , 17 of 31 patients ( 55% ) had a response rate with greater than 50% reduction in countable seizures . investigators found fatigue , vomiting , anorexia in 10% to 20% of patients but no serious adverse effects . again , this study concludes that rufinamide is effective and well tolerated in patients with refractory epilepsy , including lgs . although promising as a novel anticonvulsant in lgs , several caveats arise regarding rufinamide . in part , issues relate to the study populations . as discussed above , patients with lgs are notoriously difficult to treat , are often treated with multiple anticonvulsant medications , and seizures are of several different types and difficult to monitor and count . therefore , the question arises as to what place rufinamide will hold in overall treatment algorithm for lgs . however , a larger number of patients must be treated with this medication to establish its role in the treatment armamentarium . the role of this new medication in relationship to prior drugs with some reported effectiveness in lgs needs to be further defined . rufinamide is a novel , broad - spectrum anticonvulsant drug with promising potential for treatment of many seizure types . in particular , the multiple seizure types seen in patients with lgs may be amenable to treatment with rufinamide . rufinamide appears to have a good safety profile and is well tolerated with minimal expected side effects . some of the most common side effects ( eg , somnolence , vomiting ) could perhaps be ameliorated by slow titration . patient and caretaker satisfaction data need to be obtained . given the devastating nature of the seizures and cognitive impairments in lgs , any treatment with an even modest benefit is a welcome addition to the therapeutic armamentarium .

objective : this review summarizes the treatment of lennox - gastaut syndrome , an intractable epileptic encephalopathy of early childhood . in particular , the review focuses on rufinamide , a recently released anticonvulsant medication with reported effectiveness in this epilepsy syndrome.methods:a systematic literature search ( pubmed ) was performed to review the existing literature pertaining to the treatment of lennox - gastaut syndrome as well as studies involving rufinamide as an anticonvulsant medication.results:the published literature to date documents a beneficial effect of rufinamide on children over 4 years old with lennox - gastaut syndrome . studies indicate a significant decrease in tonic and atonic seizure frequency as well as total seizure frequency compared to placebo - treated children . rufinamide appears to be well tolerated and a safe medication , somnolence and vomiting being the most common side effects.conclusions:rufinamide is a promising adjunctive therapy for lennox - gastaut syndrome , an intractable childhood epilepsy . to ensure its optimal effectiveness , clinicians must be familiar with the medication s clinical response profile and potential for adverse effects .

pubmed

interpopulational variation in sexual chemical signals may provide the basis for premating reproductive isolation and speciation in many animals [ 1 , 2 ] . phenotypic plasticity in sexual signals could play a key role in initial signal divergence , for example , as a way to maximize the efficiency of signals for communication in different environments [ 4 , 5 ] . these differences can be later amplified by sexual selection leading to differences in mating preferences [ 68 ] , which could preclude mating between populations ( e.g. , [ 913 ] ) , and lead to speciation processes . in many lizards , intraspecific communication and sexual selection for example , chemical compounds secreted by femoral gland of males can convey information about social status [ 1822 ] and genetic quality of a male [ 2326 ] . also , differences in chemical signals may preclude interspecific mating between related sympatric species ( e.g. , [ 27 , 28 ] ) . we hypothesized that interpopulational variations in femoral gland secretions within the same species might lead to reproductive isolation and thus promote speciation processes . the iberian wall lizard , podarcis hispanica , is a small diurnal lizard , living in rocky habitats of the iberian peninsula . molecular and morphological studies suggest that this lizard is paraphyletic and forms part of a species complex , which suggests the existence of cryptic speciation within taxa previously considered to be conspecific [ 2934 ] . chemical cues of males , mainly from the femoral gland secretions , are important in male - male interactions [ 19 , 20 , 40 ] and in female mate choice decisions [ 35 , 41 , 42 ] . also , chemical cues of females , in conjunction with coloration , elicit courtship by males . this raises the possibility that p. hispanica lizards use chemical sexual signals to discriminate between populations , which might lead to reproductive isolation ( if variation of signals is discrete or there is a barrier to gene flow ) and explain the genetic and morphological differences observed between populations . in this study , we explored the role of chemical sexual signals in interpopulational recognition between five distinct populations of iberian wall lizards in central spain . in this area , several populations inhabiting different environments live close together without geographical barriers that isolate the populations , and individuals may find each other easily [ 11 , 12 , 43 ] . however , some populations maintain clear distinct morphotypes and differ genetically [ 3234 ] , which suggests that they might be , at least partly , reproductively isolated . we hypothesized that interpopulational variations in chemical signals could allow chemosensory recognition between populations and lead to premating boundaries . to test this , we first compared the morphological characteristics of these populations , and then we analyzed whether there was variation in the composition and proportions of chemical compounds in femoral gland secretions of males by using gas chromatography - mass spectrometry ( gc - ms ) . we further conducted tongue - flick experiments to analyze whether males and females discriminated by chemosensory cues alone between scent of lizards from different populations . we hypothesized that male and female lizards could be able to recognize by chemical cues alone , and maybe prefer , the scents of individuals of their own population , which may contribute to a reduced gene flow . we expected that interpopulational differences in chemical signals of males and in population recognition abilities could suggest the probable existence of reproductive isolation and cryptic speciation between these iberian wall lizard populations . during february - march 2008 , we captured by noosing male and female p. hispanica lizards at five localities within the madrid region ( central spain ) ( figure 1 ) . three of these were localized in the northern mountain area ( fuenfra , golondrina , and pedrezuela ) , and the other two were situated in the southern plain area ( belmonte and aranjuez ) . we selected these populations because lizards clearly differ in morphology and coloration [ 43 , 44 ] . in the north , we captured lizards from a population occupying different granite rock cliffs at the edge of a pine forest in the upper part of the fuenfra valley ( 4047n , 0403w ; 1750 m altitude ; 21 males and 26 females ) , on granite rocky outcrops inside a large oak forest golondrina near cercedilla village ( 4044n , 0402w ; 1250 m altitude ; 29 males and 27 females ) , and from old stone walls near crop fields close to pedrezuela village ( 4044n , 0336w ; 800 m altitude ; 19 males and 16 females ) . in the south , we captured lizards on human buildings and walls inside a public garden of the belmonte del tajo village ( 4008n , 0320w ; 735 m altitude ; 22 males and 17 females ) and on chalk and gypsum rocks in deforested bushy hills near aranjuez village ( 4002n , 0337w ; 494 m altitude ; 21 males and 32 females ) . all lizards were individually housed at el ventorrillo field station ( cercedilla , madrid ) about 5 km from the capture sites of the northern populations , in indoor 60 40 cm pvc terraria containing sand substratum and rocks for cover . cages were heated with 40 w spotlights during 6 h / day , and overhead lighted ( 36 w full - spectrum daylight tubes ) on a 10 h : 14 h light / dark cycle , and were screened from each other using cardboard . every day , lizards were fed mealworm larvae ( tenebrio molitor ) dusted with multivitamin powder for reptiles , and water was provided ad libitum . lizards were returned to their exact capture sites with good health condition at the end of experiments . we made the following morphological measurements of each individual lizard : body mass ( or weight ) ( measured with a digital balance to the nearest 0.01 g ) and body size ( snout - to - vent length , svl ; measured with a ruler to the nearest 1 mm ) . we also made morphological measurements of the head using a digital caliper ( to the nearest 0.05 mm ) . head length was the distance between the tip of the snout and the posterior side of the parietal scales . head depth was the greatest distance from the highest portion of the head to the bottom of the lower jaw . we also counted under a magnifying glass the number of femoral pores on the right and left hindlimbs of lizards and calculated an average number for both sides . finally , we noted the number of small but distinctive and conspicuous blue ocelli that runs along each of the body sides on the outer margin of the belly of males and calculated an average number for both sides . these ocelli seem to have a role in sex recognition and intrasexual social relationships between males [ 39 , 45 ] . all biometrical variables were log transformed prior to analysis to meet assumptions of normality and homoscedasticity . we used one - way analyses of variance ( anovas ) to test for differences in morphological variables between populations . immediately after capture in the field , we extracted femoral gland secretion of males by gently pressing with forceps around the femoral pores and collected secretion directly in glass vials with teflon - lined stoppers . we also used the same procedure on each sampling occasion but without collecting secretion , to obtain blank control vials that were treated in the same manner to compare with the lizard samples . before the analyses we added 250 l of n - hexane ( sigma , capillary gc grade ) to each vial . we analyzed lipophilic compounds in samples by using a finnigan - thermoquest trace 2000 gas chromatograph ( gc ) fitted with a poly ( 5% diphenyl/95% dimethylsiloxane ) column ( thermo fisher , trace tr-5 , 30 m length 0.25 mm i d , 0.25 mm film thickness ) and a finnigan - thermoquest trace mass spectrometer ( ms ) as detector . sample injections ( 2 l of each sample dissolved in n - hexane ) were performed in splitless mode using helium as the carrier gas at 30 cm / sec , with injector temperature at 250c . the oven temperature program was as follows : 50c isothermal for 5 min , then increased to 270c at a rate of 10c / min , isothermal for 1 min , then increased to 315c at rate of 15c / min , and finally isothermal ( 315c ) for 10 min . initial tentative identification of secretion components was done by comparison of mass spectra in the nist / epa / nih 1998 computerized mass spectral library . identifications were confirmed by comparison of spectra and retention times with those of authentic standards from sigma - aldrich chemical co. for unidentified or unconfirmed compounds we report here their characteristic ions , which we used together with retention times and characteristic m / z ratios to confirm whether these compounds were present in a given individual . for the statistical analyses of femoral secretions , the relative amount of each component was determined as the percent of the total ion current ( tic ) . the relative areas of the peaks were transformed following aitchison 's formula [ 4749 ] . the homogeneity of variance of these variables was tested with levene 's test , and bonferroni 's correction was applied . the transformed areas were used as variables in a principal component analysis with varimax rotation . the eight principal components ( pcs ) extracted ( all with eigenvalues > 1 , which explained 82.55% of variance ) were used as covariates in a discriminant analysis to test whether chemical compounds in femoral secretions could be used to predict the population of origin of a male lizard . then , we calculated the squared mahalanobis distances of individuals with all other individuals and compared them between populations . lizards have been shown to react to a variety of chemical stimuli with increased and differential rates of tongue extrusions . tongue - flick ( tf ) rate can , therefore , be used as a quantitative bioassay of detection of chemical cues ( e.g. , [ 11 , 38 ] ) . thus , to test for differential responses to scents , we made comparisons of tf rate by lizards ( males and females ) in response to chemical stimuli presented on cotton applicators impregnated with scents of male or female p. hispanica from each of the five different populations ( aranjuez , golondrina , fuenfra , pedrezuela , and belmonte ) or with deionized water ( odorless control ) . we obtained lizard scents from the femoral pores of males or from the cloacal area of females because these are the body areas most frequently and intensely investigated by tongue flicking during social encounters [ 19 , 37 , 39 ] . therefore , after first dipping the cotton tip ( 1 cm ) of a wooden applicator attached to a long stick ( 50 cm ) in deionized water , we rolled the tip over those body areas ( of one population and sex per applicator ) . first , males were exposed to scents from males and then to scents from females of each population tested . trials were conducted in outdoor conditions during april , which coincided with the mating season of lizards in their original natural populations ( p. lpez and j. martn , unpublished data ) , and between 11:00 and 13:00 ( gmt ) when lizards were fully active . to begin a trial , the experimenter slowly approached the terrarium and slowly moved the cotton swab to a position 1 cm anterior to the lizards ' snout . lizards usually did not flee from the swab , but explore it repeatedly by tongue flicking or ignore it after the first tfs . in all cases , lizards directed tfs to the swab in all conditions . the numbers of tfs directed at the swab were recorded for 60 s beginning with the first tf . to examine differences among treatments , previous analyses showed that responses to the different scents differed as a function of the population of the responding lizard . thus , we used separated one - way repeated measures anovas to test the effect of scent stimuli ( within factor ; fuenfra versus golondrina versus aranjuez versus pedrezuela versus belmonte versus water ) in number of tfs directed at the swab within each population of responding lizards . data were log - transformed to ensure normality . tests of homogeneity of variances ( levene 's test ) showed that in all cases variances were not significantly heterogeneous after transformation . there were significant differences between populations in all morphological measurements ( table 1 ) . in general , lizards from fuenfra and golondrina populations were significantly heavier and longer and had greater heads than lizards from aranjuez and belmonte , which did not differ . lizards from pedrezuela were intermediate in size between the other populations ( table 1 ) . however , when the effect of variation in body size between populations was removed , head size differences were significant only for head depth ( anova on residuals of head size with svl , p = 0.005 for both sexes ) , but not for head length ( p > 0.20 for both ) or width ( p > 0.05 for both ) . with respect to the number of femoral pores , both male and female lizards from aranjuez had significantly less femoral pores than lizards from belmonte , fuenfra , and golondrina , which did not differ . the number of femoral pores was not significantly related to body size ( p > 0.60 in all cases ) . finally , males from aranjuez , belmonte , and pedrezuela had significantly more blue ocelli than males from fuenfra and golondrina ( table 1 ) . we found 53 lipophilic compounds in femoral gland secretions of male p. hispanica ( table 2 ) . the lipophilic fraction of femoral gland secretions of males , all five populations pooled , is a mixture of steroids ( 83.69% of tic ) , and carboxylic acids ranged between n - c14 and n - c22 and their esters ( 10.30% ) , but we found also five alcohols between n - c16 and n - c24 ( 3.53% ) , a furanone ( 1.18% ) , four waxy esters ( 1.10% ) , squalene ( 0.60% ) , and two terpenoids ( 0.28% ) . on average , the five most abundant chemicals were cholesterol ( 63.24% of tic ) , followed by cholesta-5,7-dien-3-ol ( 5.16% ) , hexadecanoic acid ( 3.73% ) , campesterol ( 3.66% ) , octadecenoic acid ( 2.46% ) , and octadecanoic acid ( 1.77% ) . there were 34 chemical compounds shared by lizards from all populations , but we found differences between populations in the presence / absence of 19 compounds in femoral secretions ( table 2 ) . the discriminant analysis showed that the eight pcs scores describing proportions of compounds in femoral secretions could be used to predict the population of origin of a male lizard ( wilks ' = 0.0001 , f32,355 = 607.45 , p < 0.0001 ) ( figure 2 ) . all the pairwise comparisons of the mahalanobis distances between populations , which ranged between 150.35 and 1015.13 , were significant in all cases ( 210.46 < f8,96 < 1290.20 , p < 0.0001 in all cases ) . the number of tfs differed significantly between the scents presented in all cases ( table 3 ; figure 3 ) . in all populations , males discriminated between scents of any male and water ( tukey 's tests : p < 0.005 in all cases ) . males from aranjuez and belmonte directed a significantly higher number of tfs to scent of males of their own population or of the other southern population than to scent of males from the three northern populations , which did not differ ( table 3 ; figures 3(a ) and 3(b ) ) . the number of tfs directed by males from fuenfra was significantly higher in response to scent of males of their own population than to scent of males from any other population , which did not significantly differ ( table 3 ; figure 3(c ) ) . males from golondrina directed a significantly higher number of tfs in response to scent of males of their own population than to males from aranjuez , belmonte and pedrezuela ( table 3 ; figure 3(d ) ) . the number of tfs in response to scent of males of their own population and fuenfra males was not significantly different , and the latter was not significantly different from the rest of populations . finally , males from pedrezuela directed significantly more tfs in response to males of their own population than to males of the two southern populations ( aranjuez and belmonte ) , which did not significantly differ ( table 3 ; figure 3(e ) ) . however , responses to males of their own population did not significantly differ from responses to males of the other two northern populations . the number of tfs differed between treatments in all populations ( table 3 ; figure 4 ) . in all cases , males discriminated between scents of any female and water ( tukey 's tests : males from aranjuez and belmonte directed a significantly higher number of tfs to scent of females of their own population than to scent of females from all the northern populations , which did not significantly differ ( table 3 ; figures 4(a ) and 4(b ) ) . the number of tfs directed by males from fuenfra was significantly higher in response to scent of females of their own population than to females from any other population ( table 3 ; figure 4(c ) ) . males from golondrina directed a significantly higher number of tfs in response to scent of females from the three northern populations , including their own population , than to females from the two southern populations ( table 3 ; figure 4(d ) ) . males from pedrezuela directed significantly more tfs in response to scent of females of their own population than to scent of females from any other population ( table 3 ; figure 4(e ) ) . however , responses to scent of females from the two other northern populations were significantly higher than to females from the two southern populations , which did not differ . the number of tfs differed between treatments in all populations ( table 3 ; figure 5 ) . all females discriminated between scents of any male and water ( tukey 's tests : p < 0.005 in all cases ) . females from aranjuez and belmonte directed a significantly higher number of tfs in response to scent of males of their own population than to males from the three northern populations , which did not differ significantly ( table 3 ; figures 5(a ) and 5(b ) ) . females from aranjuez and belmonte did not significantly differ in their responses to scent of males of their own population or to males from the other southern population ( belmonte or aranjuez ) . the number of tfs directed by females from fuenfra was significantly higher in response to scent of males of their own population than to males from the two southern populations and from one of the northern populations ( pedrezuela ) , which did not significantly differ ( table 3 ; figure 5(c ) ) . responses to scent of males of their own population and to males from golondrina were not significantly different , nor were different the responses to males from golondrina and pedrezuela . females from golondrina directed a significantly higher number of tfs in response to scent of males from the three northern populations ( fuenfra , pedrezuela , and their own population ) than to scent of males from the southern populations ( aranjuez and belmonte ) ( table 3 ; figure 5(d ) ) . females from pedrezuela directed significantly more tfs in response to scent of males from their own population than to males from all the other southern and northern populations , which did not differ significantly ( table 3 ; figure 5(e ) ) . our study showed that different populations of iberian wall lizards p. hispanica living within a relatively small geographical area , whose environmental conditions differed between population sites , differed in morphology and in the composition and proportion of chemical compounds in femoral gland secretions of males . males of each population secreted a singular and characteristic mixture of compounds used as sexual signals . tongue - flick tests showed that these differences resulted in differential chemosensory recognition between some populations . these results suggested that there could be premating reproductive isolation between some , but not all , populations of this lizard . with respect to morphology , we could first differentiate between individuals from the south and north of the study area . lizards from fuenfra and golondrina ( i.e. , northern populations ) were characterized by being larger , heavier , and with larger , more robust heads than individuals from aranjuez and belmonte ( i.e. , southern populations ) . these differences could be explained by the different contrasting environments where these populations live , northern mountains ( with cold temperature , high humidity , and high altitude ) versus southern plains ( hot temperatures , dry conditions , and low altitude ) . variations of body size of many animals , and in particular of vertebrates , are often explained by phenotypic plasticity or local adaptation to different climatic conditions , with individuals from colder environments being larger than those from warmer areas ( e.g. , ) . lizards with a large body size have low thermal inertia ( i.e. , low cooling rates ) , and this may be an adaptation to increase effectiveness of thermoregulation in the northern populations where ambient temperatures are relatively cold , in contrast to the southern populations where temperatures are warmer and lizards are smaller . moreover , iberian wall lizard populations differ in the number of femoral pores and blue spots , with males from the northern populations having more femoral pores and less blue spots than males from the southern populations . only lizards from the pedrezuela northern population had an intermediate number of femoral pores . because femoral pores and blue spots are used in chemical and visual intraspecific communication , respectively ( e.g. , [ 37 , 39 ] ) , it is likely that the importance of these two sensory modes differ between populations . a higher number of femoral pores may be related to a higher production of chemical secretions , whereas a larger number of blue spots may represent a higher use of visual signals . the relative importance of chemical and visual signals may be explained by the effectiveness of these two types of communication in different environments [ 4 , 54 ] , which might have affected the evolution of sexual signals of different populations of p. hispanica lizards . in fact , the chemical analyses showed that , similarly to other lizard species , femoral gland secretions of p. hispanica have carboxylic acids and steroids as predominant components ( reviewed in ) . however , compounds found in femoral gland secretions of male p. hispanica varied in composition and proportions between populations , and these variations alone would allow a characterization of males from each population . these differences could be due to local adaptation to the habitats of each population [ 4 , 54 ] . selection for a better efficiency of substrate scent marks might have led to differences in composition of secretions of lizards inhabiting distinct environments , with less volatile and stable molecules being found in the southern populations where temperature and evaporation rates were higher [ 4 , 11 ] . also , differences in secretions might be related to different diets or differently available food sources . the question that arises is whether these differences in chemicals affect recognition systems and whether this may have consequences for speciation . chemosensory recognition experiments showed that individuals of p. hispanica from each population could clearly detect scents of lizards from any population in comparison with an odorless control ( i.e. , water ) . however , lizards showed different tongue - flick ( tf ) rates depending on the population of origin of the lizard 's scent presented . both females and males varied in their responses to scents from lizards from the different populations . ( i.e. , a higher tf rate ) for scents from males from their own area ( i.e. , north versus south ) ; males from the northern populations made more tfs in response to scent of males from the northern populations than to scent of southern males . similarly , southern males made more tfs in response to scents from southern males than to scent from northern males . only males from fuenfra population showed a clearly higher response to scents from males of their own population . for the rest of populations , there were not higher responses to scent of males from their own population , but there was a recognition of the area of origin ( north versus south ) of the male . males from northern populations showed more interest for scents from northern females than for scents from southern females ; similarly this occurred in southern males . however , we observed one interesting difference : males from the populations that are geographically located far away from the others ( i.e. , aranjuez , fuenfra , and pedrezuela ) showed a clear discrimination and interest ( i.e. , higher tf rates ) for scents of females from their own population against scent of females from any other population . there was also a further secondary intermediate interest for scent of females from other populations of their own area and finally a lower interest for females from the other area . in contrast , for the populations geographically located in the middle of the madrid region ( i.e. , belmonte and golondrina ) , we did not observe a discrimination nor a higher interest of males for scent of females from their own population , but only a discrimination of females from their own area . in addition , we found similar results for the males ' scents recognition by females . females from northern populations made more tfs in response to scent of northern males than to southern males , and vice versa , but there were no differences between populations within each area . we found only a clearly higher interest of pedrezuela females for scents of males from their own population against all the other populations . these results seem concordant with the previous description of morphotypes of p. hispanica using morphological and genetic data [ 29 , 3234 ] . thus , northern populations would be close to those described for the morphotype 1 , while the southern populations would be more similar to the morphotype 2 . however , we observed a particular result for lizards from pedrezuela population ; these lizards live in the north , but they have chemical and morphological differences with respect to other northern populations . moreover , the chemical signals in this population are singular in comparison to the other populations , and this chemical signature is effective in the chemosensory recognition of scent of males and female from their own population . therefore , the assignation of this population to previously described morphotypes is not clear . in summary , our results showed that male and female p. hispanica lizards from five distinct populations of the madrid region can recognize and discriminate between scents of individuals from the northern and southern populations , and have more interest for scents of lizards from their own area than for scents of lizards from the other area . moreover , males from some populations discriminate and maybe prefer scents of females from their own population than from any other populations . this clear ability of males to discriminate between some female populations might suggest that there is a cryptic speciation process , probably mediated by the role of chemical signals in sexual interactions . however , we need further mating experiments to test this . in addition , females also seem to discriminate male chemicals between areas ( north versus south ) , but not between populations . all these results support that reproductive isolation between all the distinct populations of p. hispanica is not entirely clear , but that , at least between some populations , there could be reproductive isolation and cryptic speciation , which merits further studies .

interpopulational variation in sexual signals may lead to premating reproductive isolation and speciation . genetic and morphological studies suggest that the iberian wall lizard , podarcis hispanica , forms part of a species complex with several cryptic species . we explored the role of chemical sexual signals in interpopulational recognition between five distinct populations of iberian wall lizards in central spain . results showed that these populations differed in morphology and in composition and proportion of chemical compounds in femoral gland secretions of males . tongue - flick experiments indicated that male and female lizards discriminated and were more interested in scents of lizards from their own area ( i.e. , northern versus southern populations ) , but did not discriminate between all populations . moreover , only males from the populations that are geographically located more far away preferred scent of females from their own population . these data suggest that , at least between some populations , there may be reproductive isolation mediated by chemical signals and cryptic speciation .

pubmed

the scientific activity of a group of researchers is manifested through published papers , which are validated and legitimized by their peers . these papers can also serve as indicators of the degree to which their scientific knowledge in their area of research has been developed . the formal means of releasing scientific publications , which include the results of research conducted at medical education institutions , is the collection of national and international scientific journals that have been indexed into databases representing various areas of knowledge.14 there are several major international databases that contain publications from scientific journals in the area of health science : medline / pubmed , which is organized by the national library of medicine and the national institutes of health ; the web of science , which is maintained by the institute of scientific information ( isi)/thomson reuters ; embase , which is an european database ; and current contents , which is also maintained by isi . in brazil , the main databases are lilacs ( latin american and caribbean health sciences ) and scielo ( scientific electronic library online ) , which is a database allowing free access to full texts of brazilian , latin american and caribbean scientific literature.5,6 among these databases , medline / pubmed ( http://www.pubmed.gov ) , which offers free access and has more than 5,100 titles , is the most often searched . another important tool in the recovery of available indexed articles in databases is the controlled vocabulary known as medical subject headings ( mesh),7 which allows researchers to retrieve information using a more exact , standardized description of the document in question.810 international and national databases make it possible to search for an article using keywords as well as the authors last names , without considering the order of their authorship . in some cases , however , the recovery of articles and citations is compounded by the different ways that the names of authors or institutions can be entered.1113 compound last names , names with suffixes ( e.g. , neto , jr . ) or names which include de and da are often included in publications in a non - standardized way.14 in addition , different researchers from the same group frequently list their department and institution names in a non - standardized way . small details like these make it difficult for researchers to find desired articles and for institutional evaluations to be made by the government or even by their peers.12,13 the journals selected for publication also reflect the level of productivity of a research group and , consequently , the institution to which they belong . these values are calculated from the number of citations of the articles published by the journals over a two - year period.15,16 journals with high ifs are considered to be more relevant to , or influential on , the scientific field in question.17 few brazilian journals , even those that are indexed in databases , have ifs . however , this number has recently increased , from 23 in 2006 to 30 in 2007 , despite the fact that the demand for the publication of english - language articles has favored a more visible role for them within the international community.18 in addition to the absolute number of publications , institutions are also qualitatively evaluated according to certain bibliometric indicators . one such indicator is the assessment of the number of times that the scientific articles generated in a particular institution are cited by their peers.19 the evaluation of scientific production from a university is rather complex , because it involves different areas of knowledge , each one with its own peculiarities . analysis of smaller units , like university departments , disciplines or services , can be made more easily . when the number of researchers is significantly high , a raffle system is often used to choose representatives from different functional categories , including professors and non - professors . in this current investigation , our purpose was to provide a panoramic view of publications from the universidade de so paulo , faculdade de medicina , during the period of 2001 to 2006 , by analyzing the scientific production of full professors ( level ms-6 ) at this institution and subsequently verifying their impact . research into the number of publications in the medline / pubmed database by full professors of the faculdade de medicina da universidade de so paulo ( fmusp ) who were working during 20012006 was performed using a list of referred professors . ( ataad , http://fmadm.fm.usp.br/attaad)20 provided the following data , in addition to names : job position , registration , department , discipline , kind of work and dates of admission and dismissal . the inclusion criteria of the study were full professorship and employment by fmusp during the entire established period of time . some full professors were excluded because they were dismissed during this period , mainly because of retirement , and others because they were hired through recruiting held during the same period . the survey of the number of publications in medline / pubmed was first done by individually searching for the name of each author . once published articles for that author had been identified , an excel spreadsheet was created to record relevant data : the kind of authorship ( sole author , main author , co - author ) ; the bibliographical references of each retrieved article according to the year of the search ; the if of each retrieved journal ; and the citations generated by each of the published homonyms that had been used in publication , which were determined by a search of the lattes curriculum records for each full professor . in addition , complete articles that were retrieved on the internet were confirmed . after compiling the list of articles by each professor that have been indexed in the medline / pubmed database , the if of each scientific journal was determined , using the journal citation report ( jcr , 2006 edition ) . finally , the number of citations by the authors peers that have been generated by these published articles was calculated using the march , 2009 update of the web of science database . there were 66 full professors at the fmusp during the period between 2001 and 2006 , of whom 57 ( 86.4% ) were males and only 9 ( 13.6% ) were females . we verified that 36.4% of these professors were teaching full - time ( 40 hours ) , that 60.6% of them were working intermediate shifts ( 24 hours ) , and only 3% were working partial shifts ( 12 hours ) . our survey of the journal databases revealed 1,960 bibliographic records that were published in 630 international and national scientific journals . the number of published articles for each year that were retrieved on medline / pubmed are provided in table 1 , with 31.3% overall being published in national journals and 68.7% in international journals . there was a gradual growth in the scientific production of the researchers during these years , with an average publication rate of five articles per year by each professor increasing from 3.8 in 2001 to 5.9 in 2006 . with regard to the authorship of the articles , only 2% ( 31 ) of the published articles had the professors as sole authors , and 5% ( 103 ) had them as first authors . the fmusp professors were co - authors for the majority of publications ( 93% ) , which included partnerships with other researchers from the institution . of the total number of articles that were published with other collaborators , 62.3% had 4 to 7 different authors . of the 630 journals in which the articles were published , only 103 ( 16.4% ) did not have ifs listed in the 2006 edition of the jcr . just considering those articles published in international journals ( 1,347 ) , taking into account all of the articles , 47.7% ( 935 ) were printed in journals with ifs between 1 and 5 . in addition , 6.4% of the articles were published in journals with ifs between 5 and 10 , which represented 8.2% of the journals , and 1.5% of the articles were published in journals with ifs between 10 and 52 , representing 1.7% of the journals ( table 2 ) . with regard to those articles published in brazilian journals ( 613 ) , 127 ( 20% ) were published in four journals with assigned ifs : brazilian journal medical biological research ( if = 1.075 ) , arquivos de neuropsiquiatria ( if = 0.400 ) , memrias do instituto oswaldo cruz ( if = 1.209 ) and revista de sade pblica ( if = 0.343 ) ( table 3 ) . until 2004 , the journal arquivos brasileiros de cardiologia accounted for the greatest number of published articles ( 90/613 ) , followed by revista do hospital das clnicas da faculdade de medicina usp ( 65/613 ) . starting in 2005 , the journal revista do hospital das clnicas da faculdade de medicina usp , which had its title changed to clinics , accounted for 21.4% of the articles published in brazil ( 66/613 , table 3 ) . the qualitative analysis , which was based upon the number of times that an article was cited by peers , was performed using the web of science database . almost half of the total published articles ( 45% ) were cited , which amounted to a total of 9,335 citations . these citations were not only found in articles published in brazilian journals , in which 9% of the fmusp articles generated 367 citations , but also for articles published in international journals , in which 91% of the fmusp articles generated 8,968 citations . we verified that 27 of the 1,960 published articles ( 1.4% ) were cited more than 50 times by their peers , as ( table 4 ) . the present investigation aimed at providing an overview of the scientific production of the faculdade de medicina da universidade de so paulo , with a focus on a specific group of faculty members with high - level positions at the university , who were the full professors ( level ms-6 ) . the use of only one group of professors in this study provides results that can later be confirmed for the other research professors and non - professors of the university . this group was selected to be representative of the other professors , taking into consideration the level of scientific maturity achieved and especially the possibility of collaboration with other members of the same institution , as well as other national and international institutions . analysis of the publications by the 66 full professors on staff during the period of 2001 to 2006 revealed a total of 1,960 published articles . this number represents 0.04% of worldwide scientific publications , 2.4% of national publications , 4.3% of the all - time total publications of the fmusp and 12.33% of the total publications of the fmusp during the same period.21,22 of these articles , 68.7% were published in international journals , and 31.3% were published in brazilian journals . the obligatory use of the english language in a number of the brazilian journals that published the professors articles has led to a better visibility and accessibility of research results . few brazilian journals have ifs available , and only 4 of the 24 journals represented here had ifs . this number , however , represents 17.39% of the total number of brazilian journals that have assigned ifs ( 2006 jcr ) . there is , unfortunately , little data from the fmusp that would enable a comparison of the results of this study . in a paper published in 2005 , the scientific production that was generated by the laboratrio de investigao mdica do complexo hospital das clnicas da faculdade de medicina da universidade de so paulo was evaluated . the doctors , not all full professors , who represented the 62 laboratories in the hospital complex at that time published a total of 399 articles in 2004.23 a comparison of the number of publications by full professors during the same period indicates that , although the number is small ( 326 ) , it can serve as an estimate of the number of publications of the institution over a certain period . although the estimation of scientific productivity was possible for full professors , it is interesting to note that these estimates can be lower than those that were actually generated by the institution of interest . this type of data should be evaluated using different categories in order to make comparisons with other groups of professors . during this time period , this group of full professors had an average publication rate of five published articles per year per professor . records found in the anurio estatstico da universidade de so paulo , 200722 indicate that the productivity of professors and researchers from the fmusp during the period of 2003 to 2007 was 3.1 articles per teacher per year . if we consider that the number of publications per year per teacher has remained almost constant , our study shows a higher number of publications per year per professor than what was expected from the total group of teachers at the institution . approximately 89% of the articles were published in international journals with assigned ifs , which provides high worldwide visibility . however , the analysis of these ifs should take specific research areas into account , in addition to the kind of publication , because some areas have a greater number of researchers in comparison to others . basic disciplines have a great advantage over specialized ones , in that the citation practices and publications are more focused.24 considered as a whole , the articles counted in this study were published in journals with ifs that varied from 0.063 to 51.296 . for 20.7% ( 406 ) of the articles , the ifs varied from 3.015 ( am j cardiol ) to 51.296 ( new engl j med ) . for example , research in the dentistry area carried out in 2006 revealed that a large portion of dentistry publications occurred in journals with ifs varying from 0.692 to 1.569.13 with regard to citations of these published articles , 31.3% of the citations occurred in brazilian journals and 68.7% in international ones , which resulted in a total of 9,335 citations . it is noteworthy that 20 of the evaluated articles received more than 70 citations . in a recent publication of faculdade de medicina da universidade de so paulo , the collective publications of the fmusp were reported to be among the 100 most cited in the world.25,26 despite some limitations , the bibliographic record data in this study can be useful in analyzing the scientific production of a group of professors from the fmusp and for establishing a basis for comparison with other institutions in brazil . in addition , this data could possibly be used to evaluate the output of scientific production in the entire field of health .

introductionthe scientific production of institutions of higher education , as well as the dissemination and use of this published work by peer institutions , can be assessed by means of quantitative and qualitative measurements . this type of analysis can also serve as the basis of further academic actions . variables such as the type of evaluation , the number of faculty members and the decision to include or exclude researchers who are not professors are difficult to measure when comparing different schools and institutions.objectivesthe purpose of this study was to assess the scientific production of tenured faculty from the universidade de so paulo , faculdade de medicina performed from 2001 to 2006.methodsmedline/pubmed database was considered and the impact factors ( ifs - journal citation report , 2006 ) and the number of generated citations ( web of science / isi thomson ) were also evaluated.resultsthe analysis of the scientific production of 66 full professors ( level ms-6 ) revealed 1,960 scientific articles published in 630 scientific journals , of which 31.3% were brazilian and 68.7% were from international sources . among these , 47% of the articles were published in 62.9% of the journals with ifs above 10 , although 16.4% of the journals did not have assigned if values . we verified that 45% of the published articles received 9,335 citations ( average of 11 + 17 ) , with the majority of these ( 8,968 citations ) appearing in international scientific journals.conclusionsour results indicate that it is possible to analyze the scientific production of a learning institution by the number of papers published by full professors , taking into account not only their academic position and influence , but also the fact that publication is an opportunity to stimulate joint projects with other members of the same institution .

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according to the who experts , if rubella infection occurs during the early stages of pregnancy , the potential of passing the virus through the placenta and reaching the fetus is about 90% . the most important complication of rubella is congenital rubella syndrome ( crs ) , which directly relates to lack of immunity during pregnancy . on the other hand , by vaccination of all the susceptible people or women in reproductive age , or the susceptible girls at the time of marriage and induce immunity in them , it is possible to prevent the disease in mothers and its possible serious complications in their fetus . so the primary purpose of rubella vaccination is to prevent congenital infection with rubella virus and crs , which affects an estimated 110,000 infants each year in developing countries . the disease is spread globally , and before vaccination era at the northern hemisphere the maximum of its attacks had been between february and april in the countries without effective policy for childhood vaccination or those countries where the vaccination policies only targeted the females , still rubella is endemic and in winter and early spring it causes some outbreaks . in the closed populations such as military camps or prisons , however , in those who had suffered from this disease or vaccinated before , the possibility of infection is minimized . the disease is usually rare in infants born from igg positive mothers due to antibody transfer and they will be immune for the first 46 months of their life . in general , the spread of the disease among the children has been seriously decreased , by extensive vaccination . the most commonly used vaccine globally is derived from the ra27/3 virus strain , which makes more immunity in comparison with other vaccines and the immune response is more similar to the immunity after natural infection and one dose induces seroconversion in 95% of vaccinated persons . a variety of seroepidemiological studies have been done globally to evaluate the situation of protective antibodies and need assessment to vaccinate against rubella virus . for example in australia , the rubella vaccination was established for the schoolgirls since 1971 and the crs was significantly decreased thereafter . in denmark , the vaccination policy was optional for the schoolgirls from 1974 , but in 1987 it was replaced by extensive vaccination with two doses of mmr . nine years later , the anti - rubella antibody rose to 96.5% in vaccinated people . a seroepidemiological study in chahar mahal - va - bakhtiari province ( western iran ) , on 1347 urban and rural females using the hemoagglutination inhibition ( hi ) method , indicated 90.5% immunity against rubella . in another study in tabriz ( north - western iran ) , the rubella level of immunity was 85.83% . in a study made on 400 high school girls in ghaem shahr ( northern iran ) in 1997 , and using the elisa method , 90.75% had immunity to rubella and 9.25% were susceptible . during the study in 1999 on 200 female university students in kordestan ( western iran ) , only 14.9% of them did not have anti - rubella igg . during a study made in 1999 in kermanshahi girls at marriage , 41 out of 240 studied cases did not have protective antibody and so about 20% of the referred population were susceptible , and it was then prescribed that screening for them shall be done before marriage and in susceptible cases ; they shall be vaccinated against rubella . it should be mentioned that due to these studies , finally the health plans policy makers and authorities concluded to add the rubella and mumps vaccines to the iranian national vaccination plan and remove the worry resulted from the maternal rubella . so that this plan was implemented in 2003 , and almost all of the 5 to 25 age group of iranian ( 98% ) have been vaccinated , and thereafter , the mmr vaccine was contained in the country vaccination program instead of measles vaccine alone and since then the trend associated with the studies on rubella seroepidemiology was changed to compare the situation of rubella protective antibody before and after vaccination . the national immunity of measles rubella program was commenced at december 2003 , with the target of vaccination of 33,579,082 people of 5 to 25 years old iranians and 98% of the target population were vaccinated . such successful proceeding resulted in a decrease in the appearance of measles and rubella for less than one case per million . the main goal of this survey was to evaluate and compare the susceptibility and resistance of kermanshahi girls at marriage , before and after mass rubella vaccination . the samples were randomly selected from population referred to the main marriage consultancy center in kermanshah city , west of iran . about 10,000 girls in kermanshah refer to rafatieh health centre for their premarriage medical tests , annually . since it was expected that the immunity rate after rubella vaccination to be increased from 80% in 1999 to more than 90% in 2006 , the sample size of 140 was calculated using the epiinfo software and 95% confidence . all blood samples were taken and tested under similar conditions and by the same person at the reference laboratory . for quality assurance , about 10% of the samples the results of this study were compared to those of the previous similar study conducted by the main researcher in 1999 with similar design at the same place but unvaccinated girls . age distribution indicated that the majority of the study population ( 35% ) was 2024 years old . their average age was 21.95 ( sd = 3.64 ) , and the youngest and oldest were 15 and 30 years old , respectively . with respect to educational level , 20.14% of the samples were illiterate , 19.29% were in primary school , 20% in guidance school , 32.14% in secondary school , 5% have college degree , 11.43% were bachelor , and 2.86% were not identified . a sum of 19.29% of cases was living in rural and the rest ( 80.71% ) in urban areas . positive antibody , immunity against rubella , was seen in 99.3% ( 139 out of 140 vaccinated girls , compared to 79.6% ( 191 out of 240 unvaccinated girls ) [ figure 1 ] of the previous study . therefore , we are faced to a significant rise of 19.7% ( 95% ci 12.7%26.7% ) of seropositivity and immunity against rubella in vaccinated versus unvaccinated girls ( z = 5.481 , p < 0.001 ) . situation of anti - rubella antibody and immunity in unvaccinated ( left ) and vaccinated ( right ) kermanshahi girls at marriage , 2006 this study revealed that mass vaccination of rubella caused a highly significant decrease in the rate of negative antibody from 20.42% to 0.7% . the finding is in line with reliable sources and extensive country study , which has compared immunity rate before and after iranian 2003 mass vaccination . the result also agrees with the research conducted in mashhad ( east of iran ) . in addition , our study showed higher immunity improvement than those reported from shahre - kord and hamadan cities in the west of iran . during an extensive study made by immunology department researchers on 1217 serum samples before vaccination ( on 324 susceptible and 893 immune persons ) , and 2007 serum samples after vaccination ( 24 susceptible and 1983 immune ) in 5 to 15 years age group fulfilled in ten provinces , it was identified that the immunity rate of the prevaccination was raised from 73.4% to 98.8% of the postvaccination . in another study in mashhad , the level of immunity before and after vaccination has been 70.38% and 98.5% , respectively . during a study made on 150 shahr - e - kord medical university students , it was identified that 129 ( 86% ) of them were immune before vaccination , while it increased to 96% after vaccination . during a study at hamadan health center , the level of anti - rubella antibody in hamadani girls at marriage had been 83.5% , while two years after such program it has increased to 98.4% [ table 1 ] . the effectiveness of the ra 27/3 vaccine has been demonstrated by the elimination of rubella and crs from the western hemisphere and by the several european countries that have achieved and maintained high vaccination coverage with vaccines containing ra 27/3 . mass rubella vaccination in 5 to 25 years old iranian population during 2003 , and continued thereafter as mmr in national vaccination program , has significantly resulted in increase in anti - rubella antibodies and immunity in high - risk people , as premarriage kermanshahi girls , and in comparison to many of the results specified in local and international sources , and due to the susceptibility of less than 1% of the kermanshahi girls at marriage , there is no more need to screen the vaccinated girls at marriage , which was emphasized in 1999 ; however , the screening studies is recommended every few years . as the dispersed studies associated with the rubella seroepidemiology in different locations of the country resulted in awareness of lack of immunity of about 20% of the most vulnerable society members , i.e. , the pregnant women , and after that the policy makers decided to announce the rubella vaccine as the compulsory state vaccines , it is proposed such studies to be made for the immunity status against tetanus , diphtheria , etc . additionally , due to the fact that neither rubella infection nor vaccination result in life - long immunity , it is proposed that once each couple of years the studies associated with the rubella seroepidemiology to be performed in different regions of the country and if necessary , the vaccination of vulnerable society members be made , like in 2003 .

background : rubella is a mild viral infectious disease , usually occurs subclinically without a serious complication . but if occurs during pregnancy , specially the first trimester , it can transmit and invade to fetus causing congenital rubella syndrome or fetal death . in 2003 , nearly all iranians at the age of 5 to 25 years old were vaccinated against rubella and the vaccine was included in the compulsory immunization , mmr afterwards.methods:this study was carried out on girls attended at a marriage consultancy center in kermanshah city ( west of iran ) in 2006 . considering 95% of confidence , the sample size was estimated at 140 . all samples were tested using elisa serologic method and compared with similar study conducted on 240 unvaccinated girls at the same place in 1999.results:antibody positivity was observed in 99.3% , compared to 79.58% of the previous study . a rise of 19.72% ( 95% ci 12.7%-26.7% ) of seropositivity was observed in vaccinated girls versus unvaccinated ones ( p < 0.001).conclusion : in our previous study , we had recommended to screen for susceptibility to rubella before marriage , which is no longer required since more than 99% of vaccinated girls showed immunity at the time of marriage . however , as sustainability of immunity after rubella vaccination is usually less than immunity due to illness , we recommend screening for rubella protective antibody every few years .

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with the advent of cell type specific molecular markers , description and analysis of developmental morphogenesis has become possible in a number of biological systems . gene - specific rna probes are now extensively used as they enable visualization of gene expression patterns and thus provide valuable information regarding the role of specific genes during development , as well as regarding positioning and movement of a particular cell type at different stages of development . this technique , known as whole mount in situ hybridization ( wish ) , was initially performed using radioactive probes , but was radically simplified when nonradioactive probes were used successfully [ 24 ] . it is now widely used in several biological systems , including drosophila , xenopus , quail , dictyostelium , and zebrafish . in situ hybridization ( ish ) is a quite common technique among developmental research labs , yet its uses are quite diverse and include medical and prenatal diagnostics [ 916 ] . not long after its introduction , protocols using two different labels for the rna probes and two color substrates were described , allowing the detection of more than one transcript simultaneously . however , a major limitation of these chromogenic multilabeling techniques is that the overlapping regions of expression are very difficult to discern . the use of fluorescent methods for detecting transcripts may overcome this limitation ; yet fluorophores for single- as well as double - transcript visualization in whole mounts have only been used extensively in the fly and to some extent in the zebrafish . more recently , simultaneous fluorescent detection of three transcripts was reported in the chick , and multiplexing has been used to detect up to seven transcripts in the fly [ 1921 ] . however , successful implementation of current fluorescent protocols is very rare , especially in the mouse or xenopus embryos , and even then the fluorescent images are of insufficient quality and thus can not rival the staining obtained with normal chromogenic substrates . in addition , imaging the signal is complicated due to the strong autofluorescence of the embryos , which interferes with fluorescent detection of rna and makes the use of enzymatic amplification necessary in the mouse , xenopus , chick , and other embryos . the amplification step is also necessary in the case of chromogenic protocols ; however , this step undermines the resolving ability of both methods . amplification reactions create a precipitate which is deposited in the area surrounding the rna and then diffuses out . it therefore does not remain localized within the particular intracellular region or compartment where the rna in question is localized . in fact , it often diffuses throughout the cell and sometimes even leaks outside of the cell being labeled . this problem is less pronounced in the case of tyramide amplification , where the peroxidase reaction produces tyramide radicals that react covalently with proteins at the site of the reaction , reducing the diffusion radius appreciably [ 24 , 25 ] . however , even in this case , the resolution is still limited by diffusion and depends on the time that the reaction is allowed to proceed . to combat diffusion issues , direct labeling of the probe is required , as this allows single cell , as well as , intracellular resolution of the localization of a particular mrna . in addition to improved resolution , direct labeling of the probe allows simultaneous hybridization of multiple mrnas , as long as fluorophores can be spectrally resolved . it is also extremely simple and can be quantitative since the fluorescence of the probe can be calibrated . despite successful implementation of direct labeling of messenger rnas in cell culture [ 26 , 27 ] , this has not been possible in embryos due to the low fluorescent intensities of organic fluorophores . the use of fluorescent methods for detecting transcripts is highly advantageous compared to chromogenic methods , especially because it enables higher quality three - dimensional imaging , multiplexing different rna species , and covisualization of rna with proteins . as already mentioned , the major limitations preventing widespread use of fluorescence detection for wish are the high endogenous background fluorescence of many embryos , as well as the limited brightness and photostability of organic fluorophores . a new type of inorganic fluorophore , namely , quantum dots ( qds ) have been used recently in several systems in vitro for detection of proteins as well as in vivo for protein labeling and lineage tracing [ 2842 ] . qds were also used for in situ hybridization in clinical biopsies for the detection of multiple mrnas with successful conjugation to oligonucleotide probes . qds have ideal optical properties for use in biology like strong fluorescent signal emission compared to organic and protein fluorophores [ 44 , 45 ] . in addition , due to their longer excited state lifetime , their fluorescence can be separated from the background fluorescence with time - domain imaging . using qds offers a number of other advantages over organic fluorophores including wide excitation spectra ( which makes the use of a single excitation filter possible ) , narrow and tunable emission spectra ( which reduces spectral overlap making the simultaneous use of more colors possible ) , large separation between the excitation and emission ( which increases the detection sensitivity ) , and resistance to photobleaching [ 47 , 48 ] . their unique optical properties made qds an ideal candidate for detecting multiple mrnas in ish protocols , and their high fluorescence intensity raised the possibility of using them for rna detection in whole embryos . previous efforts to do this met with aggregation issues and were time consuming since detection had to be done with rna covalently linked to hydroxylated qds . since then , we and others have reported new methods of creating hydrophilic qds and hydrophilic qd conjugates have become commercially available [ 32 , 38 , 51 ] . studies have also pointed out difficulties in using qds for fish experiments [ 52 , 53 ] , including steric hindrance , degradation of qd conjugates and adherence to tubes and tips . in this work we show that the greatest limitation of these nanocrystals is penetration , since commercially available qds are quite large and therefore fail to penetrate the many cell layers of an embryo or are significantly trapped if they do . however , we have determined that proteinase k can render xenopus embryos sufficiently permeable to allow qd penetration deep within embryonic tissues . more specifically , we produced fluorescein- ( fitc- ) , biotin- , and digoxigenin ( dig ) labeled rna probes and used qd - antibody and qd - streptavidin conjugates to visualize them . our experiments show that this is an extremely sensitive assay that significantly improved rna detection sensitivity . we then employed qds to visualize several rna probes that had been used to perform wish in xenopus embryos . we demonstrate that qd detection of endogenous messenger rnas is effective and that it can be used to provide wish data of higher resolution than current techniques . finally , we show that qds can be used to carry out two - color in situ hybridization simultaneously . therefore , the use of qds to perform nonamplified fluorescent whole mount in situs in xenopus embryos , one of the most highly autofluorescent ( and thus demanding ) vertebrate developmental organisms , suggests that qd whole - mount in situs will find successful applications in most developmental models . xenopus laevis embryos from induced spawning were staged according to nieuwkoop and faber . xenopus embryos were fertilized in vitro and dejellied using 2% cysteine - hcl , ph 7.8 , then maintained in 0.1x marc 's modified ringer 's ( mmr ) . xenopus laevis embryos were fixed in 3.7% formaldehyde in memfa ( 2 hours at room temperature ) , and the vitelline envelope was manually removed with forceps . permeabilization of embryos was carried out several ways : ( 1 ) overnight in 1x pbs supplemented with 0.5% triton , and 1% dmso ( pbdt ) , ( 2 ) overnight in 1x pbs supplemented with 5% triton and 1% dmso , ( 3 ) two hours in 1x pbs supplemented with 0.2% sds , and ( 4 ) for four hours in 1x pbs supplemented with 0.2% sds or ( 5 ) for 25 minutes in 10 g / ml proteinase k. embryos were then blocked for 2 hours in 1x pbs with 0.5% triton , 5% bsa , and 1% normal goat serum . embryos were incubated with biotin - conjugated phosphotyrosine ( anti-4g10 , millipore ) antibody overnight at 4c at a dilution of 1 : 500 ( in block solution ) . embryos were then washed ( 3 10 min ) in pbdt and incubated for 2 hours at room temperature with streptavidin conjugated cy3 or qds 655 nm at 1 : 500 dilution in fresh block solution . after incubation , embryos were washed in pbdt ( 3 10 min ) . for negative control experiments , clearing of embryos was performed by immersion of the embryos in two parts benzyl benzoate and one part benzyl alcohol after methanol dehydration ( murray 's clearing medium , bb : ba ) . the refractive index of bb : ba closely matches the refractive index of yolk thereby rendering xenopus embryos nearly transparent . the embryos were imaged on a zeiss axio imager z1 using a zeiss axiocam mr3 , the axiovision software 4.7 . antisense digoxigenin - ( dig- ) , biotin- , and fluorescein - ( fitc- ) labeled xbra ( in cs2++ ) , edd , myod ( in cs2++ ) , amylase ( in pcr4blunt - topo ) , xa-1 ( in pbsk+ ) , cardiac actin ( in psp64 ) , and ltbp1 ( in cs2++ ) probes were synthesized by in vitro transcription from linearized plasmid using rna polymerase sp6 or t3 and ribonucleotide mixture which results in rna transcripts containing bio - utp , fitc - utp , or dig - utp ( roche ) . the manufacturer 's protocol was followed with a modification in the total reaction volume which was scaled down to 20 l . biotin- , fitc- , and dig - labeled rna probes ( transcribed as described above ) were used to perform in situ hybridization using the protocol reported by harland . with some modification methanol was substituted with ethanol and 4% paraformaldehyde in pbs was used to fix the embryos instead of formaldehyde . after proteinase k treatment ( 5 min for chromogenic wish and 25 minutes for qd - based wish ) embryos were blocked with 0.1% bsa , 10% sheep serum in 0.1% tween in 1 x pbs(pbt ) solution , and then washes were performed in pbs . after blocking , embryos were refixed for one hour in 4% paraformaldehyde followed by prehybridization at 65c . for chromogenic wish experiments , the original protocol was followed . however , for qd - based fluorescent wish experiments the protocol was modified . after the last 0.2x ssc wash at 65c the embryos were blocked with 1 x pbs + 0.1% bsa + 0.1% tween for one hour and then transferred to a new vial which contained 1 ml of a 1 : 500 dilution qdot - streptavidin 705 nm , qd - anti - fitc or qd - anti - dig 655 nm ( invitrogen ) conjugates in blocking solution . the addition of 0.1% bsa and 0.1% tween into the incubation buffer significantly improved penetration and decreased background without appreciably affecting the qd colloidal stability or the signal intensity . after the incubation , the embryos were washed in pbt ( 4 30 min ) at room temperature . embryos were then cleared in bb : ba ( as described above ) . the fluorescent signal remains localized after clearing , and this allows data acquisition from different planes within the embryo without the need for sectioning . the embryos were imaged on an upright zeiss fluorescent microscope with a zeiss axiocam and the axiovision 4.7 software ( using a custom filter set ; excitation 300460 nm , emission 500 nm longpass , dichroic 475 nm ) . when detecting weak signals , the axiovision software allowed white balancing of the camera so that that the green background ( in embryos viewed with a 420 long pass filter due to the bias of the autofluorescence towards shorter wavelengths ) appears white , resulting in a major boost of the qd signal ( 605 peak emission ) , decrease of the threshold of detection and significantly better contrast . if the calibration of the camera is done properly , control embryos that are not labeled with qds appear completely white under uv excitation without any traces of red . this color separation method has to be performed carefully and control embryos need to appear white otherwise the risk of generating false staining increases significantly . due to the fact that the embryo has several distinct regions where the background fluorescence changes not only in terms of intensity but also in terms of spectral balance the region chosen for assignment of white was the region in which the background had the longest average wavelength . in this manner we ensured that long - average wavelength background regions would appear white , and shorter average wavelength regions we first wanted to examine whether qds could penetrate xenopus embryos to a sufficient depth so as to allow specific deep tissue staining . we initially compared streptavidin - conjugated qds to streptavidin - conjugated cy3 in whole - mount immunostaining experiments for their ability to detect a biotinylated antibody against phosphotyrosine . this antibody was purposely selected due to its strong and specific staining pattern of cell - cell boundaries that allows easy visual confirmation upon successful labeling . not surprisingly , the staining pattern obtained using qds was very similar to the one obtained using cy3 in the superficial cell layer of the embryo ( upper area of figure 1(a ) showing superficial cells of the fin of a tadpole and data not shown ) . however , and in contrast to cy3 , qd staining could not be detected beyond the first cell layer indicating that qds encountered penetration issues , most likely due to size restrictions ( figure 1(a ) lower area showing the somites of the tadpole ) . several approaches were employed to increase qd embryo permeability , including the use of harsh detergents like sds , with limited success . however , use of proteinase k treatment enabled penetration of qds into deep tissues and resulted in specific deep tissue staining ( figure 1(b ) and table 1 ) . however , proteinase k treatment is not suitable for use with most whole - mount immunostaining antibodies as it may lead to degradation of the target antigen . it should be noted that the best results were obtained using newly opened qd - streptavidin conjugates from invitrogen . unfortunately though , the performance of these conjugates was batch dependent and significantly diminished if qd - streptavidin conjugates were stored for more than two - three months , despite a six - month shelf life stated by the manufacturer . in addition , it was evident that use of qds with emissions in the nir ( 700 and 800 nm ) provided a big improvement in detection sensitivity due to a significant reduction of tissue autofluorescence in this region of the spectrum . however , commercially available qds with peak emissions in these wavelengths are quite large and suffer from even greater permeability problems . consequently , there is an increased need for more reliable and smaller nir qds to become commercially available . given that proteinase k treatment facilitated qd penetration in xenopus embryos and allowed specific deep tissue staining in whole - mount immunostaining experiments , we wanted to determine whether qds could be used in wish experiments in which proteinase k treatment is a standard permeabilization approach . we initially tested whether anti - digoxigenin- conjugated qds could detect a dig - labeled rna probe specific for xenopus latent transforming growth factor binding protein 1 ( ltbp1 ) . as shown in figure 1 , anti - dig qds ( see figures 1(c ) and 1(d ) ) gave a similar staining pattern of xenopus ltbp1 to the published pattern obtained using the same probe but developed using a chromogenic reaction . ltbp1 signal was detected in the head region including branchial arches and around the eye ( shown as inset of figure 1(c ) ) and the somites in agreement with the published expression pattern . furthermore , qd - labeling of ltbp1 exhibited high signal intensity and resolution confirming that a regular fluorescence microscope is sufficient for detection ( see figures 1(c ) and 1(d ) . to further establish that the qd in situ protocol is capable of successful and specific staining , we sectioned and imaged whole - mount qd - stained embryos . as shown in figure 2(a ) , qds can penetrate deep into the embryo following proteinase k treatment and stain structures independently of their proximity to the free qds in solution . importantly , the use of qds for in situ staining could result in more accurate determination of transcript expression patterns . figure 2(b ) shows a series of optical section images taken from a z - stack movie , using a fluorescence microscope , starting at the head region and moving posteriorly . as indicated , qd labeling survives the clearing protocol used to render xenopus and other embryos transparent . more importantly , however , is that the optical sections demonstrate that the staining does not originate from the notochord as originally published , but rather from the somitic mesoderm flanking the notochord . therefore , the additional spatial cues provided by optical sectioning can lead to a more accurate determination of an expression domain , further emphasizing the advantages of qd in situs . the above results suggest that use of anti - dig conjugated qds in wish can give highly specific staining of dig labeled mrna probes , even in deep tissues . to further determine the utility of this approach we tested whether qds could also label alternative modified oligonucleotide probes through biotin - streptavidin and fitc- anti - fitc interactions , using a similar protocol . figure 3(a ) depicts in situ staining performed on a dissected xenopus tadpole gut , against a biotin labeled amylase probe using either streptavidin - conjugated qds or the chromogenic reaction . it is evident , from the images , that similar staining patterns are obtained ; both the qds and the chromogenic staining were restricted to the pancreas , where amylase rna is expressed . non - stained areas appear white due to background fluorescence that is present in all visible wavelengths . despite high background , we obtained a good signal to noise ratio ( sufficient to allow clear visualization and delineation of the expressing region ) and excellent contrast in the most highly autofluorescent organ of the xenopus tadpole . we went on to test three well - known mrnas , namely , ( a ) endodermin ( edd : a pan - endodermal marker ) , ( b ) xbra ( an early mesodermal marker ) , and ( c ) myod ( a gene encoding a dna - binding protein that can activate muscle gene expression ) , which stain the gut , the mesodermal belt , and the muscle somites , respectively . as indicated in figures 3(b ) and 3(d ) , the use of qds in in situs gives staining patterns that closely match the ones obtained using standard enzymatically amplified chromogenic reaction methods , while maintaining high resolution . the degree of resolution , however , varies with the transcript of interest and its respective expression pattern . for example , in the case of myod ( figure 3(b ) , fitc - labeled probe used ) , the posterior somites look fused when using the chromogenic protocol but are clearly distinct when using qds . in contrast , qd staining of a biotin - labeled probe against edd , which is expressed in the highly autofluorescent gut appears weak except at the anterior , where the gene is expressed at higher levels ( figure 3(c ) ) . alternatively , in the case of xbra ( figure 3(d ) ) , the staining of the chromogenic and the qd ( against biotin - labeled probe against xbra ) in situ is almost identical . hoechst was used to counter - stain the nuclei blue in this experiment , and the cleared embryo was visualized from the animal pole . the fact that all three common modifications of rna probes could be detected successfully with qds raised the possibility that qds could be used to detect two or more transcripts simultaneously . achieving multiple transcript labeling using chromogenic protocols is a time consuming , stepwise process which , as explained earlier , results in the inability to distinguish areas of coexpression . in order to determine whether two transcripts can be visualized simultaneously , with the use of qds , we generated two probes ; a fitc - labeled probe against xa-1 and a biotin - labeled probe against xenopus cardiac actin . the two probes were hybridized at the same time and detected using spectrally distinguishable anti - fitc - conjugated and streptavidin - conjugated qds , respectively . as seen in figures 4(a ) and 4(b ) both probes were visualized successfully demonstrating the ability of simultaneous detection of multiple transcripts using the qd in situ protocol . in addition , due to the lack of enzymatic amplification , the resolving ability of this method is substantially better than that of amplified protocols . this is evident in figures 4(c ) and 4(d ) , where we present high - magnification images of whole - mount - stained embryos showing the intracellular mrna localization for xa-1 and ltbp1 , respectively . as can be seen , qd labeling of transcripts is of extremely high resolution and enables distinction of intracellular localization patterns of mrna . while ltbp1 localized in the cytoplasm , the xa-1 transcript appeared to be concentrated near the plasma membrane at the cell - cell contact areas . even though there is no evidence proving that the presented mrna distribution coincides with the true intracellular localization of these transcripts , the fact that there are such dramatic differences in the signal patterns from different probes suggests that this is indeed the case . future work will have to focus on closely examining the resolution of this method in model systems where direct , nonamplified in situs can be performed , using traditional fluorophores for comparison . nevertheless , it is clear that this level of resolution can not be achieved with existing methods for rna transcript detection in xenopus , which are mostly based on enzymatic amplification . we thus propose that use of the qd approach can simultaneously give macroscopic and intracellular data regarding the distribution of mrnas in vertebrate embryos . herein we describe a new application of qds in nonamplified whole - mount fluorescent in situ detection of endogenous mrnas . the ideal optical properties of qds provide unprecedented resolution and strong signal intensities that have not been possible to attain using traditional fluorophores . in fact , even though wish has been available for more than a decade , the requirement for an enzymatic amplification step significantly limited the resolution of this method . additionally , limitations of current fluorescent protocols have prevented widespread use of fluorescent in situs in most developmental models , with the exception of drosophila , and to some extent zebrafish ; direct visualization of highly abundant transcripts is possible in drosophila , but not in most vertebrate models , in which an enzymatic amplification step is required . fluorescent detection of a messenger rna opens exciting possibilities in terms of imaging and can eliminate the need for sectioning samples . it can also be used to create three - dimensional maps of expression , at a previously unattainable resolution , especially if the amplification step is eliminated . the study of mrna localization is limited by the current in situ methods , both fluorescent and chromogenic , due to their relatively low resolving ability . chromogenic reactions have been used successfully to localize mrna transcripts in cultured cells but only for transcripts of very high abundance [ 6063 ] . the method we describe is the first nonamplified fluorescent detection of mrna in situ in xenopus . our protocol results in signal intensities sufficient for imaging on a regular epifluorescence microscope without the need for confocal microscopy . of great significance , in terms of xenopus and other opaque embryos like the chick , is the fact that the qd in situ staining is capable of remaining localized and fluorescent for more than an hour after the embryo is cleared . this has not been possible using traditional chromogenic or fluorescent methods . in the case of c. elegans , where background is not a major issue , use of fluorescent antibodies to detect labeled rna probes has to be carried out in conjunction with chromogenic amplified detection of the probes in order to get a comprehensive picture of the overall expression of a gene . the amplified reaction detects low expressing regions and overall expression , whereas the fluorescent antibodies are used to resolve intracellular localization . the fact that our qd in situ protocol can do both in the highly autofluorescent xenopus embryo , which is highly demanding , makes us confident that the implementation of qd in situs in other less demanding model systems will be met with equal or more success . more importantly , these results suggest that qds could be introduced as alternative fluorophores in other fluorescent in situ hybridization assays where their spectral properties can offer significant advantages . overall , our results demonstrate that qd in situs are a viable alternative to current ish protocols , and they expand the uses of qds in biology .

the photostability and narrow emission spectra of nanometer - scale semiconductor crystallites ( qds ) make them desirable candidates for whole - mount fluorescent in situ hybridization to detect mrna transcripts in morphologically preserved intact embryos . we describe a method for direct qd labeling of modified oligonucleotide probes through streptavidin - biotin and antibody - mediated interactions ( anti - fitc and anti - digoxigenin ) . to overcome permeability issues and allow qd conjugate penetration , embryos were treated with proteinase k. the use of qds dramatically increased sensitivity of whole - mount in situ hybridization ( wish ) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification . therefore , this method offers significant advantages both in terms of sensitivity , as well as resolution . specifically , the use of qds alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos . it also offers new imaging possibilities , including intracellular localization of mrnas , simultaneous multiple - transcript detection , and visualization of mrna expression patterns in 3d .

pubmed

pancreatic cancer is a devastatingly lethal disease , with the highest mortality and the lowest one , three , and five - year relative survival rates of all cancers in the united states.1,2 although pancreatic cancer accounts for a relatively small number of new diagnoses at approximately 44,000 per year , with over 37,000 deaths per year,1 pancreatic cancer ranks only behind breast , colon , and lung cancer in total us cancer mortality.3 pancreatic cancer is a heterogeneous disease classified by cancer subtypes according to their cell type of origin , structure , and behavior . the majority of pancreatic cancer cases are pancreatic ductal adenocarcinoma ( pdac),2 which has the most aggressive tumor biology and metastatic potential among pancreatic cancer histopathologic subtypes.4,5 the median survival time after pdac diagnosis is about four months.6 only about 19% of patients with this disease survive past one year and only about 2% survive past five years.2 however , the survival of patients diagnosed with pdac may vary significantly depending on many factors , including demographic characteristics and the pathologic nature of the tumor , as well as the type of performed treatment . to estimate the relative impact of the distinct risk factors on the survival of patients diagnosed with pdac , cox proportional regression models have been used.68 it was shown that the following demographic factors have a significant impact on the survival of pdac : gender , race , age at diagnosis,6 marital status,8 and the time period of the pdac diagnosis.6 in addition , the following clinical factors have a significant influence on the survival of pdac : tumor size , nodal status , and distant metastases.7 the most dramatic influence on the survival of patients diagnosed with pdac is a surgical resection of the tumor ( with our without adjuvant chemotherapy and radiation).9 unfortunately , less than 20% of patients diagnosed with pdac are suitable for surgical resection.7 for the remaining patients , the extent of disease precludes the use of surgical procedures . the combination of surgery with radiation and chemotherapy has been shown by some to improve survival compared to surgery alone.9 the estimated parameters ( values of the relative impact of risk factors on the survival of cancer patients ) obtained by the cox proportional regression models are largely varied , depending on the quantity and quality of data used , the set of the considered variables , and the technical approaches utilized in developing these models . the obtained estimates depend on the underlying cox models , the credibility of which can be estimated by values of their concordance indexes ( c - indexes ) , which present the probability that for any pair of randomly drawn patients , the patient with the worst outcome estimated by this model dies earlier than one with the better estimated outcome . a c - index value equal to 0.5 indicates that the predictive power of the model is not better than random chance , while a level of 1.0 of this index signifies perfect discrimination . the cox proportional hazards regression models for estimation of survival for patients diagnosed with pdac were used for the development of two nomograms.10,11 one nomogram estimates the survival for patients who have undergone pancreatectomy of the malignancy and accounts for the following variables ( covariates ) : tumor size , grade , stage , lymph node data , resected tissue amount , weight loss , and pancreatic subsite.10 this nomogram was constructed using data from highly selected patients treated at a single , specialized center . the other nomogram predicts the risk of perioperative mortality in pdac patients scheduled to undergo pancreatectomy and accounts for patient demographics ( age , gender , race ) , type of pancreatectomy ( distal , radical , and other ) , hospital characteristics ( size , type ) , as well as the presence of co - morbidities in pc patients ( renal failure , neurological disorders , hypothyroid , chronic heart failure ( chf ) , liver disease , uncomplicated hypertension , cardiac arrhythmia , diabetes , and chronic obstructive pulmonary disease).11 the utility of these nomograms to patients diagnosed with pdac , however , is limited to the relatively small fraction of patients with resectable pdac ( less than 20%).12,13 recently , two new cox regression models were developed to estimate the survival for patients with resectable and unresectable pdac.9 these models use the following variables : age , gender , race , tumor stage , tumor site , tumor grade , and radiotherapy . based on these models , an interactive , online clinical calculator was developed to estimate survival probability for patients diagnosed with pdac , based on an individual s unique clinicopathological profile . the calculator is also capable of estimating conditional survival probabilities ( defined as the future survival probability or duration that is calculated after a given period of survival that increases over time elapsing since the diagnosis of pdac).14 because this calculator can estimate personalized survival probability , which can influence personal decision making , disease - related anxiety , and quality of life of cancer patients , it has been proposed for clinical use.9 however , this calculator does not provide the standard errors of the survival estimates and its reliability and generalizability were not checked ( or at least not disclosed in that work ) with the use of c - indexes . the reliability and generalizability of a model are essential when a model s predictions are considered for clinical practice.15 in the present work , we used seer data on 18,352 pathologically confirmed cases of pdac , diagnosed during 20042009,16 and developed cox proportional hazards regression models adjusted by the following covariates : race , gender , marital status , time period , age at diagnosis , tumor site , tumor size , t - stage , m - stage , n - stage , tumor grade , type of surgery , and radiotherapy . based on these models , we developed a web - based , real - time prognostic tool for estimating survival and conditional survival probabilities ( as well as their corresponding standard errors ) for patients with pdac . the reliability and generalizability of the developed models were proven by their calibration and discrimination abilities , validated by the use of both in this work , we used cancer data from the surveillance epidemiology and end results ( seer ) database from 18 geographical areas ( san francisco - oakland , connecticut , detroit , hawaii , iowa , new mexico , seattle , utah , atlanta , san jose , los angeles , alaska natives , rural georgia , greater california , kentucky , louisiana , new jersey , and greater georgia).16 these data were published by seer in april 2012 and contain cases diagnosed up to 2009 . from this release , we extracted survival data for pancreatic cancer patients diagnosed with adenocarcinoma ( international classification of diseases for oncology , 3rd edition histology codes 8140 - 8149 ) . we initially queried 33,252 cases of pdac diagnosed between 2004 and 2009 . from this subset we excluded : 5,583 cases that did not have pancreatic cancer as the primary cancer for the patient , 999 cases that were not pathologically confirmed ; one case diagnosed at an age younger than 20 years ( ages 019 are customarily used to classify childhood cancers ) ; 2,032 cases of races that were not black or white ( cases for asian / pacific islanders , native americans , and individuals of unknown race were not available in sufficient quantities ) ; 906 cases with unknown marital status ; 271 cases with unknown radiation therapy status ; 259 cases with unknown surgery status or having non - therapeutic procedures ; 176 cases marked t0 or tis stage ; and 4,673 cases with unknown tumor extension . this left 18,352 cases prior to stratification into subgroups for t1/t2 stage and t3/t4 stage . data on the remaining 18,352 pdac cases were described by 13 categorical variables ( five demographic covariates , six clinical covariates , and two medical treatment covariates ) . as demographic categorical covariates ( the corresponding categories are presented in brackets ) , we used : ( i ) race ( white , black ) , ( ii ) gender ( male , female ) , ( iii ) age at diagnosis ( quartile groups : 2059 , 6069 , 7077 , 78 + years ) , ( iv ) year ( time period ) of diagnosis ( january 1 , 2007 through december 31 , 2009 ; and january 1 , 2004 through december 31 , 2006 ) , and ( v ) marital status ( married , single ) . as the clinical covariates , we used : ( i ) tumor site , i.e. , pancreatic subsite ( head , body , tail , other , unknown ) , ( ii ) tumor size ( separated by the median size into 0.04.0 and > 4 cm groups ) , ( iii ) ajcc 6th edition t - stage ( t1 , t2 , t3 and t4 ) , ( iv ) ajcc 6th edition n - stage ( n0 , n1 , and nx ) , ( v ) ajcc 6th edition m - stage ( m0 , m1 , and mx ) , and ( vi ) tumor grade ( low , high , and unknown ) . finally , as medical treatment covariates we used : ( i ) pancreatic resection ( pancreatoduodectomy / total pancreatectomy , partial pancreatectomy / pancreatic excision , and no surgery ) , and ( ii ) radiation therapy ( yes or no therapy ) . it should be noted that comprehensive chemotherapy data are not available in the seer database , and therefore the effects of chemotherapy could not be studied in this work . the prognostic effects of these variables were studied in univariate models with the kaplan - meier method and in multivariate cox proportional hazards models , in which the survival function , s , for the failure time , t , associated with a p 1 column covariate vector , z , takes the form : where s0(t ) is the baseline hazard function and is a p 1 column vector of the regression coefficients ( the is the transpose of the ) . the proportional hazard assumptions were assessed by visual inspection of log - log plots.17 all tests were two - sided with a significance level set at p < 0.05 . , cary , nc ) was utilized to estimate the regression coefficients , , of the multivariate cox models . the estimates , ( here and below the sign , ^ , designates an estimate ) and the estimated covariance matrix of the parameter estimates , cv( ) , were obtained using the phreg sas procedure . estimates of the baseline survival function , 0 and their standard errors , s(0 ) , were obtained by the phreg sas procedure using the baseline statement . outputs of the sas phreg procedure were used to calculate estimates of the survival probability , , and standard error , s( ) . for a given time , t , survival is a function of the two variables:18,19 the baseline survival function , 0 , and the prognostic index , l : estimates of the standard errors of the estimates of the prognostic index , s(l ) , were obtained by the formula that is used in sas phreg procedure : estimates of the standard errors of the estimates of the survival , s( ) , were calculated by the following formula , obtained by standard rules of error propagation,19 and implemented in the phreg sas procedure : in ( 3 ) , [ / 0 ] and [ /l ] are partial derivatives , which can be easily obtained from ( 2 ) , and the covariance cv ( 0 , l ) can be evaluated by numerical experiments . we found that cv ( 0 , l ) s(0 ) s(l ) ; this approximation can be further used in formula ( 5 ) . by definition , the conditional survival , s(t2 | t1 ) , represents the probability that a patient with cancer will survive an additional t2 months , given that the patient has already survived a given t1 months . in this case , the estimate of the conditional survival , ( t2| t1 ) , can be presented by formula : where 2 and 1 designate the corresponding estimates of the survival probabilitie ( t2| t1 ) and ( t1 ) . survival function estimates 1 and 2 in formula ( 6 ) are dependent random variables with covariance depending on survival time t1 end t2 . neglecting this covariance while using the standard rules of error propagation , we can calculate the conservative ( upper ) estimate of the standard error of the estimate of the conditional probability , s [ ( t2| t1 ) ] , by formula : wher s ( 1 ) and s ( 2 ) are determined by formula ( 5 ) . the accuracy of the cox models developed in this work were assessed by the c - indexes ( to estimate the discrimination power ) and calibration plots ( to estimate an agreement between observed outcomes and predictions).20 the reproducibility and transportability of these models were also checked , as we describe below . the reproducibility is defined as the performance of a model on a sample of similar patients not included in the development of the model , while transportability reflects a model s ability to predict among patients from different but plausibly related populations.21 to estimate the reproducibility of the parameters of the developed models , cross - validation with 10 iterations was done . for this purpose , the systematic sample splits were performed in such a way that all patients have served once in the model evaluation dataset . the end digit in the unique patient i d number , by which each case is coded in the seer database , was used as an exclusion criterion . thus , the systematic sample split was done by varying the exclusion criterion , starting from the digit 0 and sequentially changing it to digits 1 , 2 , ... , and 9 . for each iteration , cases not satisfying the considered exclusion criterion were used as the model training dataset , while the excluded cases were used as a testing ( validation ) dataset . the discrimination power ( c - indexes ) of the models , obtained with the use of the training dataset , was checked using the testing dataset . the generalizability or transportability of the models ( i.e. , goodness of using the same variables and same coefficients ) was checked by utilizing the models for predicting the survival of the cancer patients living in different geographical areas . for this purpose , the c - indexes of the models , developed by using the cases from distinct geographic areas as training datasets , were validated on testing ( external ) datasets exclusively containing cases from the geographical areas that were not included in the training datasets . estimates of observed survival , stratified by each variable , were obtained using the kaplan - meier method . for each variable , the proportional hazards assumption was assessed graphically ( data not shown ) by log - log plots.17 we determined that for the following 12 categorical variables the log - log plots are nearly parallel : race , gender , marital status , time period , age at diagnosis , tumor site , tumor size , t - stage , m - stage , n - stage , tumor grade , type of surgery , and radiation therapy . for the t - stage variable , however , the corresponding curves on the log - log plots were not parallel ( and , as a result , the ajcc 6th edition stage group also demonstrates non - proportional hazards ) . in fact , figure 1 shows that the curves related to the t1- and t2-stages ( tumor extension localized to the pancreas ) are not parallel to the curves related to the t3- and t4-stages ( tumor extension not localized ) . data on 18,352 pdac cases were stratified in two groups by degree of pdac localization . the first group ( which we called localized pdac , lpdac ) contained 5,422 cases , in which the tumor is localized within the pancreas ( t1- and t2-stages ) . the second group ( called extended pdac , epdac ) contained 12,930 cases , in which the tumor is extended beyond the pancreas ( t3-stage ) or is locally advanced and involves the celiac axis or the superior mesenteric artery ( t4-stage ) . after separation of all pdac cases into these two groups , the proportional hazard assumptions for all categorical variables describing the cases in each of these groups were satisfied . this allowed us to perform the multivariate cox regression analysis for the lpdac and epdac groups separately . in the multivariate cox regression analysis of 5,422 cases for the lpdac group , we initially did not use the t - stage variable , which is not needed for this group of cases . this is because the lpdac group contained only cases with t1- and t2-stages that are different from one another only by the tumor size ( cases with tumor size equal or less than 2 cm are t1-stage , and cases with tumor size larger than 2 cm are t2-stage),22 but to categorize tumor size , the tumor size variable can be used . when performing the cox analysis , we found that the variable tumor subsite ( head , body , tail , and other ) does not have a significant influence on the survival of the lpdac patients , and thus we did not use this variable for the development of the final lpdac cox model . in multivariate cox regression analysis of 12,930 cases for the epdac group , we initially utilized all the variables described in materials and methods . however , the cox modeling of the epdac cases showed that the variable tumor subsite ( head , body , tail , and other ) did not have a significant influence on the survival of the epdac patients . analogously , the variable t - stage , which for the epdac cases varies by degree of tumor extension , also did not have a significant influence on the survival of the epdac patients . therefore , we did not use the variables tumor subsite and t - stage for the development of the final epdac cox model . tables 1 and 2 present the final results of multivariate cox regression analysis for the lpdac and epdac cases , correspondingly . as can be seen from these tables , the vast majority of the considered covariates have significant influence on the survival of patients diagnosed with pdac . there is also a good qualitative agreement in the estimated contributions of similar covariates on the survival of patients diagnosed with the localized and extended pdac . in both cases , the performed treatments ( surgery and/or radiation therapy ) were the most beneficial for survival . among the clinical characteristics of a tumor , the largest risk factors accelerating death from pdac include the presence of metastasis ( m1 ) , poor tumor differentiation ( high grade ) , and large ( > 4 cm ) tumor size . the involvement of lymph nodes ( n1 ) makes a small ( likely due to the simultaneous effect of other , larger contributors , such as m1-stage ) , but significant contribution to survival for patients diagnosed with lpdac , but not with epdac . within the demographic characteristics of patients , age at diagnosis gender , race , and marital status make modest and comparable contributions to the survival of patients diagnosed with lpdac and epdac . the accuracy of the cox models developed for the lpdac and epdac cases were assessed by the c - indexes and calibration plots.20 the obtained c - indexes of these models were equal to 0.702 and 0.712 , respectively . the calibration plots presented in figures 2 and 3 show how close the survival estimates are to the observed values , predicted by these models at the time point ( chosen to be equal to 12 months ) . as can be seen from these figures , the reproducibility of the cox models developed for the lpdac and epdac cases were analysed by cross - validation with 10 repeats ( table 3 ) . as can be seen from table 3 , the cross - validated c - indexes of the lpdac and epdac models vary within the small intervals , ( 0.6870.720 ) and ( 0.696 0.732 ) , correspondingly . the averaged values of the cross - validated c - indexes were 0.700 and 0.711 , respectively , which are very close to the c - indexes ( 0.702 and 0.712 ) initially obtained for lpdac and the epdac models . this suggests high reproducibility of the cox models for the lpdac and epdac cases presented in table 1 and table 2 . the generalizability ( transportability ) of the lpdac and epdac models to the pdac patients living in different geographical areas were also checked . specifically , the cases from one of 18 geographic areas were considered as external datasets and used for validation of the lpdac and epdac cox models trained by cases collected in other 17 geographic areas . note that this table shows data for the geographical areas having more than 100 cases of white and black patients , which could be used as external datasets . because the utah , hawaii , alaska , and rural georgia seer registries had less than 100 cases to be used for external testing of the lpdac models , we did not perform validation for these areas . as can be seen from table 4 , the c - indexes of the lpdac and epdac cox models for individual geographical areas vary within the intervals , ( 0.651 0.739 ) and ( 0.6990.739 ) , respectively . the averaged values of these indexes for the lpdac and epdac models were 0.700 and 0.714 , respectively . these numbers are very close to the c - indexes ( 0.702 and 0.712 ) initially obtained for the lpdac and the epdac cox models . this suggests that the lpdac and epdac models , the parameters of which are presented in tables 1 and 2 , are sufficiently generalized to be transportable for applications to different geographical areas of the us . overall , the data presented in tables 3 and 4 confirm high reproducibility and generalizability of the lpdac and epdac cox models developed with the use of 18,352 pathologically confirmed cases of pdac diagnosed between 2004 and 2009 . this allowed us to use the parameters of these models , which are presented in tables 1 and 2 , to develop a real - time prognostic estimator of survival for patients diagnosed with pdac . this tool allows one to estimate the conditional survival (t2| t1 ) and its standard error se[(t2| t1 ) ] , using , cov( ) , s0 and se(s0 ) as unchangeable datasets and z ( the personalized demographic and clinical data on patients diagnosed with pdac , as well as information on medical treatment ) , t1 ( number of months that the patient has already survived after the pdac diagnosis ) , and t2 ( number of additional months that the patient will survive ) as variable input data . depending on the value of the t - stage variable , the estimator uses the lpdac ( for t1 and t2 stages ) or epdac ( for t3 and t4 stages ) models with the corresponding unchangeable datasets . the conditional survival probability and its standard error have been calculated by formulas ( 6 ) and ( 7 ) , respectively . the estimator is a real - time , web - based computerized tool , compatible with major browsers and user devices , including tablets and smart phones . as with any other cox models , the lpdac and epdac models developed in this work have several potential limitations that may explain why predictions performed by the lpdac and epdac models may be inaccurate in up to 30% of patients ( discriminative power of these models , or c - index ~ 0.70 ) . specifically , the lpdac and epdac models use variables provided by seer , while other variables , for instance , co - morbidities in pdac patients ( such as renal failure , neurological disorders , liver disease , and cardiac arrhythmia ) that may significantly contribute to mortality risk from pdac are not utilized.11 the second limitation is due to the fact that although the public - use seer data on pdac contains information on adjuvant radiation therapy , information on chemotherapy data is not provided.23,24 however , about 85% of the pdac patients who received radiation therapy also received chemotherapy.25 nevertheless , the absence of information on chemotherapy may lead to a bias in the estimation of the efficiency of radiation therapy when seer data is used.25 the third limitation is due to the use of the sum of the model variables , weighted by their coefficient values ( log hazard ratio ) as the prognostic index . in fact , estimation of the prognostic index , which is at the heart of cox models , may be accurate for the majority of the observed pdac population , but it may be inaccurate for relatively small , yet very important patient subpopulations . for instance , for lpdac patients who have undergone pancreatectomy for a small sized tumor , the benefits of radiotherapy , as determined by our lpdac cox model , can be overestimated . nevertheless , our newly developed lpdac and epdac models and computing tool ( estimator ) have distinct advantages over the existing models and tools , aimed at predicting survival probabilities for patients diagnosed with pdac . some of these advantages are similar to those that were highlighted in the work recently published by katz et al.9 these advantages are : ( i ) the developed models are applicable for the vast majority of pdac patients ( not only for those who are qualified for pancreatectomy ) and ( ii ) the developed web - based computing tools are capable of the real - time prediction of conditional and unconditional survival probabilities for the patients diagnosed with pdac . however , lpdac and epdac models have additional advantages that can be seen by exploring differences between the models and computing tools developed in our work and the katz work , as presented below . the models presented in the katz work were stratified by surgery status , while our lpdac and epdac models are stratified by tumor extension . our models use the following covariates ( which were not used in the katz work ) : marital status , time period , t - stage , m - stage , n - stage , and type of surgery . on the other hand , tumor subsite ( which we found did not have significant influence on survival ) and a stage covariate ( which we found did not satisfy the proportional hazards assumption ) , further , our models were tested using c - indexes and cross - validated with the use of internal and external datasets , while the accuracy , reproducibility and generalizability of the corresponding models were not disclosed in the katz work . finally , our models were trained using data collected in 20042009 and account for time period effects , while the models of that work were trained on data collected in 19882005 and ignore possible time period effects . there are also differences in performances of the estimator , developed in our work , and the calculator , developed in the katz work . in fact , the calculator assesses survival time without adjusting for time period effects and thus implicitly makes predictions anchored to the middle of the 19882005 time interval , while our estimator predicts survival , projected to the later time period ( 20072009 ) . in addition , the estimator provides standard errors for the predicted survival probabilities , while the calculator does not have this capability . we showed that the lpdac and epdac models developed in this work fit the observed survival data well , are well - calibrated , and have good discrimination ( c - indexes ) between cases . these models were used in the development of a web - based tool ( estimator ) to assess survival probability , conditional survival probability , and their standard errors . this tool uses the personalized demographic and clinical data on patients diagnosed with pdac , as well as information on ( performed , ongoing , or planned ) medical treatment as input data . the estimator performs all calculations anchored to the latest time period , 2007 2009 , which contains the latest seer data available at the time of the preparation of this work . in fact , clinicians mainly rely on personal experience , while the estimator is based upon the combined survival experience and clinical care of many patients and account for the effect of dozens of risk factors simultaneously . the estimator removes preconceived bias and offers assessments based on the personalized demographic and clinical data of a given patient . by toggling different treatment options , clinicians will be able to compare the predicted impacts of possible treatments on survival of the patient . a treatment that will not provide any significant advantage in survival , but would instead be detrimental to the patient s quality of life , should not be considered as an appropriate option . using the computer - generated assessments and carefully considering the patient s co - morbidities , clinicians will be able to assist patients diagnosed with pdac in making a more educated decision regarding potential treatments .

the 18,352 pancreatic ductal adenocarcinoma ( pdac ) cases from the surveillance epidemiology and end results ( seer ) database were analyzed using the kaplan - meier method for the following variables : race , gender , marital status , year of diagnosis , age at diagnosis , pancreatic subsite , t - stage , n - stage , m - stage , tumor size , tumor grade , performed surgery , and radiation therapy . because the t - stage variable did not satisfy the proportional hazards assumption , the cases were divided into cases with t1- and t2-stages ( localized tumor ) and cases with t3- and t4-stages ( extended tumor ) . for estimating survival and conditional survival probabilities in each group , a multivariate cox regression model adjusted for the remaining covariates was developed . testing the reproducibility of model parameters and generalizability of these models showed that the models are well calibrated and have concordance indexes equal to 0.702 and 0.712 , respectively . based on these models , a prognostic estimator of survival for patients diagnosed with pdac was developed and implemented as a computerized web - based tool .

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we recruited japanese patients who underwent pta for cli in kansai rosai hospital , hyogo , japan , between 2003 and 2009 . all patients with chronic ischemic rest pain and/or foot ulcer or gangrene were evaluated for limb ischemia by angiography . the diagnosis and management of cli were compliant with the transatlantic inter - society consensus ( tasc ) ( 10 ) or its revised consensus , tasc ii ( 1 ) . once they were diagnosed as having cli , we utilized pta as the first - choice procedure for revascularization , within the recommendations in tasc ii . the indication of pta was judged by consensus among vascular specialists , including vascular surgeons . pta was not considered to be feasible when lesions were heavily calcified and/or with diffuse occlusions , whereas poor - risk patients , having relative contraindications for bypass surgery , those with advanced age , expected high mortality under general anesthesia , and morbidity setting , for instance , would not necessarily be contraindications for pta . major amputation , namely , above - the - ankle amputation , was indicated when revascularization failed to relieve patients from rest pain or control their foot lesions . the indication for amputation was judged by consensus among plastic surgeons as well as vascular specialists . insufficient blood flow even after revascularization and uncontrollable limb infection were considered to be indications for amputation . in the current study , we analyzed all of the recruited patients as well as those with diabetes . the preoperative variables considered in the analyses were sex , age , activities of daily living ( adls ) , fontaine stage , the presence of infection , diabetic condition , hypertension , dyslipidemia , smoking , receiving regular hemodialysis , and serum albumin level as a nutritional marker . diabetic condition was determined as the presence of diabetes in the whole population and as a glycemic control in the diabetic population . the diagnosis of diabetes was based on world health organization criteria , whereas glycemic control of diabetic patients was evaluated by a1c . hypertension was diagnosed as systolic blood pressure 130 mmhg or diastolic blood pressure 80 mmhg or having been treated for hypertension . dyslipidemia was defined as serum ldl cholesterol 100 mg / dl or hdl cholesterol < 40 mg / dl or triglycerides 150 mg / dl or having been treated for dyslipidemia . partial or total dependence in transferring ( requiring some help in moving in and out of bed / chair or a complete transfer ) were regarded as impaired adls . data are given as means and sds for continuous variables or as percentages for dichotomous variables . limb salvage and survival were plotted using the kaplan - meier method , and , if necessary , the differences between the two groups were assessed by a log - rank test . cox proportional hazards regression model was used to determine the unadjusted association of each variable with the outcome . the statistically significant variables in the univariate analyses , as well as sex and age , were entered into multivariate models to reveal the independent impact on the outcome . statistical analyses were performed using spss version 15.0j ( spss , chicago , il ) . data are given as means and sds for continuous variables or as percentages for dichotomous variables . limb salvage and survival were plotted using the kaplan - meier method , and , if necessary , the differences between the two groups were assessed by a log - rank test . cox proportional hazards regression model was used to determine the unadjusted association of each variable with the outcome . the statistically significant variables in the univariate analyses , as well as sex and age , were entered into multivariate models to reveal the independent impact on the outcome . statistical analyses were performed using spss version 15.0j ( spss , chicago , il ) . a total of 278 cli patients undergoing primary pta procedures were recruited ; aorto - iliac lesions were revascularized in 23% of the population , femoro - popliteal in 63% , and infrapopliteal in 68% . diabetic patients had a significantly higher prevalence of fontaine stage iv and hemodialysis than those without diabetes ( p = 0.006 and p = 0.001 , respectively ) . baseline characteristics of the study population data are n ( % ) or means sd . differences between continuous variables of diabetic and nondiabetic groups were evaluated by unpaired t tests , whereas dichotomous variables between the two groups were compared by fisher exact tests . follow - up period was 90 72 weeks ( 1.7 1.4 years ) , and 48 patients underwent major amputations and 89 died . figure 1 shows the cause of death and kaplan - meier estimates of major amputation and mortality . the leading cause of death was cardiac and vascular disease ( n = 40 ) ; followed by infection ( n = 31 ) , including pneumonia ( n = 16 ) , sepsis secondary to infective gangrene of the foot ( n = 8) , bed sore infection ( n = 1 ) , and catheter - related infection ( n = 1 ) ; and unspecified causes ( n = 5 ) . diabetic patients had a higher rate of major amputation compared with nondiabetic patients ( p = 0.008 , log - rank test ) , whereas they had a similar life prognosis ( p = 0.717 ) . the cause of death in the whole population ( a ) and in the population with diabetes ( b ) . kaplan - meier estimates of major amputation ( c and d ) and survival ( e and f ) in the whole population . the prognosis was compared between diabetic patients ( solid line ) and nondiabetic patients ( dotted line ) in d ( p = 0.008 , log - rank test ) and f ( p = 0.717 , log - rank test ) . cv , cardiac and vascular disease ; mn , malignant neoplasm ; dm , diabetes . table 2 shows the influence of preoperative variables on the prognosis in the whole population . the presence of diabetes was independently associated with major amputation , and the adjusted hr was 3.101 ( 95% ci 1.2627.621 ) ( p = 0.014 ) . rather , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level ; the adjusted hrs were 1.036 ( 1.0101.063 ) in the 1-year increment ( p = 0.007 ) and 2.302 ( 1.4073.605 ) ( p < 0.001 ) , 2.699 ( 1.6824.332 ) ( p < 0.001 ) , and 1.788 ( 1.1502.782 ) in the 1-g / dl decrement ( p = 0.010 ) , respectively . association of preoperative variables with the prognosis of cli patients undergoing pta data are hr ( 95% ci ) . we also performed analyses with a focus on the patients with diabetes ( table 3 ) . here again , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level . a1c level was , however , independently associated with major amputation , whose adjusted hr was 1.349 ( 95% ci 1.1031.650 ) in the 1% increment ( p = 0.004 ) . its association was still significant in the multivariate models in which every other peripheral vascular factor available in our dataset was substituted for fontaine stage iv ( online appendix table a , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0939/dc1 ) . association of preoperative variables for the prognosis with cli patients with diabetes undergoing pta data are hr ( 95% ci ) . next , we divided the a1c dataset into quartiles to analyze the relationship between the increment of a1c level and the risk for major amputation in the diabetic patients . 5.9% was categorized into the first quartile ( q1 ) , 6.06.7% into the second quartile ( q2 ) , 6.87.6% into the third quartile ( q3 ) , and 7.7% into the fourth quartile ( q4 ) . in a stepwise multivariate model , the categorized a1c level was independently associated with major amputation , with adjustment for fontaine stage iv , hemodialysis , and infection . the adjusted hrs of q2 , q3 , and q4 relative to q1 were 2.030 ( 95% ci 0.6576.266 ) ( p = 0.218 ) , 3.398 ( 1.2279.412 ) ( p = 0.019 ) , and 3.983 ( 1.39811.35 ) ( p = 0.010 ) , respectively . we further analyzed the influence of the presence of diabetes on major amputation according to their a1c level ; each a1c quartile of the diabetic group , defined above , was compared with the nondiabetic group in a stepwise multivariate model . as shown in fig . 2a , the two higher a1c quartiles of the diabetic group ( that is , the diabetic group with a1c 6.8% ) had a significantly higher risk than the nondiabetic group , whereas the two lower a1c quartiles ( that is , a1c < 6.8% ) did not . based on these findings , we reanalyzed with substitution of diabetes with a1c 6.8% for the presence of diabetes in the original multivariate model shown in table 2 . the result was that diabetes with a1c 6.8% , infection , and hemodialysis were significantly associated with major amputation ; their adjusted hrs were 2.907 ( 95% ci 1.6065.264 ) ( p < 0.001 ) , 2.375 ( 1.1984.711 ) ( p = 0.014 ) , and 3.530 ( 1.7727.029 ) ( p note that these three variables were independent of one another and therefore were expected to have additive influences on major amputation . in fact , a kaplan - meier model showed that those with the accumulation of these prognostic factors had an increased risk of major amputation ( fig . when they had all of these three risk factors , their prognosis was extremely poor . their estimated median time to limb loss was only 23 weeks , which would be rarely different from natural course of nonrevascularized cli patients ( 1 ) . data are the adjusted hrs and 95% cis of each a1c quartile of the diabetic group relative to the nondiabetic group in the stepwise multivariate model . they were adjusted for impaired activity of daily living , fontaine stage iv , infection , and receiving hemodialysis . the quartiles of a1c were as follows : q1 : 5.9% , q2 : 6.06.7% , q3 : 6.87.6% , and q4 : 7.7% . b : kaplan - meier estimates of major amputation according to the number of risk factors ( p < 0.001 , log - rank test ) . risk factors considered here are the following three variables : diabetes with a1c 6.8% , the presence of infection , and receiving hemodialysis , all of which had independent associations in the stepwise multivariate cox proportional hazards regression model . table 2 shows the influence of preoperative variables on the prognosis in the whole population . the presence of diabetes was independently associated with major amputation , and the adjusted hr was 3.101 ( 95% ci 1.2627.621 ) ( p = 0.014 ) . rather , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level ; the adjusted hrs were 1.036 ( 1.0101.063 ) in the 1-year increment ( p = 0.007 ) and 2.302 ( 1.4073.605 ) ( p < 0.001 ) , 2.699 ( 1.6824.332 ) ( p < 0.001 ) , and 1.788 ( 1.1502.782 ) in the 1-g / dl decrement ( p = 0.010 ) , respectively . association of preoperative variables with the prognosis of cli patients undergoing pta data are hr ( 95% ci ) . we also performed analyses with a focus on the patients with diabetes ( table 3 ) . here again , mortality was associated with age , impaired adls , hemodialysis , and serum albumin level . a1c level was , however , independently associated with major amputation , whose adjusted hr was 1.349 ( 95% ci 1.1031.650 ) in the 1% increment ( p = 0.004 ) . its association was still significant in the multivariate models in which every other peripheral vascular factor available in our dataset was substituted for fontaine stage iv ( online appendix table a , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0939/dc1 ) . association of preoperative variables for the prognosis with cli patients with diabetes undergoing pta data are hr ( 95% ci ) . next , we divided the a1c dataset into quartiles to analyze the relationship between the increment of a1c level and the risk for major amputation in the diabetic patients . 5.9% was categorized into the first quartile ( q1 ) , 6.06.7% into the second quartile ( q2 ) , 6.87.6% into the third quartile ( q3 ) , and 7.7% into the fourth quartile ( q4 ) . in a stepwise multivariate model , the categorized a1c level was independently associated with major amputation , with adjustment for fontaine stage iv , hemodialysis , and infection . the adjusted hrs of q2 , q3 , and q4 relative to q1 were 2.030 ( 95% ci 0.6576.266 ) ( p = 0.218 ) , 3.398 ( 1.2279.412 ) ( p = 0.019 ) , and 3.983 ( 1.39811.35 ) ( p = 0.010 ) , respectively . we further analyzed the influence of the presence of diabetes on major amputation according to their a1c level ; each a1c quartile of the diabetic group , defined above , was compared with the nondiabetic group in a stepwise multivariate model . as shown in fig . 2a , the two higher a1c quartiles of the diabetic group ( that is , the diabetic group with a1c 6.8% ) had a significantly higher risk than the nondiabetic group , whereas the two lower a1c quartiles ( that is , a1c < 6.8% ) did not . based on these findings , we reanalyzed with substitution of diabetes with a1c 6.8% for the presence of diabetes in the original multivariate model shown in table 2 . the result was that diabetes with a1c 6.8% , infection , and hemodialysis were significantly associated with major amputation ; their adjusted hrs were 2.907 ( 95% ci 1.6065.264 ) ( p < 0.001 ) , 2.375 ( 1.1984.711 ) ( p = 0.014 ) , and 3.530 ( 1.7727.029 ) ( p < 0.001 ) , respectively ( online appendix table b ) . note that these three variables were independent of one another and therefore were expected to have additive influences on major amputation . in fact , a kaplan - meier model showed that those with the accumulation of these prognostic factors had an increased risk of major amputation ( fig . when they had all of these three risk factors , their prognosis was extremely poor . their estimated median time to limb loss was only 23 weeks , which would be rarely different from natural course of nonrevascularized cli patients ( 1 ) . data are the adjusted hrs and 95% cis of each a1c quartile of the diabetic group relative to the nondiabetic group in the stepwise multivariate model . they were adjusted for impaired activity of daily living , fontaine stage iv , infection , and receiving hemodialysis . the quartiles of a1c were as follows : q1 : 5.9% , q2 : 6.06.7% , q3 : 6.87.6% , and q4 : 7.7% . b : kaplan - meier estimates of major amputation according to the number of risk factors ( p < 0.001 , log - rank test ) . risk factors considered here are the following three variables : diabetes with a1c 6.8% , the presence of infection , and receiving hemodialysis , all of which had independent associations in the stepwise multivariate cox proportional hazards regression model . as is mentioned in the tasc ii ( 1 ) , a primary outcome in cli patients would be amputation - free survival , although it remains unclear so far , especially in the population undergoing pta , which preoperative variables are associated with their prognosis and whether morality and limb loss have the same predictive factors . in the current study , a1c level the multivariate model , however , showed that a1c level had a significant association with major amputation independently of infection . the current study also confirmed that the presence of diabetes was significantly associated with major amputation in the whole population and that its significant association was dependent on the glycemic control . some investigators already reported the association between diabetes and limb loss ( 11,12 ) , but they did not take glycemic control into consideration . interestingly , when compared with nondiabetes , diabetes a1c 6.8% , but not a1c < 6.8% , was significantly associated with major amputation . this finding suggests that the increased risk of major amputation for diabetic patients is mainly attributed to their poor glycemic control . it is well known , however , that poor glycemic control is associated with decreased immune response and delayed wound healing , as well as the progression of microangiopathy including neuropathy , all of which play an important part of foot lesions ( 1315 ) ; the existence of poor glycemic control might reflect these underlying conditions . in discussion of limb prognoses of pad patients exist several ways of assessing preoperatively their peripheral arterial severity . in the current study , we adopted fontaine stages , a widely used clinical classification , and found that it was not significantly associated with limb salvage in a multivariate model , as was poor glycemic control . to examine this paradoxical result , we further analyzed with two other preoperative peripheral arterial factors available in our dataset , namely ankle - brachial index , which is physiological , and runoff below the ankle before pta , which is anatomical ( or imaging ) . yet , the results were similar ; no preoperative peripheral arterial factors had independent associations with limb loss ( online appendix table a ) . in contrast , a postoperative variable , the runoff below the ankle after pta , was significantly associated with major amputation . one interpretation of these findings is that the limb prognosis depends on the peripheral blood flow after , rather than before , revascularization . successful revascularizations would improve peripheral blood flow and could lead to avoidance of limb loss , even if the preoperative peripheral flow is severely impaired . the severity of preoperative peripheral flow would not always mean the similar severity of postoperative peripheral flow . given that peripheral blood flow can be changed by revascularization procedures , it is not surprising that limb prognosis after pta is associated with postoperative , rather than preoperative , peripheral arterial factors . we also surveyed the influence of preoperative variables on survival , which confirmed that diabetes failed to have a significant association with morality . , most studies surveyed cli patients undergoing pta together with those undergoing bypass surgery and/or nonrevascularized patients , whereas we limited our study population to those undergoing pta . in addition , we revealed that a1c level in diabetic patients had no significant association with mortality . the prognostic factors for mortality were , rather , age , impaired adls , hemodialysis , and serum albumin level . these significant associations were affirmed even with additional adjustment for cardiac function or ejection fraction evaluated with echocardiography ( online appendix table c ) . previous reports ( 9,18,19 ) investigated some of these prognostic variables , but they did not mention nutritional status in the population . as is well known , malnutrition affects life prognosis in various clinical situations ( 20,21 ) . we adopted serum albumin level as a nutritional marker in the current study because of its great advantages in ease and cost and its wide use in clinical practice . it is possible that serum albumin level is affected by clinical factors other than malnutrition . but we confirmed the relationship between malnutrition and mortality by the analyses with other nutrition - related markers , such as lymphocyte count and serum cholinesterase level ( online appendix table d ) . in conclusion , we investigated the association of preoperative variables with the prognosis of cli patients undergoing pta , which suggests that prognostic indicators are somewhat different between survival and limb salvage . diabetes and poor glycemic control , for instance , was significantly associated with limb loss but not mortality . diabetes with poor glycemic control , infection , and hemodialysis was independently associated with limb loss , and the accumulation of these prognostic factors increased the amputation risk . especially , those with all of the three risk factors had such poor limb prognosis that they might fail to receive a prognostic benefit of pta . it is possible , however , that correction of poor glycemic control by adequate interventions leads to prognostic improvement . a further prospective investigation is required to determine whether the intervention on glycemic control subsequently improves their prognosis .

objectiveto reveal the influence of preoperative factors on the prognosis of patients undergoing percutaneous transluminal angioplasty ( pta ) for critical limb ischemia ( cli).reseach design and methodswe recruited 278 japanese patients who underwent pta for cli between 2003 and 2009 . the outcome measures were mortality and major amputation . cox proportional hazards regression analyses were performed.resultsthe prevalence of diabetes was 71% , and a1c was 7.0 1.4% . the follow - up period was 90 72 weeks , and 48 patients underwent major amputations and 89 died . the presence of diabetes in the whole population and a1c level in the diabetic population had no influence on morality ; rather , mortality was associated with age ( p = 0.007 ) , impaired activities of daily living ( p < 0.001 ) , hemodialysis ( p < 0.001 ) , and albumin level ( p = 0.010 ) . in contrast , the presence of diabetes and a1c level had significant association with major amputation ( p = 0.012 and p = 0.007 , respectively ) . the quartile analysis showed that diabetic subjects with an a1c 6.8% , but not < 6.8% , had a significantly higher risk of major amputation than nondiabetic subjects . the adjusted hazard ratio of diabetes with a1c 6.8% was 2.907 ( 95% ci 1.6065.264 ) ( p < 0.001).conclusionsdiabetes with poor glycemic control is associated with major amputation , but not mortality , in cli patients undergoing pta . prognostic indicators seem somewhat different between survival and limb salvage in the population .

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human cystic echinococcosis ( ce ) , caused by echinococcus granulosus , is one of the most important and widespread parasitic zoonoses ( 1 ) . humans acquire infection by accidental ingestion of e. granulosus eggs voided in the faeces of infected dogs and the disease is common in parts of the world where there is close contact between the intermediate and definitive hosts , usually sheep and dogs , respectively ( 2 ) . cystic echinococcosis in humans usually presents with symptoms associated with the presence of fluid - filled cysts in the liver , lungs , or other viscera and diagnosis is usually established by a combination of radiology and serology ( 3 ) . one of the problems that can be encountered after treating ce patients is the risk of postsurgical relapses or treatment failure due to nonradical surgical procedures or perisurgical spillage of parasite material , especially protoscoleces . relapses in the form of newly developing cysts have been reported and may affect between 2 and 25% of cases after therapy , according to previous studies ( 46 ) . therefore , postsurgical follow - up of ce patients for years is necessary , with the aim of detecting newly growing cysts as soon as possible . a posttreatment follow - up method to prognostically determine the efficacy of treatment should therefore include markers that allow the detection of newly growing or relapsing cysts and tracking of previously undetected but still viable cysts . serology has been one of the methods selected for the post - operative control of hydatidosis . however , the long persistence of anti - e . granulosus antibodies after recovery makes difficult the diagnosis of relapse by serology ( 7 ) . in this sense , many serologic techniques have been evaluated ( latex agglutination , passive hemagglutination , immunoelectrophoresis and specific ige , igm , igg enzyme - linked immunosorbent assay ) in the post - operative monitoring of hydatid disease patients . therefore , there is still a need to develop or improve immunodiagnostic tools in order to meet the requirements expressed by clinicians . one approach consists of searching for and identifying new antigens that specifically allow the classification of patients into cured and noncured categories . this study examined the pattern of antigenic bands essential for the serologic diagnosis of ce , revealed by immunoblotting analysis . we also report on the post - operative evolution of patients treated for this disease and also determined the diagnostic performance of western blot . blood samples were obtained from 50 patients ( 16 males and 34 females ; mean sd age was 31.111.2 years , range 969 years ) with clinically / radiologically diagnosed ce , 40 non - ce patients with different parasitic infections and malignancy and from 20 sex and age - matched healthy controls . samples from all subjects were centrifuged at 2000g for 10 minutes at 4 c to obtain the serum . the sera was divided in to 3 tubes for each subject and stored immediately at 70 c until analysis . all ce patients were sampled 1 week before treatment and received antihelminthic treatment of albendazole , 400 mg twice a day for 3 months , plus praziquantel , 40 mg / kg / day for two weeks as per standard guidelines ( 8) and underwent surgical procedures . all procedures were approved by the local ethical committee and all subjects gave their informed consent to the study . the results were compared between preoperative and post - operative group and were evaluated statistically using paired t test . hydatid cyst fluid antigen ( hcf ) was prepared according to standard procedure as described earlier ( 9 ) . briefly , the hydatid cyst fluid ( hcf ) was aseptically aspirated from fertile hydatid cysts obtained from livers of naturally infected sheep slaughtered at the local abattoir . the aspirated fluid was centrifuged at 2000g for 20 min at 4 c to remove the protoscolices . the supernatant was then filtered through a whatman wcn type membrane filter ( cellulose nitrate , 47 mm diameter , 0.45 m pore size ) and dialyzed against distilled water overnight at 4c using dialysis tubing ( sigma aldrich , usa ) with molecular weight cut off 2000 da ( dalton ) . the antigen protein concentration was estimated by the lowry method ( 10 ) with bovine serum albumin ( bsa ) as a reference standard . hydatid sheep antigen ( 40 g ) was subjected to discontinuous sds - page , using 12.5% gel in a bio - rad apparatus at 50 ma / gel for 1 hour . the proteins separated were transferred from un - stained gels to nitrocellulose membranes ( schleicher & schuell , inc . , keene , u.s.a . ) by means of the phasttransfer ( pharmacia lkb ) following standardized procedures ( 11 ) . efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloro acetic acid ) . the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05% tween 20 ; ph 7.4 ) for 2 hours . the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 2 hours at room temperature . after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti - human igg ( sigma ) at a dilution of 1/2000 ( in washing buffer+1% bsa ) for 2 h at room temperature . after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 + 6 mg / ml dab in 50 mm tris - hcl , ph 7.6 ) . molecular weight estimates were made by comparing the mobility of the different antigen fractions with these of prestained protein marker , broad range ( 8250 kda ) , ( new england biolabs , usa ) which were separated in the same gel of a hydatid fluid sample and electrotransferred to the same sheet . hydatid cyst fluid antigen ( hcf ) was prepared according to standard procedure as described earlier ( 9 ) . briefly , the hydatid cyst fluid ( hcf ) was aseptically aspirated from fertile hydatid cysts obtained from livers of naturally infected sheep slaughtered at the local abattoir . the aspirated fluid was centrifuged at 2000g for 20 min at 4 c to remove the protoscolices . the supernatant was then filtered through a whatman wcn type membrane filter ( cellulose nitrate , 47 mm diameter , 0.45 m pore size ) and dialyzed against distilled water overnight at 4c using dialysis tubing ( sigma aldrich , usa ) with molecular weight cut off 2000 da ( dalton ) . the antigen protein concentration was estimated by the lowry method ( 10 ) with bovine serum albumin ( bsa ) as a reference standard . hydatid sheep antigen ( 40 g ) was subjected to discontinuous sds - page , using 12.5% gel in a bio - rad apparatus at 50 ma / gel for 1 hour . the proteins separated were transferred from un - stained gels to nitrocellulose membranes ( schleicher & schuell , inc . , keene , u.s.a . ) by means of the phasttransfer ( pharmacia lkb ) following standardized procedures ( 11 ) . efficacy of transfer was checked by staining the membrane with ponceau s stain ( 0.001 g / ml in 3% trichloro acetic acid ) . the membranes with blotted antigen were cut into strips and blocked with 5% ( w / v ) of skimmed milk in washing buffer ( 10 mm tris , 150 mm nacl , and 0.05% tween 20 ; ph 7.4 ) for 2 hours . the strips were incubated with test sera ( 1/100 dilution in washing buffer with 1% bovine serum albumin ( bsa ) for 2 hours at room temperature . after 3 washes ( each 15 min ) , the strips were incubated with horseradish peroxidase conjugated anti - human igg ( sigma ) at a dilution of 1/2000 ( in washing buffer+1% bsa ) for 2 h at room temperature . after 3 washes as before , bound antigens was developed using diaminobanzidine ( dab ) substrate ( 0.1% h2o2 + 6 mg / ml dab in 50 mm tris - hcl , ph 7.6 ) . molecular weight estimates were made by comparing the mobility of the different antigen fractions with these of prestained protein marker , broad range ( 8250 kda ) , ( new england biolabs , usa ) which were separated in the same gel of a hydatid fluid sample and electrotransferred to the same sheet . in the present study the age of patients varied between 969 years . the mean sd age of the patients was 31.1211.24 years . the highest incidence of disease was recorded among patients between 20 to 49 years age . the predominance of hydatidosis was in females ( 68% ) than in males ( 32% ) . thirty four ( 34 ) of the radiologically and surgically confirmed cases had hepatic cysts while 16 had extrahepatic cysts ( 12-lung cysts , 3-liver and lung and 1-thigh cyst ) . all patients responded to pharmacological and surgical treatment except for two women ( 32 and 36 years old ) in whom multiple cysts ( 12 and 7 cysts ) were detected in liver and lung two years after the first operation . igg reactivity from patients with cystic echinococcosis revealed multiple immunoreactive bands ranging from 18239 kda ( fig . igg immunoreactivity of hydatid sera against different antigenic fractions in pre - operative samples the major immunoreactive bands were 24 , 39 , 46 , 52 , 57 , 6061 and 63 kda ( table 1 ) . sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . comparative evaluation of igg immunoreactivity with antigenic fractions in pre and post - operative samples antigenic fractions of 24 kda and 39 kda decreased significantly in six month ; one year and two year follow up samples the antigenic fractions of 46 , 52 , 57 , 6061 and 63 kda present before surgery in higher number of samples persisted significantly in post surgical six month , one year and two year follow up samples the healthy control sera were not reactive to any antigenic fraction ( table 2 ) . therefore the antigenic bands with molecular weight of 52 , 24 , 39 , 46 , 63 and 6061 kda which were reactive to 72.09% , 62.79% , 60.46% , 48.83% , 34.88% and 30.23% samples of pre - operative patients were specific for hydatid disease , suggesting thereby that theses antigenic fractions have higher diagnostic value . similarly the 39 kda band reactivity decreased significantly from 60.46% in pre - operative samples to 35.71% with six month , 21.42% with one year and 7.5% with two year follow - up samples ( table 1 , fig . however 24 kda and 39 kda persist in 2 patients who had a relapse at two year follow - up . igg antibody immunoreactivity with antigenic fractions in control serum samples igg immunoreactivity of hydatid sera against different antigenic fractions in post - operative samples the antigenic fractions of 52 , 46 , 39 and 24 kda in combination may serve as useful diagnostic markers . immunoreactive bands of 52 , 46 and 57 kda present in pre - operative samples persisted in higher number of post - operative samples . the percentage immunoreactivity of 24 and 39 kda decreased significantly within two year post - operative samples . the 24 kda immunoreactivity decreased from 62.79% in pre - operative samples to 30.95% with six month , 19.04% with one year and 7.5% with two year follow - up samples . igg reactivity from patients with cystic echinococcosis revealed multiple immunoreactive bands ranging from 18239 kda ( fig . igg immunoreactivity of hydatid sera against different antigenic fractions in pre - operative samples the major immunoreactive bands were 24 , 39 , 46 , 52 , 57 , 6061 and 63 kda ( table 1 ) . sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . comparative evaluation of igg immunoreactivity with antigenic fractions in pre and post - operative samples antigenic fractions of 24 kda and 39 kda decreased significantly in six month ; one year and two year follow up samples the antigenic fractions of 46 , 52 , 57 , 6061 and 63 kda present before surgery in higher number of samples persisted significantly in post surgical six month , one year and two year follow up samples the healthy control sera were not reactive to any antigenic fraction ( table 2 ) . therefore the antigenic bands with molecular weight of 52 , 24 , 39 , 46 , 63 and 6061 kda which were reactive to 72.09% , 62.79% , 60.46% , 48.83% , 34.88% and 30.23% samples of pre - operative patients were specific for hydatid disease , suggesting thereby that theses antigenic fractions have higher diagnostic value . similarly the 39 kda band reactivity decreased significantly from 60.46% in pre - operative samples to 35.71% with six month , 21.42% with one year and 7.5% with two year follow - up samples ( table 1 , fig . 2 ) . however 24 kda and 39 kda persist in 2 patients who had a relapse at two year follow - up . igg antibody immunoreactivity with antigenic fractions in control serum samples igg immunoreactivity of hydatid sera against different antigenic fractions in post - operative samples the antigenic fractions of 52 , 46 , 39 and 24 kda in combination may serve as useful diagnostic markers . immunoreactive bands of 52 , 46 and 57 kda present in pre - operative samples persisted in higher number of post - operative samples . the percentage immunoreactivity of 24 and 39 kda decreased significantly within two year post - operative samples . the 24 kda immunoreactivity decreased from 62.79% in pre - operative samples to 30.95% with six month , 19.04% with one year and 7.5% with two year follow - up samples . different conventional serological techniques for the diagnosis of human cystic echinococcosis have been developed , but their diagnostic value is limited because of the variable sensitivity and specificity ( 12 ) . in recent years , western blotting has created a new era in immunodiagnosis , which greatly reduces cross - reactions . this technique has been reported to give specific results in human studies ( 13 , 14 ) . therefore , the present study was designed to use this technique for the diagnosis and post - operative follow - up of ce patients . the igg reactivity in the sera of pre - operative patients identified multiple immunoreactive bands with the molecular weight ranging from 18239 kda . our results are in agreement with previous study , where the predominance of 24 , 3234 , 4446 and 5254 kda was observed in sera of patients with hepatic and pulmonary cystic echinococcosis . kanwer et al . , ( 16 ) also noticed the antigenic bands of high molecular weight ranging from 8116 kda with the frequent occurrence of 8 kda , 16 , 24 , 38 , 45 , 58 kda in ce patients . the antigenic bands of 1214 , 16 , 20 , 2426 , 34 , 39 and 42 kda in molecular weight enabled the detection of antibodies in the pre - surgical samples and antibodies specific to 39 and 42 kda disappeared in less than one year in the patients cured after surgery in ce patients ( 17 ) . al - olayan and helmy , ( 18 ) observed the antigenic bands of 22 , 24 , 35 , 38 , 40 , 45 , 50 , 55 , 6065 , 80 , 97 and 110 kda were recognised by sera from hydatidosis patients . there are not enough studies about the immunoreactivity of igg with high molecular mass antigenic bands in patients with ce ( 16 , 19 , 20 ) . the results revealed that 57 kda band cross - reacts with patient sera containing antibodies of other parasitic infections such as ascariasis , amoebiasis , toxoplasmosis and malignancy . the non - specificity may be caused mainly by a sharing of hydatid antigen with those of other parasites ( 21 ) but other possible reasons are interaction with some blood group antigen ( 22 ) or with non - specific host proteins in hydatid fluid ( 23 ) . the variability in the results could be due to many reasons , the source of antigen used as sheep hydatid fluid ( 15 ) , camel hydatid fluid ( 18 ) , human hydatid fluid ( 24 ) , the differences in purification protocols may also change the ability of antigens to recognized the specific antibodies ( 25 ) , differences in experimental condition , differences in echinococcus strains and geographical / clinical characteristics of patients ( 26 ) . the results verified all the purified antigenic bands except 57 kda were specific to hydatid antibodies as they did not cross - react with the antibodies of other parasitic infections . the application of western blotting for monitoring the surgical treatment is a subject that is not available in the literature . with the use of different antigenic bands , it was verified that western blotting technique shows a disappearance of some bands in the post - operative case , as well as the persistence of some bands . this latter situation agrees with the differences in the reactivity of the different isotypes of immunoglobulins with the hydatid antigens , observed with the western blotting technique applied to sera of patients suffering hepatic hydatidosis ( 20 ) . the western blotting appears to be a useful method for diagnosing hydatidosis and for post - surgical monitoring , incapable at the moment of being detected by any other immunologic technique , given that the other techniques react with all the antigens without the ability of observing the appearance or disappearance of the bands observed only with the western blotting . in this sense , the antibodies against proteins of 24 , and 39 kda constitute a good marker for post - surgical monitoring , since in the cases of surgically cured patients these disappear in post - operative samples , while they remain as long as cysts persist . further studies are also required to analyze the behaviour of these antibodies after medical treatment . the western blotting appears to be a useful method for post - surgical monitoring because the antibodies against proteins of 24 and 39 kda constitute a good marker for post - surgical monitoring , since in the cases of cured patients these disappear in postoperative samples , while they remain as long as cysts persist . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

background : cystic echinococcosis ( ce ) is one of the most important parasitic zoonosis worldwide . due to the high recurrence rate of the disease after surgery , follow up of the patient is necessary . the aim of current research was to assess the performance of western blotting ( wb ) , using sheep hydatid fluid , for serological diagnosis and post - treatment monitoring of human ce.methods:serum samples obtained from 50 clinically / radiologically proven cases of ce along with serum samples from non - ce patients and healthy persons were tested by wb , using sheep hydatid fluid as antigen.results:the wb test enabled the detection of antibodies in the pre - operative samples for proteins of 18239 kda in molecular weight . from 50 sera of ce patients , 31 cases ( 72.09% ) detected 52 kda subunit , 27 cases ( 62.79% ) detected 24 kda band , 26 cases ( 60.46% ) recognised 39 kda band and 21 cases ( 48.83% ) identified 46 kda component of sheep hydatid antigen . sera from patients with other parasitic infections and malignancy showed cross - reactivity with the cluster of 5459 kda bands . the healthy control sera were not reactive to any antigenic fraction . the antigenic bands with molecular weight of 52 , 24 , 39 and 46 kda were specific for ce , and may serve as useful diagnostic markers . the antibodies specific to proteins 24 and 39 kda significantly decreased in the patients cured after surgery , while in patients with recurrent parasitism the bands present before surgery persisted.conclusion:the wb with sheep hydatid antigen might be useful in the diagnosis and post - surgical monitoring of ce patients .

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obesity is a common metabolic disease world - wide and dyslipidemias among the most common metabolic disorders associated with obesity [ 17 ] . obesity is often described by classical parameters such as body mass index ( bmi ) , waist circumference ( wc ) , and waist - to - hip ratio ( whr ) [ 13,7,8 ] . results of meta - regression analysis of prospective , randomized studies showed that whr and wc may play important roles in assessment of increased risk of cardiovascular ( cv ) events . in recent years , attention has been drawn to new obesity indices ( e.g. , waist - to - height ratio ( whtr ) , visceral adiposity index ( vai ) , and body adiposity index ( bai ) , which also take into account adipose tissue distribution [ 1316 ] . results of the some recently published studies suggest that whtr , the newer obesity index , may be helpful for assessing risk of metabolic complications , including dyslipidemia , and it may also be a predictor of cardiovascular events in obese patients [ 1,8,1620 ] . nevertheless , there is little data about the associations between other newer obesity indices ( e.g. , vai and bai ) and lipid profile in obese patients . therefore , the aim of this study was to examine the relationship between obesity parameters or indices ( bmi , wc , whr , whtr , bai , vai ) and serum lipid levels of total cholesterol ( tc ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , triglycerides ( tg ) , and lipid indices such as tc / hdl - c , ldl - c / hdl - c , and tg / hdl - c ratios in obese non - diabetic patients . this cross - sectional study was conducted in a population of 66 consecutively enrolled obese non - diabetic patients ( bmi 30 kg / m ) with mean age of 55.811.6 years , including 40 women ( mean age 57.510.8 years ) and 26 men ( mean age 53.312.3 years ) treated in the outpatient department of hypertension and lipid disorders of wam university hospital in lodz , poland . patients with fasting hyperglycemia 5.6 mmol / l ( 100 mg / dl ) underwent a glucose tolerance test ( 75 g ) to exclude diabetes mellitus ( dm ) . patients with dm , pregnancy , cancer , acute stroke , acute hepatic or renal diseases and acute cardiovascular events or with history of abdominal surgery , which could have an impact on abdominal fat distribution , were excluded from the study . patients with psychiatric disorders receiving antipsychotic agents that might impact appetite and lead to weight gain were also excluded . based on calculated bmi values , patients were divided into 3 groups according to their obesity level : group i consisted of patients with obesity class i with bmi of 30.034.9 kg / m ; group ii consisted of patients with obesity class ii with bmi of 35.039.9 kg / m ; and group iii consisted of patients with obesity class iii with bmi 40 kg / m . the study was conducted after receiving approval from the bioethics committee of the medical university of lodz , resolution no . all the patients underwent a complete blood count , lipid and liver profile , tsh , glucose , renal function indices ( bun and creatinine ) , and general urinalysis . the serum ldl - c level was calculated based on friedewald s formula ( in mmol / l ) : measurements accurate to within 0.1 kg were made of body mass , and measurements accurate to within 0.5 cm were made of height , wc , and hc . wc was measured at mid - distance between the last rib and the iliac crest , while hc was measured at the level of the greater trochanter . based on these measurements , the following body mass - related indices were calculated using the following formulas : bmi = body mass ( kg ) / [ height ( m ) ] whr = wc ( cm ) / hc ( cm ) whtr = wc ( cm ) / height ( cm ) men vai = [ wc / 39.68 + ( 1.88 bmi ) ] ( tg / 1.03 ) ( 1.31 / hdl ) ; women vai = [ wc / 36.58 + ( 1.89 bmi ) ] ( tg / 0.81 ) ( 1.52 / hdl ) . bai = [ hip circumference ( cm ) / height ( m ) ] 18 [ 1315,18 ] . in addition , based on the lipid profile results obtained , the following lipid metabolism indices were calculated : tc / hdl - c ratio values > 5 indicate increased cardiovascular risk ; ldl - c / hdl - c ratio increased cardiovascular risk with values > 3 ; tg / hdl - c ratio increased cardiovascular risk with values 3 . the results are presented as mean values with standard deviation ( normality of distribution was tested using the shapiro - wilk test ) . significance of differences between groups was assessed using the t - test for independent samples , and in the case of multiple comparisons ( more than 2 study groups ) , univariate analysis of variance ( anova ) was applied with subsequent use of post - hoc multiple comparison tests ( tukey s test ) . for statistical analysis of discontinuous results , values of p<0.05 were assumed to be statistically significant . to assess the relationship and correlation between obesity indices and lipid metabolism parameters , pearson s linear correlation analysis was used , along with a linear regression model with a 95% confidence interval . this cross - sectional study was conducted in a population of 66 consecutively enrolled obese non - diabetic patients ( bmi 30 kg / m ) with mean age of 55.811.6 years , including 40 women ( mean age 57.510.8 years ) and 26 men ( mean age 53.312.3 years ) treated in the outpatient department of hypertension and lipid disorders of wam university hospital in lodz , poland . patients with fasting hyperglycemia 5.6 mmol / l ( 100 mg / dl ) underwent a glucose tolerance test ( 75 g ) to exclude diabetes mellitus ( dm ) . patients with dm , pregnancy , cancer , acute stroke , acute hepatic or renal diseases and acute cardiovascular events or with history of abdominal surgery , which could have an impact on abdominal fat distribution , were excluded from the study . patients with psychiatric disorders receiving antipsychotic agents that might impact appetite and lead to weight gain were also excluded . based on calculated bmi values , patients were divided into 3 groups according to their obesity level : group i consisted of patients with obesity class i with bmi of 30.034.9 kg / m ; group ii consisted of patients with obesity class ii with bmi of 35.039.9 kg / m ; and group iii consisted of patients with obesity class iii with bmi 40 kg / m . the study was conducted after receiving approval from the bioethics committee of the medical university of lodz , resolution no . all the patients underwent a complete blood count , lipid and liver profile , tsh , glucose , renal function indices ( bun and creatinine ) , and general urinalysis . the serum ldl - c level was calculated based on friedewald s formula ( in mmol / l ) : measurements accurate to within 0.1 kg were made of body mass , and measurements accurate to within 0.5 cm were made of height , wc , and hc . wc was measured at mid - distance between the last rib and the iliac crest , while hc was measured at the level of the greater trochanter . based on these measurements , the following body mass - related indices were calculated using the following formulas : bmi = body mass ( kg ) / [ height ( m ) ] whr = wc ( cm ) / hc ( cm ) whtr = wc ( cm ) / height ( cm ) men vai = [ wc / 39.68 + ( 1.88 bmi ) ] ( tg / 1.03 ) ( 1.31 / hdl ) ; women vai = [ wc / 36.58 + ( 1.89 bmi ) ] ( tg / 0.81 ) ( 1.52 / hdl ) . bai = [ hip circumference ( cm ) / height ( m ) ] 18 [ 1315,18 ] . in addition , based on the lipid profile results obtained , the following lipid metabolism indices were calculated : tc / hdl - c ratio values > 5 indicate increased cardiovascular risk ; ldl - c / hdl - c ratio increased cardiovascular risk with values > 3 ; tg / hdl - c ratio increased cardiovascular risk with values 3 . the results are presented as mean values with standard deviation ( normality of distribution was tested using the shapiro - wilk test ) . significance of differences between groups was assessed using the t - test for independent samples , and in the case of multiple comparisons ( more than 2 study groups ) , univariate analysis of variance ( anova ) was applied with subsequent use of post - hoc multiple comparison tests ( tukey s test ) . for statistical analysis of discontinuous results , values of p<0.05 were assumed to be statistically significant . to assess the relationship and correlation between obesity indices and lipid metabolism parameters , pearson s linear correlation analysis was used , along with a linear regression model with a 95% confidence interval . there were no statistically significant differences in clinical characteristic between compared groups ( table 1 ) . among persons enrolled in the study , the largest group consisted of dyslipidemic patients , accounting for 90.9% of the general study population , while hypercholesterolemia was present in 43.9% , mixed hyperlipidemia in 36.4% , and hypertriglyceridemia in 10.6% of subjects ( table 1 ) . hypertension was diagnosed in 83.3% patients from the whole study population ( table 1 ) . patients from group iii had greater mean wc compared to group ii and i ( table 2 ) . patients in obesity class ii also had greater mean wc than in group i. mean whtr , bmi , and bai values were higher in group iii compared to group ii and group i ( table 2 ) . higher mean whtr and bmi were observed in group ii compared to group i ( table 2 ) . mean serum tc and ldl - c in group iii were significantly lower than in group i ( table 2 ) . negative significant correlations were found in the whole study population between : bmi and tc , ldl - c , hdl - c ; wc , whr , vai and hdl - c ( table 3 ) . by contrast , positive significant correlations were found between wc , whr , and tg / hdl - c , as well as between vai and : tc , tg , tc / hdl - c , ldl / hdl - c , and tg / hdl - c ( table 3 ) . lipid disorders were the most frequent concomitant disorders in our study population . the high incidence of dyslipidemia in obese patients was also shown in 2 large polish epidemiological studies interestingly , the group of patients with class iii obesity had lower mean serum levels of tc and ldl - c compared with patients from the other 2 groups , and this difference was statistically significant when compared to the patients with class i obesity . this result was not related to lipid - lowering treatment because of lack of statistically significant differences between compared groups . similar results were obtained in the study lipidogram 2004 , where higher serum levels of tc , ldl - c , and tg were found in overweight people compared with the obese . on the other hand , the negative correlations observed in our study between bmi , wc , and ldl - c might have resulted from a lower ldl - c level in patients with morbid obesity as previously observed by others [ 2628 ] . found a 2-phase relationship between bmi , wc , and ldl - c levels , especially in women . increase in bmi and wc values initially resulted in increases in ldl - c levels , subsequently leading to reductions in the most obese persons . in native americans , increase in ldl - c levels was positively correlated with bmi only for the first 3 quartiles , then this relationship was reversed . the authors suggest that increase in adipose tissue in this population may decrease ldl - c levels in 2 ways : through increased activity of ldl - c receptors in adipose tissue or because of diluting the circulating pool of ldl - c through the increased circulating blood volume in more obese people . it must also be noted that levels of small , dense ldl ( sdldl ) fractions , as in our study , were not measured and a preponderance of sdldl induced by insulin resistance can lead to significant reductions in ldl - c . howard et al . concluded , based on results of clinical studies , that the relationship between bmi and ldl - c serum levels is complex and depends on numerous factors such as age or sex . it was demonstrated that higher ldl - c levels were seen in young ( 2044 years of age ) obese women , and were positively correlated with bmi . the authors did not observe significant differences between patients from different obesity classes with regards to levels of hdl - c and tg . in the strong heart study conducted in 773 women and 739 men of native american origin , hu et al . found significant negative correlation between bmi and hdl - c in women . wc values were positively correlated with serum tg levels and negatively correlated with hdl - c levels , and the relation between wc and assessed lipid fraction levels in women was much stronger than in the case of bmi . in men , positive correlations were observed between bmi , wc , and tg levels , as well as a negative correlation between bmi , wc , and hdl - c levels , although , in contrast with women , the strength of correlation was comparable for both obesity parameters . data obtained from the available literature suggest that increasing bmi results in decreasing hdl - c levels and increasing tg levels . these studies , however , were conducted in general populations rather than selected groups of patients with obesity . these observations are also supported by the results of a study conducted in 1518 adult peruvians , where the authors found a very strong negative correlation between bmi , vai , and the hdl - c fractions , as well as a positive correlation with tg ; however , this was irrespective of gender . we also found a negative correlation between bmi , wc , whr , and hdl - c , but not with tg . similar findings were observed in the prospective epidemiological study of myocardial infarction ( prime ) and by mojiminiyi et al . . the negative correlation between wc and hdl - c levels observed in our study is supported by results presented by hu et al . ; however , unlike the present authors , no statistically significant correlation between bmi , wc , and tg was found , although a similar tendency was present . the above - mentioned negative correlation between wc and hdl - c was also reported by chehrei et al . in 750 non - obese people . the results of our study suggest that obesity , especially abdominal obesity , is significantly associated with dyslipidemia , which manifests itself mainly with reductions in hdl - c . it is especially evident with the vai index , which demonstrated the strongest correlation with tg and hdl - c levels and with the lipid indices tc / hdl - c , ldl - c / hdl - c , and tg / hdl - c . this is not surprising , however , given the fact that the vai formula includes both the hdl - c and tg levels . the positive correlation observed in our study between whr and tg / hdl - c ratio is supported by results obtained by marrotta et al . results of our study demonstrate a relationship between some selected obesity indices , especially those pertaining to visceral obesity , and lipid parameters . this is especially the case with new , recently proposed obesity indices , such as vai , whtr , and bai , which have been investigated in only a few studies to date [ 1,1316 ] . one strength of this study is that there is relatively little information on obese patients grouped according to obesity level , especially among the morbidly obese ( bmi 40 kg / m ) . most studies have assessed the relationship between obesity indices and lipid metabolism parameters in the general population , and sometimes in non - overweight or healthy people . obesity is associated with lipid disturbances , especially with hdl - c reduction , in obese non - diabetic patients . vai is strongly related with lipid profile and thus may be the most valuable obesity index in obese patients with dyslipidemias .

backgroundthe aim of this cross - sectional study was to examine the relationship between obesity and lipid markers.material/methodswe divided 66 non - diabetic adult obese patients ( mean age : 55.811.6 years ) into 3 groups according to body mass index ( bmi ) . all patients were measured for waist circumference ( wc ) , hip circumference ( hc ) , body mass index ( bmi ) , waist - to - hip ratio ( whr ) , waist - to - height ratio ( whtr ) , body adiposity index ( bai ) , and visceral adiposity index ( vai ) . serum levels of total cholesterol ( tc ) , high - density lipoprotein cholesterol ( hdl - c ) , low - density lipoprotein cholesterol ( ldl - c ) , and triglycerides ( tg ) were determined , and lipid indices tc / hdl , ldl / hdl , and tg / hdl were also estimated.resultstc and ldl - c in group iii were lower than in group i ( 5.01.0 vs. 6.01.0 mmol / l , and 2.90.9 vs. 3.81.2 mmol / l ; p<0.05 for both ) . negative correlations were found between : bmi and tc , ldl , and hdl ( r=0.291 ; r=0.310 , r=0.240 , respectively ) ; and wc , whr , vai , and hdl ( r=0.371 , r=0.296 , r=0.376 , respectively ) . positive correlations were found between wc , whr , and tg / hdl ( r=0.279 , r=0.244 , respectively ) and between vai and : tc ( r=0.327 ) , tg ( r=0.885 ) , tc / hdl ( r=0.618 ) , ldl / hdl ( r=0.480 ) , and tg / hdl ( r=0.927).conclusionsobesity is associated with lipid disturbances , especially with hdl - c reduction , in obese non - diabetic patients . vai is strongly related to lipid profile and thus may be the most valuable obesity index in obese patients with dyslipidemias .

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there has been an explosive growth in the use of internet not only in india but also worldwide in the last decade . there were about 42 million active internet users in urban india in 2008 as compared to 5 million in 2000 . the internet is used by some to facilitate research , to seek information , for interpersonal communication , and for business transactions . on the other hand , it can be used by some to indulge in pornography , excessive gaming , chatting for long hours , and even gambling . there have been growing concerns worldwide for what has been labeled as internet addiction . griffith considered it a subset of behavior addiction and any behavior that meets the 6 core components of addiction , i.e. , salience , mood modification , tolerance , withdrawal , conflict , and relapse . while davis avoided the term internet addiction , referring it as a dependency on psychoactive substances , he instead preferred the term pathological internet use ( piu ) . young linked excessive internet use most closely to pathological gambling , a disorder of impulse control in dsm iv and adapted the dsm iv criteria to relate to internet use in the internet addiction test developed by her . according to her , various types of internet addiction are cyber - sexual addiction , cyber - relationship addiction , net compulsions , information overload , and computer addiction . his findings indicated that social isolation plays a greater role in behavioral symptoms of piu than does the presence of psychopathology . hence , caplan suggested replacing the term pathological internet use with problematic internet use . the prevalence found by greenfield was about 6% among the general population , while scherer found it to be 14% among the college - based population . surveys conducted online revealed that 4 - 10% of the users meet the criteria for internet addiction . the addicted averaged 38.5 h / week on a computer , whereas the non - addicted averaged 4.9 h / week . measuring goldberg developed the internet addictive disorder ( iad ) scale by adapting the dsm iv . brenner developed the internet - related addictive behavior inventory ( irabi ) comprising of 32 true and false questions . young initially developed 8-question internet addiction diagnostic questionnaire ( dq ) based on dsm iv . later , she included 12 new items in addition to the 8 items to formulate an internet addiction test ( iat ) . young 's iat is the only available test whose psychometric properties have been tested by widyanto and mcmurran . in india , hence , it was found necessary to study pattern of internet usage in young adults in indian setting and its relationship with their mental and physical health . with this background the aim of the study was to study the prevalence of internet addiction and association of any psychopathology in college going student population in the city of mumbai . the aim of the study was to study the prevalence of internet addiction and association of any psychopathology in college going student population in the city of mumbai . the cross - sectional survey was carried out in 3 different colleges in the city of mumbai during the period of august it covered about 1000 college students ( aged 16 - 18 years ) having access to the internet from the past 6 months . the semi - structured proforma along with the scales were distributed in classes , each with roughly 40 students , and necessary instructions were given . the study was conducted after obtaining the approval from the institutional review board and permission was sought from the college authorities of all the colleges . of the total 1000 students , 13 could not be included in the study as they were not using internet . the tools used in this study were as follows : semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . data was collected from those using internet for at least since last 6 months.the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study.according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use.the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . there are 11 scales with maximum score for each scale being 100 and minimum being 0 . six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study . according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use . the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . there are 11 scales with maximum score for each scale being 100 and minimum being 0 . six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . the cross - sectional survey was carried out in 3 different colleges in the city of mumbai during the period of august it covered about 1000 college students ( aged 16 - 18 years ) having access to the internet from the past 6 months . the semi - structured proforma along with the scales were distributed in classes , each with roughly 40 students , and necessary instructions were given . the study was conducted after obtaining the approval from the institutional review board and permission was sought from the college authorities of all the colleges . of the total 1000 students , 13 could not be included in the study as they were not using internet . the tools used in this study were as follows : semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . data was collected from those using internet for at least since last 6 months.the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study.according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use.the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . there are 11 scales with maximum score for each scale being 100 and minimum being 0 . six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . semi - structured proforma that contained details of demographics , educational qualification and status , purpose of using the internet ( by choosing among the options like education , entertainment , business transactions , or social networking ) , money spent per month , place of access ( home , cybercaf , or workplace if working part - time ) , the time of day when the internet is accessed the most ( by choosing between morning , afternoon , evening , or night ) , and the average duration of use per day . the internet addiction test ( iat ; young , 1998 ) is a 20-item 5-point likert scale that measures the severity of self - reported compulsive use of the internet . total internet addiction scores are calculated , with possible scores for the sum of 20 items ranging from 20 to 100 . the scale showed very good internal consistency , with an alpha coefficient of 0.93 in the present study . according to young 's criteria , total iat scores 20 - 39 represent average users with complete control of their internet use , scores 40 - 69 represent over - users with frequent problems caused by their internet use , and scores 70 - 100 represent internet addicts with significant problems caused by their internet use . the duke health profile is a 17-item generic questionnaire instrument designed to measure adult self - reported functional health status quantitatively during a 1-week time window . there are 11 scales with maximum score for each scale being 100 and minimum being 0 . six scales ( i.e. , physical health , mental health , social health , general health , perceived health , and self - esteem ) measure function , with high scores indicating better health . five scales ( i.e. , anxiety , depression , anxiety - depression , pain disability ) measure dysfunction , with high scores indicating greater dysfunction . most extensive use was in family practice patients with the broadest spectrum of diagnoses , but it was used in patient populations with specific diagnoses such as insulin - dependent diabetes mellitus , end - stage renal disease , ischemic disease , and impotence . both internal consistency ( cronbach 's alpha ) and temporal stability ( test - retest ) testing have supported reliability of the duke . validity has been supported for the duke scales by ( a ) comparison of the duke scores with scores of other health measures for the same patients , ( b ) comparison of duke scores between patient groups having different clinical diagnostic profiles and severity of illness , and ( c ) prediction of health - related outcomes by duke scores . in the present study , out of 1000 students , 13 were excluded and the remaining 987 adolescents who participates included 681 ( 68.9% ) females and 306 ( 31.1% ) males . subjects had more girls than boys as the data was collected during routine lectures and the attendance of girls might have been more the subjects belonged to different streams : 33.5% to science , 30.3% to commerce , and 36.2% to arts . using young 's original criteria , the users were divided into groups : 74.5% as moderate users , 24.8% as possible addicts , and 0.7% as addicts . males in comparison to females were significantly more likely to be addicted ( x2=10.2 , p=0.006 ) . moderate users and the possible addicts used the internet mostly for social networking , academic purposes , chatting , emailing , gaming , and downloading media files and pornography . they indulged more in social networking , chatting , and downloading media files ( x2=76 , p<0.001 ) . significantly , most of the addicts used the internet mostly in the evening and nights as compared to other users who used it in the mornings and afternoons as well ( x2=26.4 , p=00019 ) . interesting findings were noted with respect to the place of accessing internet . about half of the adolescents with addiction were also working part - time and this finding too was statistically significant ( x2=144 , p<0.001 ) . in this study , no significant relationship was found between internet addiction and the hours of use per day . moreover , the criteria used in iat does not take into consideration the exact duration of use . losing track of time while being online and staying online longer than intended are more likely to be seen in addicts . using duke 's health profile , it was found that addicts have poor mental , physical , and mental health score [ figure 1 ] . addicts had high anxiety , depression , and anxiety depression score ( x2=12.26 , p<0.0022 ) [ table 1 ] . internet use and psychopathology a number of studies have been conducted across the world , especially among adolescents with respect to internet addiction . this study is a preliminary step toward understanding the extent of internet addiction among college students in india . the findings of the present study corroborates with previous studies stating that addiction is more common in males than in females(anderson et al . ) . also , the finding that addicts are more likely to use their workplace to access internet is comparable to internet abuse at workplace reported by young and case . this could be a confounding factor for comparison as our study had students as subjects . employers have found that employees with access to the internet at their desks spend a considerable amount of their working day engaging in non - work - related internet use ( beard , 2002 ) . in a study done previously , young concluded that depression assessment should be done in suspected cases of piu . although it is not clear whether depression precedes the development of internet abuse or it is a consequence , yet assessment of the same is imperative . in 2005 , a study by nemiz et al . dealt with british university students showed that those students who were pathological internet users had low self - esteem and were socially inhibited online . young showed that withdrawal from significant real - life relationships is a consequence of pathological internet users . in the last one decade , internet has become an integral part of our life . it can put into three groups using young 's original criteria : 74.5% as moderate users , 24.8% as possible addicts , and 0.7% as addicts . those towards the addict part of spectrum reported had high anxiety , depression , and anxiety depression score . in the emerging era , where young people have been more exposed to the internet and use online activity as an important form of social interaction . however , it may still remain a matter of debate whether to call internet addiction a distinct disorder by itself or a behavioral problem secondary to another disorder . at present whether or not we will have any such diagnosis included in the future is yet to be seen . in future , if it is added , it is more likely to be classified as an impulse control disorders not elsewhere classified rather than in the diagnostic criteria for substance dependence . by studying the association of internet usage and its effects on human behavior , we can formulate interventions like setting boundaries and detecting early warning signs of underlying psychopathology at the earliest . in the emerging era , where young people have been more exposed to the internet and use online activity as an important form of social interaction . however , it may still remain a matter of debate whether to call internet addiction a distinct disorder by itself or a behavioral problem secondary to another disorder . at present whether or not we will have any such diagnosis included in the future is yet to be seen . in future , if it is added , it is more likely to be classified as an impulse control disorders not elsewhere classified rather than in the diagnostic criteria for substance dependence . by studying the association of internet usage and its effects on human behavior , we can formulate interventions like setting boundaries and detecting early warning signs of underlying psychopathology at the earliest .

background : there has been an explosive growth of internet use not only in india but also worldwide in the last decade . there is a growing concern about whether this is excessive and , if so , whether it amounts to an addiction.aim:to study the prevalence of internet addiction and associated existing psychopathology in adolescent age group.materials and methods : a cross - sectional study sample comprising of 987 students of various faculties across the city of mumbai was conducted after obtaining institutional ethics committee approval and permission from the concerned colleges . students were assessed with a specially constructed semi - structured proforma and the internet addiction test ( iat ; young , 1998 ) which was self - administered by the students after giving them brief instructions . dukes health profile was used to study physical and psychosocial quality of life of students . subjects were classified into moderate users , possible addicts , and addicts for comparison.results:of the 987 adolescents who took part in the study , 681 ( 68.9% ) were female and 306 ( 31.1% ) were males . the mean age of adolescents was 16.82 years . of the total , about 74.5% were moderate ( average ) users . using young 's original criteria , 0.7% were found to be addicts . those with excessive use internet had high scores on anxiety , depression , and anxiety depression.conclusions:in the emerging era of internet use , we must learn to differentiate excessive internet use from addiction and be vigilant about psychopathology .

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the frequency response of the resonators was measured using a downmixing method , described in detail in . the device was electrostatically actuated , and the driving voltage was applied to a side - gate parallel to the resonator . to reduce parasitic signals , the drive signal was set to half the actuation frequency 2 , thus the amplitude of motion of the resonator was proportional to the square of the actuation voltage . a bias voltage at ( + ) through the gauges was used to down - mix their resistance change ( occurring at the actuation frequency ) , and the low - frequency readout signal at was detected using a lock - in amplifier . typical measurement values were 1.5 v for the bias voltage at a measurement frequency of 500 khz . all measurements were performed in a vacuum chamber at a pressure of 10 mbar and at room temperature . thermomechanical noise was measured using the same set - up , with the drive electrode disconnected . measurements were taken with a lock - in amplifier , which also generated the drive and bias signals . the allan deviation was measured in open - loop configuration , and the frequency stability was extracted from the response of the resonator actuated at resonance frequency with a fixed driving frequency . the phase of the measured signal , (t ) , was monitored for a certain amount of time , and then transformed into frequency fluctuations using the phase response of the resonator . close to the resonance frequency , this phase response was linear , f2qf0 . using the complete phase response of the resonator instead of this linearization does not significantly alter the allan deviation . harmonics appearing at the frequency of the electricity supply ( multiples of 50 hz ) were filtered out of data during post - processing . using this method , we obtained n samples of the resonance frequency of the resonator f1fn , each averaged over an integration time , 0 . the allan deviation for this integration time could then be defined as : ( 2)a(0)=12(n1)1n1(fi+1fif0)2 to obtain the frequency stability for higher integration times from the same set of frequency samples , we followed the standard method . initial samples were averaged in groups of n samples , and the allan deviation for the new array was calculated using equation ( 2 ) to determine a(n0 ) . this process was repeated multiple times until the number of samples was too low to provide a statistically significant result . correlation measurements were performed by simultaneously measuring the response of the resonator at different frequencies within the resonator s bandwidth . the measurement set - up was based on the one described in supplementary section 3 , but here each signal was doubled , using two drive signals at different frequencies , two bias signals , and two measurement signals ( supplementary figure s9 shows a detailed measurement scheme ) . particular care was taken when choosing the drive signal amplitudes so that the resonator remained in the linear regime . moreover , the two measurement frequencies were chosen to avoid cross - talk ( e.g. 302 khz and 367 khz ) . although here we used a down - mixing set - up , correlation could also be measured with a homodyne method . the phase traces were converted to frequency traces corresponding to the different integration times , as described above . here , the complete phase response of the resonator was used rather than the linear approximation , as the frequencies for phase samples can be quite different from the resonance frequency . with this method we obtained two frequency sample arrays with an integration time 0 . the graph in figure 4b shows the correlation of these frequency traces versus the integration time . we processed the signals so that the correlation for a given only depends on frequency variations with characteristic time close to . for each of the plot , we filtered the two frequency traces with a band - pass filter centered on . for a consistent correspondence between allan deviation and correlation integration times , we chose the allan deviation transfer function as the band - pass filter , defined as : ( 3)ha(f)2=2sin4f(f)2 finally , the correlation coefficient of the filtered frequency traces f1 and f2 , each of length n , was defined by : ( 4)corrf1f2=i=1n(f1,if1)(f2,if2)nsf1sf2 where f1 and f2 are the sample means of f1 and f2 , respectively , and sf1 and sf2 are their standard deviations . the frequency response of the resonators was measured using a downmixing method , described in detail in . the device was electrostatically actuated , and the driving voltage was applied to a side - gate parallel to the resonator . to reduce parasitic signals , the drive signal was set to half the actuation frequency 2 , thus the amplitude of motion of the resonator was proportional to the square of the actuation voltage . a bias voltage at ( + ) through the gauges was used to down - mix their resistance change ( occurring at the actuation frequency ) , and the low - frequency readout signal at was detected using a lock - in amplifier . typical measurement values were 1.5 v for the bias voltage at a measurement frequency of 500 khz . all measurements were performed in a vacuum chamber at a pressure of 10 mbar and at room temperature . thermomechanical noise was measured using the same set - up , with the drive electrode disconnected . measurements were taken with a lock - in amplifier , which also generated the drive and bias signals . the allan deviation was measured in open - loop configuration , and the frequency stability was extracted from the response of the resonator actuated at resonance frequency with a fixed driving frequency . the phase of the measured signal , (t ) , was monitored for a certain amount of time , and then transformed into frequency fluctuations using the phase response of the resonator . close to the resonance frequency , this phase response was linear , f2qf0 . using the complete phase response of the resonator instead of this linearization does not significantly alter the allan deviation . harmonics appearing at the frequency of the electricity supply ( multiples of 50 hz ) were filtered out of data during post - processing . using this method , we obtained n samples of the resonance frequency of the resonator f1fn , each averaged over an integration time , 0 . the allan deviation for this integration time could then be defined as : ( 2)a(0)=12(n1)1n1(fi+1fif0)2 to obtain the frequency stability for higher integration times from the same set of frequency samples , we followed the standard method . initial samples were averaged in groups of n samples , and the allan deviation for the new array was calculated using equation ( 2 ) to determine a(n0 ) . this process was repeated multiple times until the number of samples was too low to provide a statistically significant result . correlation measurements were performed by simultaneously measuring the response of the resonator at different frequencies within the resonator s bandwidth . the measurement set - up was based on the one described in supplementary section 3 , but here each signal was doubled , using two drive signals at different frequencies , two bias signals , and two measurement signals ( supplementary figure s9 shows a detailed measurement scheme ) . particular care was taken when choosing the drive signal amplitudes so that the resonator remained in the linear regime . moreover , the two measurement frequencies were chosen to avoid cross - talk ( e.g. 302 khz and 367 khz ) . although here we used a down - mixing set - up , correlation could also be measured with a homodyne method . the phase traces were converted to frequency traces corresponding to the different integration times , as described above . here , the complete phase response of the resonator was used rather than the linear approximation , as the frequencies for phase samples can be quite different from the resonance frequency . with this method we obtained two frequency sample arrays with an integration time 0 . the graph in figure 4b shows the correlation of these frequency traces versus the integration time . we processed the signals so that the correlation for a given only depends on frequency variations with characteristic time close to . for each of the plot , we filtered the two frequency traces with a band - pass filter centered on . for a consistent correspondence between allan deviation and correlation integration times , we chose the allan deviation transfer function as the band - pass filter , defined as : ( 3)ha(f)2=2sin4f(f)2 finally , the correlation coefficient of the filtered frequency traces f1 and f2 , each of length n , was defined by : ( 4)corrf1f2=i=1n(f1,if1)(f2,if2)nsf1sf2 where f1 and f2 are the sample means of f1 and f2 , respectively , and sf1 and sf2 are their standard deviations .

frequency stability is key to performance of nanoresonators . this stability is thought to reach a limit with the resonator s ability to resolve thermally - induced vibrations . although measurements and predictions of resonator stability usually disregard fluctuations in the mechanical frequency response , these fluctuations have recently attracted considerable theoretical interest . however , their existence is very difficult to demonstrate experimentally . here , through a literature review , we show that all studies of frequency stability report values several orders of magnitude larger than the limit imposed by thermomechanical noise . we studied a monocrystalline silicon nanoresonator at room temperature , and found a similar discrepancy . we propose a new method to show this was due to the presence of frequency fluctuations , of unexpected level . the fluctuations were not due to the instrumentation system , or to any other of the known sources investigated . these results challenge our current understanding of frequency fluctuations and call for a change in practices .

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cardiovascular disease ( cvd ) is the leading cause of death in the united states . in 2010 , cvds killed over 700 000 americans , and accounted for 29.4% of total deaths . moreover , cvd accounts for a significant portion of the financial burden of medical care , costing > $ 312.6 billion in healthcare spending and lost productivity in 2010 . aspirin therapy is a prevention measure that can reduce the risk of major cardiovascular events such as heart attack and stroke , and is recommended by the us preventive services task force to prevent heart attack and ischemic stroke . aspirin is used as a primary prevention measure to aid in the prevention of a first occurrence of cvd . it can also be used as a secondary prevention measure among individuals who have experienced a heart attack or stroke to prevent additional cardiovascular events . the american heart association recommends the use of lowdose aspirin daily for people at high risk of a heart attack and regular use of lowdose aspirin for heart attack survivors . a variety of studies have examined whether individuals at risk of having a heart attack in the united states were taking aspirin . however , many of these studies failed to ascertain whether a person was recommended to take aspirin by their physician or whether they were taking it on their own initiative . thus , these studies may be not an accurate assessment of physician recommendations for aspirin use . further , much of the past research has used various risk factors to determine whether people were at high risk of cvd , rather than using the framingham risk score ( frs ) as recommended by the us preventive services task force in 2009 , the 2010 american diabetes association / american heart association / american college of cardiology recommendations , and the 2008 american college of chest physician guidelines . prevention of cvd events is particularly important , and understanding physician recommendations for aspirin therapy is critical for delivering quality health care . our current understanding of who is being encouraged to take aspirin for cvd prevention is limited . we sought to evaluate patient use of aspirin and reported physician recommendations of aspirin therapy for cvd prevention , in a nationally representative sample . we analyzed the national health and nutrition examination survey ( nhanes ) for the years 20112012 . the nhanes samples the noninstitutionalized population of the united states by using a stratified multistage probability sample design . the national center for health statistics uses a multilevel weighting system to account for survey design and nonresponse . the current study is focused on adults 40 years of age and older , because the nhanes asked this sequence of questions about aspirin use only to adults aged 40 and older . the respondents were asked if their doctor recommended that they take lowdose aspirin for prevention of cvd , stroke , or cancer . individuals who reported a recommendation to take aspirin were also asked if they were following this advice . individuals who did not report a recommendation to take aspirin were asked if they were taking lowdose aspirin on their own . for the purposes of this analysis , these individuals were combined with those who were taking aspirin on their physician 's recommendations , in order to get a better understanding of who is taking aspirin . individuals who had never been told by a physician that they had a stroke or heart attack were considered candidates for primary prevention . their eligibility for guidelinerecommended use of aspirin was based on the assessment of their risk for coronary heart disease ( chd ) . high risk for chd was classified as having a 10year chd risk of > 10% according to the frs with no history of heart attack or stroke . low risk for chd was considered a risk of 10% according to the frs with no history of heart attack or stroke as stated in the 2010 american diabetes association / american heart association / american college of cardiology guidelines . the frs utilizes gender , age , total cholesterol , highdensity lipoprotein cholesterol , systolic blood pressure , use of blood pressure medications , and smoking status . for nhanes participants , systolic blood pressure was calculated by averaging 3 readings taken during the medical examination portion of the nhanes . the scores were grouped into 2 categories for this analysis : low risk ( having a 10 year risk of heart attack be < 10% on the framingham risk calculator ) and high risk ( 10% or above ) . for people with diabetes ( defined as having been told by a physician that the respondent had diabetes [ excluding gestational diabetes ] ) , as per the american diabetes association / american heart association / american college of cardiology guidelines , individuals scoring < 5% were considered low risk , and individuals scoring > 10% were considered high risk . individuals with diabetes who had an intermediate risk score between 5% and 10% were removed from the analysis because there is no firm guideline for this group regarding aspirin use from the american diabetes association / american heart association / american college of cardiology guidelines . we did , however , examine whether there were differences in being told to take aspirin and whether they were taking it , and how they compared to low and highrisk diabetes patients . individuals who reported that a doctor had previously told them that they had been diagnosed with a stroke or a heart attack were candidates for secondary prevention . although obesity is not a specific characteristic that would justify a recommendation of aspirin for primary prevention , it is possible that both patients and physicians may interpret the cvd risk inherent in obesity as justifying aspirin . the nhanes 20112012 does not ask questions regarding history of peptic ulcer or other gastrointestinal conditions that could increase risk of bleeding . it does , however , collect information about stomach cancer , a condition for which aspirin use is contraindicated . to control for this , we removed individuals with a history of stomach cancer from the analysis . age was selfreported and divided into 2 categories : 40 to 64 and 65 and older . race / ethnicity was also selfreported and was categorized as nonhispanic whites , nonhispanic blacks , hispanics , and asians / other . education was examined because of its potential impact on selfinitiation of aspirin use . as having access to care may affect whether patients receive appropriate care for ambulatorysensitive conditions including diabetes and cvd , we examined both whether the respondent had health insurance and a regular source of health care . insurance status was defined as whether or not the individual reported having health insurance , of any form , at the time of the interview . regular access to care was defined by whether the respondent reported having a place to regularly go to receive health care . the nhanes uses a stratified multistage probability sample design . to account for this complex design the analysis utilized the nesting capability in sudaan with variables provided in nhanes ( that account for stratum level differences and primary sampling unit differences ) for all analysis , along with the appropriate weight identified by national center for health statistics for the type of data being used in our analysis . utilizing these weights and sampling design variables allows us to account for the complexity of the sampling design in performing univariate analyses , tests , and logistic regression models and make population estimates for the noninstitutionalized adult population of the united states . we examined obesity status , demographics , and access to care characteristics of the respondents . we also examined the bivariate relationship between being told to take aspirin / taking aspirin on doctor 's orders / taking aspirin on their own with the individual 's high / low risk for chd based on frs / previous diagnosis of diabetes using tests to test for significant differences . we examined both primary prevention ( those who have not been told by their physician that they have had either a stroke and/or heart attack ) and secondary prevention ( have been told by their physician that they have had either a heart attack or stroke ) . we examined the bivariate relationship between being told to take aspirin and taking aspirin with risk level using tests to test for significant differences . we also conducted separate analyses stratified by race using tests to examine the impact of race on other independent variables as well as on the outcome variables . bivariate estimates for stratifying secondary prevention by heart attack or stroke were examined , but the sample size was too small for stratified estimates to produce reliable estimates . all bivariate calculations used the standard method of pairwise deletion to account for missing data . we computed forcedinclusion logistic regression to examine the relationship of risk status , age , gender , race , education level , obesity status , having insurance , and having a regular source of care on the likelihood of whether patients were told to take aspirin by their physicians . we conducted the regression for the primary prevention sample and again separately for individuals at high and low risk . we were unable to conduct logistic regressions for individual racial / ethnic groups because the sample size was too small to generate reliable estimates . we also conducted tests for multicollinearity of risk status ( based on the frs ) with obesity , age , and gender . specifically , we computed spearman 's correlations as well as examined the tolerance and variance inflation factors of independent variables in the regressions . the frs uses gender and age as variables in its construction , and obesity is a known risk factor for cvd . the respondents were asked if their doctor recommended that they take lowdose aspirin for prevention of cvd , stroke , or cancer . individuals who reported a recommendation to take aspirin were also asked if they were following this advice . individuals who did not report a recommendation to take aspirin were asked if they were taking lowdose aspirin on their own . for the purposes of this analysis , these individuals were combined with those who were taking aspirin on their physician 's recommendations , in order to get a better understanding of who is taking aspirin . individuals who had never been told by a physician that they had a stroke or heart attack were considered candidates for primary prevention . their eligibility for guidelinerecommended use of aspirin was based on the assessment of their risk for coronary heart disease ( chd ) . high risk for chd was classified as having a 10year chd risk of > 10% according to the frs with no history of heart attack or stroke . low risk for chd was considered a risk of 10% according to the frs with no history of heart attack or stroke as stated in the 2010 american diabetes association / american heart association / american college of cardiology guidelines . the frs utilizes gender , age , total cholesterol , highdensity lipoprotein cholesterol , systolic blood pressure , use of blood pressure medications , and smoking status . for nhanes participants , systolic blood pressure was calculated by averaging 3 readings taken during the medical examination portion of the nhanes . the scores were grouped into 2 categories for this analysis : low risk ( having a 10 year risk of heart attack be < 10% on the framingham risk calculator ) and high risk ( 10% or above ) . for people with diabetes ( defined as having been told by a physician that the respondent had diabetes [ excluding gestational diabetes ] ) , as per the american diabetes association / american heart association / american college of cardiology guidelines , individuals scoring < 5% were considered low risk , and individuals scoring > 10% were considered high risk . individuals with diabetes who had an intermediate risk score between 5% and 10% were removed from the analysis because there is no firm guideline for this group regarding aspirin use from the american diabetes association / american heart association / american college of cardiology guidelines . we did , however , examine whether there were differences in being told to take aspirin and whether they were taking it , and how they compared to low and highrisk diabetes patients . individuals who reported that a doctor had previously told them that they had been diagnosed with a stroke or a heart attack were candidates for secondary prevention . although obesity is not a specific characteristic that would justify a recommendation of aspirin for primary prevention , it is possible that both patients and physicians may interpret the cvd risk inherent in obesity as justifying aspirin . the nhanes 20112012 does not ask questions regarding history of peptic ulcer or other gastrointestinal conditions that could increase risk of bleeding . it does , however , collect information about stomach cancer , a condition for which aspirin use is contraindicated . to control for this , we removed individuals with a history of stomach cancer from the analysis . age was selfreported and divided into 2 categories : 40 to 64 and 65 and older . race / ethnicity was also selfreported and was categorized as nonhispanic whites , nonhispanic blacks , hispanics , and asians / other . education was examined because of its potential impact on selfinitiation of aspirin use . as having access to care may affect whether patients receive appropriate care for ambulatorysensitive conditions including diabetes and cvd , we examined both whether the respondent had health insurance and a regular source of health care . insurance status was defined as whether or not the individual reported having health insurance , of any form , at the time of the interview . regular access to care was defined by whether the respondent reported having a place to regularly go to receive health care . the nhanes uses a stratified multistage probability sample design . to account for this complex design the analysis utilized the nesting capability in sudaan with variables provided in nhanes ( that account for stratum level differences and primary sampling unit differences ) for all analysis , along with the appropriate weight identified by national center for health statistics for the type of data being used in our analysis . utilizing these weights and sampling design variables allows us to account for the complexity of the sampling design in performing univariate analyses , tests , and logistic regression models and make population estimates for the noninstitutionalized adult population of the united states . we examined obesity status , demographics , and access to care characteristics of the respondents . we also examined the bivariate relationship between being told to take aspirin / taking aspirin on doctor 's orders / taking aspirin on their own with the individual 's high / low risk for chd based on frs / previous diagnosis of diabetes using tests to test for significant differences . we examined both primary prevention ( those who have not been told by their physician that they have had either a stroke and/or heart attack ) and secondary prevention ( have been told by their physician that they have had either a heart attack or stroke ) . we examined the bivariate relationship between being told to take aspirin and taking aspirin with risk level using tests to test for significant differences . we also conducted separate analyses stratified by race using tests to examine the impact of race on other independent variables as well as on the outcome variables . bivariate estimates for stratifying secondary prevention by heart attack or stroke were examined , but the sample size was too small for stratified estimates to produce reliable estimates . all bivariate calculations used the standard method of pairwise deletion to account for missing data . we computed forcedinclusion logistic regression to examine the relationship of risk status , age , gender , race , education level , obesity status , having insurance , and having a regular source of care on the likelihood of whether patients were told to take aspirin by their physicians . we conducted the regression for the primary prevention sample and again separately for individuals at high and low risk . we were unable to conduct logistic regressions for individual racial / ethnic groups because the sample size was too small to generate reliable estimates . we also conducted tests for multicollinearity of risk status ( based on the frs ) with obesity , age , and gender . specifically , we computed spearman 's correlations as well as examined the tolerance and variance inflation factors of independent variables in the regressions . the frs uses gender and age as variables in its construction , and obesity is a known risk factor for cvd . a total of 3435 individuals answered questions about aspirin use , representing 142 677 272 americans . demographic characteristics of the sample , primary prevention group , lowrisk primaryprevention group , and highrisk primaryprevention group are shown in table 1 . characteristics of individuals eligible for primary prevention , individuals at high risk , and individuals at low risk na indicates not applicable . table 1 shows that among the total sample eligible for aspirin as primary prevention , less than a third reported being recommended by their doctor to take aspirin . table 1 also indicates that over three fourths of primaryprevention patients were classified as low risk . less than half of the primaryprevention patients who had a high chd risk level received a recommendation to take aspirin while > one fourth of lowrisk patients were also recommended to take aspirin ( table 1 ) . of those told to take aspirin by their physicians , the vast majority complied with the recommendation . specifically , 77.4% of the total sample , 79.0% of the highrisk patients , and 76.5% of the lowrisk patients reported taking aspirin ( table 1 ) . table 2 shows the bivariate relationships between the demographic variables , health insurance , having a regular place to go for health care , and having a diabetes diagnosis and being told by the physician to take aspirin as primary prevention . analysis examining differences between low , intermediate , and highrisk diabetes showed that among lowrisk diabetes patients , 54.6% were told to take aspirin , compared to 72.8% of intermediaterisk diabetes patients and 56.2% of highrisk diabetes patients ( p = 0.17 ) . bivariate results for patients told to take aspirin , for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk table 3 presents bivariate relationships among respondents who were taking aspirin after having received a recommendation to do so by their physician . in an additional subanalysis , there were no statistically significant differences for taking aspirin for low , intermediate , and highrisk diabetes patients , with 51.5% of lowrisk diabetes patients taking aspirin , 42.2% of intermediaterisk diabetes patients taking aspirin , and 47.1% highrisk diabetes patients taking aspirin ( p = 0.53 ) . bivariate results for taking aspirin among respondents told by their physicians to take aspirin , for individuals eligible for primary prevention there were a number of significant differences in demographic and accesstocare variables by race / ethnic group among primaryprevention patients , as shown in table 4 . bivariate results for demographic differences by race , for individuals eligible for primary prevention of the 8.3% of the total sample eligible for secondary prevention , 75.9% were told to take aspirin by their physician . of those who were told by their physicians to take aspirin , table 5 shows the results of 3 logistic regression models for being told to take aspirin among individuals eligible for primary prevention , only individuals at high risk , and only individuals at low risk . the logistic regression examining all patients without a history of heart attack or stroke that included risk level as an independent variable indicated that the frs was not predictive of receiving a physician recommendation for aspirin . among all primaryprevention patients , education level , insurance status , and having a regular source of care were predictive of receiving a recommendation for aspirin for cvd prevention . among highrisk individuals , age , health insurance , and being of asian / other descent were predictive of receiving a recommendation for aspirin for cvd prevention . among lowrisk individuals , age , health insurance status , education level , and obesity status were all predictive of receiving a recommendation for aspirin therapy . logistic regression results predicting aspirin recommendation for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk or indicates odds ratio . table 1 shows that among the total sample eligible for aspirin as primary prevention , less than a third reported being recommended by their doctor to take aspirin . table 1 also indicates that over three fourths of primaryprevention patients were classified as low risk . less than half of the primaryprevention patients who had a high chd risk level received a recommendation to take aspirin while > one fourth of lowrisk patients were also recommended to take aspirin ( table 1 ) . of those told to take aspirin by their physicians , the vast majority complied with the recommendation . specifically , 77.4% of the total sample , 79.0% of the highrisk patients , and 76.5% of the lowrisk patients reported taking aspirin ( table 1 ) . table 2 shows the bivariate relationships between the demographic variables , health insurance , having a regular place to go for health care , and having a diabetes diagnosis and being told by the physician to take aspirin as primary prevention . analysis examining differences between low , intermediate , and highrisk diabetes showed that among lowrisk diabetes patients , 54.6% were told to take aspirin , compared to 72.8% of intermediaterisk diabetes patients and 56.2% of highrisk diabetes patients ( p = 0.17 ) . bivariate results for patients told to take aspirin , for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk table 3 presents bivariate relationships among respondents who were taking aspirin after having received a recommendation to do so by their physician . in an additional subanalysis , there were no statistically significant differences for taking aspirin for low , intermediate , and highrisk diabetes patients , with 51.5% of lowrisk diabetes patients taking aspirin , 42.2% of intermediaterisk diabetes patients taking aspirin , and 47.1% highrisk diabetes patients taking aspirin ( p = 0.53 ) . bivariate results for taking aspirin among respondents told by their physicians to take aspirin , for individuals eligible for primary prevention there were a number of significant differences in demographic and accesstocare variables by race / ethnic group among primaryprevention patients , as shown in table 4 . of the 8.3% of the total sample eligible for secondary prevention , 75.9% were told to take aspirin by their physician . of those who were told by their physicians to take aspirin , 89.9% were taking aspirin . table 5 shows the results of 3 logistic regression models for being told to take aspirin among individuals eligible for primary prevention , only individuals at high risk , and only individuals at low risk . the logistic regression examining all patients without a history of heart attack or stroke that included risk level as an independent variable indicated that the frs was not predictive of receiving a physician recommendation for aspirin . among all primaryprevention patients , education level , insurance status , and having a regular source of care were predictive of receiving a recommendation for aspirin for cvd prevention . among highrisk individuals , age , health insurance , and being of asian / other descent were predictive of receiving a recommendation for aspirin for cvd prevention . among lowrisk individuals , age , health insurance status , education level , and obesity status were all predictive of receiving a recommendation for aspirin therapy . logistic regression results predicting aspirin recommendation for individuals eligible for primary prevention , individuals at high risk , and individuals at low risk or indicates odds ratio . the results of this study indicate that recommendations for aspirin for cvd prevention do not match up to the objectively computed future risk . for individuals at high risk , fewer than half report receiving recommendations for aspirin . moreover , in a multivariate analysis , objective risk level was not associated with an increased risk of recommendation for aspirin . this suggests that decisions for treatment recommendations seem to be being made on criteria other than objectively calculated future chd risk . only 40.9% of patients at high risk reported having been told by their physician to take aspirin . patient reports of recommendations for aspirin use among secondary prevention patients was 75.9% , but still suboptimal . respondents who were at low risk of developing cvd and had body mass indexes over 30 were more likely to be told to take aspirin for primary prevention than normalweight patients . the benefit of using aspirin in lowrisk patients who do not have clinical cvd is negated by the risk of harms from gastrointestinal bleeding . it is possible that the seemingly suboptimal rates of recommendations to take lowdose aspirin are the result of the provider weighing the benefits of aspirin against the very real risks of upper gastrointestinal bleeding . lin et al have shown that the risk of upper gastrointestinal bleeding is 90% higher among lowdose aspirin users who are using aspirin for primary prevention compared to nonusers and 40% higher for secondaryprevention patients . the number needed to harm per 1 year of lowdose aspirin use was 601 for primaryprevention patients and 391 for secondaryprevention patients . patients with a history of upper gastrointestinal bleeding and peptic ulcers also have a higher incidence of upper gastrointestinal bleeding , as do patients with concomitant use of clopidogrel or nonsteroidal antiinflammatory drugs . all of these factors may have contributed to the lack of recommendations for aspirin use to highrisk patients . first , nhanes does not account for hemorrhagic stroke ( 13% of all strokes ) and aspirin use would be inappropriate for these patients . second , secondaryprevention patients could be taking other blood thinners that would obviate the need for lowdose aspirin . in order to better understand physician prescribing practices of aspirin and patient usage of aspirin , additional research of aspirin use in the context of other antiplatelet and anticoagulant use the apparent disconnect between risk for future cvd and aspirin recommendations may benefit from improved clinical decision support . helping to compute the actual risk rather than simply identifying general risk factors should aid in making accurate decisions . actual risk could be computed in electronic health records , which could also provide the physician with the recommendation for treatment . in addition , such tools could provide other affiliated treatment recommendations such as use of gastricprotective agents in select patient populations . in addition to a significant disconnect between who needs and who receives recommendations for aspirin use , the analysis indicated a persistent problem with access to care and receipt of recommendations for aspirin use . lack of health insurance can be a significant barrier to accessing health care , especially for preventive services . previous research has found no relationship between insurance status and aspirin use among patients who accessed clinical preventive services . these results suggest that access to care may play a role in recommendations for the advice of aspirin for cvd prevention , and the use of it by patients . the present results may differ from previous studies in that the previous studies did not assess whether the provider recommended aspirin for cvd prevention . race and ethnicity did have an impact among primaryprevention patients , with nonhispanic whites more likely to receive a recommendation for aspirin than were asians / others . additional analyses indicated that among primaryprevention patients , a greater percentage of nonhispanic whites had health insurance compared with other racial / ethnic groups . as with curative medical care , the ability to access preventive medical care is in large part influenced by insurance status . therefore , part of the racial / ethnic disparities in primary prevention that we observed are possibly attributable to lack of health insurance and a usual source of care . that a higher level of education for primaryprevention and lowrisk patients was predictive of lower likelihood of receiving a recommendation for aspirin was somewhat surprising , particularly that the finding emerged after controlling for race , age , gender , obesity , insurance status , and access to a regular source of care . it is possible that physicians may perceive more highly educated people as having a lower risk of heart disease , based on the known connection between educational attainment and health outcomes . this investigation relies on selfreport of whether or not an individual 's doctor recommended aspirin as prevention of heart attack , stroke , and cancer , as well as selfreport of a number of variables for analysis . selfreported data are very common in studies of health behaviors , but there are questions regarding the reliability of the data . patient selfreport can be unreliable , especially regarding behaviors and risk factors relevant to cvd and cancer . the national center for health statistics conducts rigorous testing of its survey instruments to ensure validity through the questionnaire design research laboratory , ensuring that all questions utilized are of high reliability and validity . in addition , lab and physical examination values were used when possible to minimize bias introduced by selfreport . an additional limitation is that the study only examines adults age 40 and over , as this is the only group that nhanes asks about aspirin use . there could be important patterns of use among younger individuals that this analysis can not look for . another limitation of this study is that we do not know how many respondents were recommended to take aspirin for heart attack or stroke prevention versus for cancer prevention . thus , it is possible that some proportion of aspirin recommendations may be for cancer prevention , rather than heart attack or stroke prevention . however , the us preventive services task force currently recommends against the use aspirin for the prevention of cancer , thereby suggesting that recommendations to take aspirin based on cancer prevention should be unlikely . in addition , this study did not control for use of warfarin or other anticoagulant / antiplatelet therapy among respondents . anticoagulant use could explain some of the deficit in the use and recommendations to take aspirin among primaryprevention patients . finally , because of limitations in what information is collected by the nhanes , we do not know what role the risk of gastrointestinal bleeding played in the decision of physicians to recommend aspirin . gastrointestinal bleeding is a significant issue that we would expect to have an impact on the likelihood of a physician recommending aspirin use for patients who may otherwise benefit from it . the available data from nhanes do not allow us to elucidate who is at greater risk for gastrointestinal bleeding and for whom a recommendation to take aspirin would be inappropriate . highrisk patients who are not receiving the recommendation are put at increased risk of cvd events . lowrisk patients are exposed to adverse risk with unnecessary use of aspirin based upon a physician 's recommendation . it is clear that quality of care for primary prevention falls far short of ideal . further research on clinical decision support systems and interventions designed to reinforce the use of appropriate risk calculation is necessary to ensure that patients receive appropriate preventive care .

backgroundaspirin use has been shown to be an effective tool in cardiovascular disease ( cvd ) prevention among highrisk patients . the patientreported physician recommendation for aspirin as preventive therapy among high and lowrisk patients is unknown.methods and resultswe conducted an analysis of the national health and nutrition examination survey 20112012 to examine the use of aspirin for cvd prevention . patients without previously diagnosed cvd were classified into high and low risk based on their framingham risk score ( 10year coronary heart disease risk ) . among patients without previously diagnosed cvd , 22.5% were classified as high risk . of the highrisk individuals , 40.9% reported being told by their physician to take aspirin , with 79.0% complying . among those who were at low risk , 26.0% were told by their physician to take aspirin , with 76.5% complying . logistic regression analysis indicated that age , access to a regular source of care , education , and insurance status were significant predictors of patientreported physician recommendations for aspirin use for primary prevention . among highrisk patients , age , race , and insurance status were significant predictors of reported recommendations for aspirin use . among lowrisk patients , age , education , obesity , and insurance status were significant predictors of reported recommendations for aspirin use.conclusionspatient reports indicate nonideal rates of being told to take aspirin , for both high and lowrisk patients for primary prevention . clinical decision support tools that could assist physicians in identifying patients at risk may increase patient reports of physician recommendations for aspirin use .

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mitochondria produce reactive oxygen species ( ros ) during oxidative metabolism through the single - electron reduction of molecular oxygen ( o2 ) . the resulting superoxide anion ( o2 ) , is converted to hydrogen peroxide ( h2o2 ) by cellular superoxide dismutases ( sods ) . similar to phosphorylation , ubiquitination , and acetylation , oxidation of protein cysteine residues represents a form of reversible , posttranslational protein modification which can regulate signaling . oxidation of cysteine thiol groups ( sh ) by h2o2 results in formation of sulphenic acid ( soh ) which quickly forms disulfide ( ss ) or sulfenyl amide ( sn ) bonds , changing protein function . the cellular thioredoxin and glutathione reductase systems restore these oxidatively modified residues back to their reduced forms in a manner analogous to phosphatases , deubiquitinating enzymes , or histone deacetylases . mitochondrial ros ( mros ) generation is required for the propagation of numerous cellular signaling pathways including those regulating tumorgenesis , immune responses , and cellular adaptation to stresses such as hypoxia . in most cases it is unknown exactly what oxidative events are required for the effects of ros on these pathways and it is likely that multiple oxidative targets potentiate transduction through a given pathway . increasing evidence suggests that mitochondria and mros generation play key roles in cellular differentiation programs . cellular mitochondrial content and oxidative capacity are increased when mouse embryonic stem cells ( escs ) , induced pluripotent stem cells ( ipscs ) , or mesenchymal stem cells are induced to differentiate . furthermore , increased cellular ros levels and oxidation products correlate with the differentiation of escs and ipscs as well as mesenchymal , neural , and epithelial stem cells . hematopoietic stem cells ( hscs ) contain significantly lower levels of ros than do the more - differentiated common myeloid progenitor cells . thus increased mitochondrial and oxidant content appears to correlate with stem cell differentiation while low mitochondrial mass and oxidant content correlates with stem cell maintenance . in our recent report , we used the mammalian epidermis as a model system to test the hypothesis that mitochondrial metabolism and ros production promote signaling through pathways required for differentiation of mammalian stem cell populations in vivo . the epidermis is a self - renewing stratified squamous epithelium and is thus regulated by stem cell populations . to maintain epidermal homeostasis , cells within the proliferative basal layer withdraw from the cell cycle and differentiate as they process outward through the suprabasal layers , compensating for cellular loss via desquamanation from the outermost epidermal layer . the epidermis also elaborates appendages such as hair follicles ( hfs ) which are themselves regulated by stem cell populations . multiple transcriptional networks are associated with differentiation within the epidermis including notch , p63 , c / ebp , and ap2 ( interfollicular epidermis ) and -catenin ( hf ) . it is incompletely understood how these various factors are regulated to promote epidermal homeostasis and what role cellular metabolism might play in this regulation . to test the hypothesis that mitochondria play an active role in the regulation of epidermal homeostasis , we conditionally deleted tfam ( transcription factor a , mitochondrial ) in basal , undifferentiated epidermal keratinocytes . tfam is required for the replication and transcription of the mitochondrial genome , and cells lacking tfam are unable to conduct oxidative phosphorylation or produce mros . mice conditionally lacking tfam in epidermis ( tfam cko mice ) lacked hair and developed an epidermal barrier defect which contributed to their perinatal mortality . similar to in vivo , primary keratinocytes derived from tfam cko mice displayed impaired differentiation in vitro . differentiation of wild - type keratinocytes was inhibited by antioxidant treatment , and differentiation marker expression in tfam cko cells could be partly restored by treatment with exogenously applied h2o2 , clearly demonstrating that oxidative signaling promotes keratinocyte differentiation . we went on to demonstrate that mros generation is required for activation of the notch and -catenin transcriptional programs that promote epidermal differentiation and hair growth respectively . together , our results show that mros act as pro - differentiation signals and are key upstream regulators of stem cell fate decisions . while the role for mros in signal transduction is becoming increasingly accepted , the identities of the exact targets of oxidation which promote signaling remain unknown in most situations . our work identified nucleoredoxin ( nxn ) as a putative target of mros - mediated wnt--catenin signaling . nxn is a member of the thioredoxin family which has previously been demonstrated to regulate -catenin - dependent transcription in a redox - sensitive manner . our results show that nxn becomes oxidized when wild - type keratinocytes are treated with the -catenin activator wnt-3a . this did not occur in tfam cko keratinocytes , suggesting that mros - mediated nxn oxidation plays a causal role in the transduction of signals between wnt receptors and -catenin . further studies will be required to determine how mros regulate notch transcriptional activity during keratinocyte differentiation . this raises the interesting possibility that aberrant keratinocyte metabolism may be associated with , and presents targets for , the treatment of epidermal disease . while the genetic causes of these diseases are increasingly studied , little is known about how disease - causing mutations might affect cellular metabolism . more generally , the interactions between cellular differentiation programs and cellular metabolism will be increasingly studied in years to come . the development of regenerative therapies depends on the ability to maximize the renewal capacity of stem cells , and subsequently , to promote complete differentiation into desired cell types . cellular metabolism represents an intriguing rheostat which may be used to modulate these ends . a further understanding of the mechanisms by which differentiation programs both regulate , and are regulated by metabolism will prove useful in this regard . proper regulation is likely dependent on cell type - specific factors as well as environmental influences such as hypoxia . in keratinocytes , as an epidermal calcium gradient exists in vivo , it is hypothesized that calcium concentrations regulate epidermal differentiation in vivo as well . calcium uptake into mitochondria is known to stimulate the activity of tca cycle enzymes ( pyruvate- , isocitrate- , and oxoglutarate - dehydrogenases ) , promoting mitochondrial respiration . we found that inhibition of mitochondrial calcium uptake prevented keratinocyte differentiation in vitro , suggesting that mitochondrial calcium uptake might promote the mitochondrial metabolism and ros production required for epidermal differentiation . a recent study suggests that mitochondrial uncoupling proteins may play a role in regulating differentiation - dependent metabolism . uncoupling protein 2 ( ucp2 ) is highly expressed in ipscs , with reduced expression in differentiated cells . ectopic ucp2 expression inhibited oxidative metabolism of pyruvate , reduced cellular ros content , and prevented differentiation suggesting that this protein could act as a metabolic switch that controls stem cell differentiation . how ucp2 expression is repressed during differentiation remains to be determined and it will also be of interest to determine if ucp2 regulates the metabolism of less - pluripotent , tissue - specific stem cell populations . beyond promotion of oxidant - dependent signaling , the mechanisms by which mitochondrial metabolism promotes cellular differentiation remain poorly understood . some differentiated lineages may require the efficient atp production provided by oxidative phosphorylation ; however , another intriguing possibility involves the nutrient - dependent regulation of chromatin ( fig . acetyl - coa , a key fuel for oxidative metabolism , is also a substrate used by histone acetyltransferases to acetylate histones and other cellular proteins . global increases in histone acetylation are associated with differentiation of escs , 3t3-l1 preadipocytes , and keratinocytes . treatment of primary human keratinocytes with histone deacetylase inhibitors is sufficient to induce cell cycle arrest and expression of several differentiation markers . although differentiation programs likely regulate histone acetylation in a gene and time - dependent manner ; these reports suggest that cellular differentiation may depend on availability of acetyl - coa to carry out acetylation reactions . in mammalian cells , most cytosolic acetyl - coa is produced through the cleavage of tca - cycle - derived citrate by the enzyme atp - citrate lyase ( acl ) . inhibition of acl expression prevents global increases in histone acetylation and differentiation of 3t3-l1 cells . electrons are transferred from tca cycle substrates to complexes i and ii of the electron transport chain via nadh and fadh2 respectively . these electrons are transferred to complex iii ( via coenzyme q ) and subsequently to complex iv ( via cytochrome c ) where they are used to reduce molecular oxygen to water . complexes i , ii , and iii of the respiratory chain contain sites in which electrons can prematurely react with oxygen , producing ros . our recent report demonstrates that mitochondrial ros production is required for activation of notch and -catenin transcriptional programs which promote epidermal differentiation and hair development respectively . mitochondrial metabolism may also promote keratinocyte differentiation by providing substrates for histone acetyltransferases ( hats ) and histone demethylases ( such as jmjd3 ) . histone methylation status is regulated by histone methyltransferases and demethylases with various methylation codes associated with either transcriptional activation or repression . one particular methyl modification trimethylation of histone 3 on lysine 27 ( h3k27me3)is associated with transcriptional repression of epidermal differentiation markers . genes such as keratin 1 and involucrin display high levels of h3k27me3 in undifferentiated keratinocytes , and lose this mark as cells differentiate . demethylation of h3k27me3 requires the demethylase jmjd3 and cells lacking jmjd3 expression are unable to differentiate in culture . jmjd3 is a member of the jumanji - c domain containing family of demethylases which use oxygen , fe(ii ) , and the tca cycle intermediate -ketoglutarate as cofactors and are thus potentially sensitive to cellular metabolic state . many other nutrient - sensitive mechanisms are likely to regulate stem cell maintenance and differentiation in both the epidermis and in other compartments . cancer researchers are currently striving to identify cancer - specific metabolic signatures which can be therapeutically targeted . metabolic study of cellular differentiation is an emerging area which , in concert with genetic approaches , has the potential to lead to strategies for targeted control of cell fate decisions and therapies for diseases associated with aging .

mitochondrial metabolism has traditionally been thought of as a source of cellular energy in the form of atp . the recent renaissance in the study of cellular metabolism , particularly in the cancer field , has highlighted the fact that mitochondria are also critical biosynthetic and signaling hubs , making these organelles key governors of cellular outcomes.1 - 5 using the epidermis as a model system , our recent study looked into the role that mitochondrial metabolism and ros production play in cellular differentiation in vivo.6 we showed that conditional deletion of the mitochondrial transcription factor , tfam within the basal cells of the epidermis results in loss of mitochondrial ros production and impairs epidermal differentiation and hair growth . we demonstrated that mitochondrial ros generation is required for the propagation of notch and -catenin signals which promote epidermal differentiation and hair follicle development respectively . this study bolsters accumulating evidence that oxidative mitochondrial metabolism plays a causal role in cellular differentiation programs . it also provides insights into the role that mitochondrial oxidative signaling plays in a cell type - dependent manner .

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hepatitis c virus ( hcv ) is a single - stranded rna virus , a member of the flaviviridae family . since the first identification of the hcv genome by choo et al . , hcv study has progressed mainly in the field of hcv functional analysis and therapeutic implications . because of low viral levels in the serum , the diagnosis of hcv infection has been made with a branched chain dna signal amplification assay and reverse - transcription polymerase chain reaction ( rt - pcr ) [ 24 ] . pathologists are often requested to detect viral pathogens within the routinely prepared biopsy tissue samples , and therefore simple and reliable histochemical techniques are needed . reports have described the detection of hcv in human liver tissue by using immunohistochemistry and in situ hybridization ( ish ) [ 414 ] , as well as by in situ rt - pcr [ 4 , 6 , 1520 ] . at the moment , hcv detection with immunohistochemistry and ish is not yet reproducible enough , we believe . in situ rt - pcr reliable histochemical techniques for detecting hcv in formalin - fixed , paraffin - embedded ( ffpe ) liver tissues are needed , particularly for routine diagnostic purpose . in the present study , we established an ish technique using a locked nucleic acid ( lna ) probe and biotin - free tyramide amplification system ( csaii ) for detecting hcv - rna in ffpe tissue sections of the liver . as positive controls , we sampled the humanized liver of hcv - infected chimeric mice [ 21 , 22 ] , and ffpe tissue sections were prepared . the chimeric mice were maintained by m. kohara , department of microbiology and cell biology , tokyo metropolitan institute of medical science , tokyo . a total of 13 humanized liver specimens from hcv - infected chimeric mice were used ( noninfected 3 , genotype 1a - infected 3 , genotype 1b - infected 4 , and genotype 2a - infected 3 ) . a total of 11 needle biopsy liver specimens of chronic hepatitis c were culled from the computer file at the diagnostic pathology division of fujita health university hospital , toyoake , japan , and ffpe sections were cut . needle biopsy specimens of hepatitis - b - virus- ( hbv-)infected chronic hepatitis , virus - negative fatty liver and autoimmune hepatitis ( n = 1 , resp . ) , as well as two surgically removed hcc lesions caused by hcv or hbv infection , were also examined . the nonneoplastic part of the liver tissue ( chronic hepatitis b ) in the hbv - related hcc case was also evaluated . non - neoplastic liver tissue was scarcely included in the hcv - related hcc case . the activity of inflammation and the degree of fibrosis were evaluated according to the new inuyama classification ( 1996 ) . serum hcv - rna levels in the patients were measured using an rt - pcr assay , the amplicor hcv monitor assay ( roche diagnostics ) , and serum hcv subtypes were determined with nested rt - pcr ( roche diagnostics ) . the sequence of an hcv - common probe was designed according to the previous description . the target was within the hcv 5-untranslated region ( hcv-5utr ) , which is a highly conserved portion in the hcv genome . an lna - modified oligonucleotide probe labeled with digoxygenin at the 3-end was prepared in gene design inc . , the nucleotide sequence of the 45-mer probe was 5-altttlggglcgtlgcclccclgcglagalctglctalgcclgagltagltgtltgglgt-3 , in which la , lt , lc , and lg represent lna monomers corresponding to the bases a , t , c , and g , respectively . endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol for 60 minutes at room temperature ( rt ) . after rinsing thrice in diethyl - pyrocarbonate- ( depc-)treated water , sections were digested with 40 g / ml proteinase k ( roche diagnostics , tokyo , japan ) at 37c for 15 minutes , washed thrice in depc - treated water , submerged in 95% ethanol for 1 minute , and air - dried completely . then , sections were heat - treated for 5 minutes at 95c on a hot plate and hybridized with 0.01 mol / l lna - modified oligonucleotide probe diluted with in situ hybridization buffer ( enzo life science , bulter pike , pa , usa ) in an incubation chamber overnight at 37c . after rinsing in 1x saline sodium citrate ( ssc ) for 30 minutes at 50c , sections were rinsed twice in 50 mm tris - hcl - buffered saline ( tbs ) , ph 7.6 , at rt . a horseradish - peroxidase - labeled antidigoxygenin antibody ( roche diagnostics ) diluted at 1 : 100 with 1% bovine serum albumin in tbs was applied for 60 minutes at rt . for amplifying signals , fluorescein - isothiocyanate- ( fitc-)conjugated tyramide enclosed in csaii ( dako , glostrup , denmark ) was applied to the sections for 15 minutes at rt . finally , a horseradish - peroxidase - labeled anti - fitc antibody equipped in the csaii kit was reacted for 30 minutes at rt . reaction products were visualized in 50 mm tris - hcl buffer , ph 7.6 , containing 20 mg / dl diaminobenzidine tetrahydrochloride and 0.006% hydrogen peroxidase . the nuclei were lightly counterstained with mayer 's hematoxylin . in order to verify the specificity of the ish technique for hcv - rna detection , we carried out the treatment with dnase and rnase before the hybridization with the lna probe on consecutive sections . namely , either 10 iu / ml dnase i ( wako , osaka , japan ) or 100 g / ml rnase ( sigma - aldrich , st . louis , mo , usa ) was incubated for 30 minutes at 37c . in order to distinguish mouse hepatocytes from chimeric human hepatocytes in hcv - infected chimeric mice , we utilized a human - specific ck8/18 monoclonal antibody ( clone : ncl 5d3 , mp biochemicals , santa ana , ca , usa ) . after blocking endogenous peroxidase activity , hydrated heat - assisted epitope retrieval was employed using a pressure pan cooker ( delicio 6l , t - fal , clichy , france ) for 10 minutes . preliminary study revealed that as a soaking solution , 1 mm ethylenediamine tetraacetic acid solution , ph 8.0 , was optimal to retrieve the antigenicity . after pressure cooking , sections were left for 30 minutes at rt for cooling . in order for avoiding nonspecific signals in mouse hepatocytes , a mouse stain kit ( nichirei bioscience , tokyo ) was applied before and after the primary antibody incubation . the human - specific ck8/18 monoclonal antibody at a 1 : 100 dilution was incubated overnight at rt . after rinsing in 10 mm phosphate - buffered saline ( pbs ) , ph 7.2 , the sections were reacted with a secondary polymer reagent , novolink ( novocastra , newcastle , uk ) . total rna was extracted from dewaxed sections using recoverall total nucleic acid isolation kit ( applied biosytems , austin , tx , usa ) , according to the manufacturer 's protocol , and stored at 80c until use . the present study was approved by the institutional ethical review board for clinical and epidemiological investigations at fujita health university , toyoake . in he - stained sections , mouse hepatocytes showed densely eosinophilic cytoplasm , while chimeric human hepatocytes were weakly eosinophilic with frequent deposition of fat droplets . immunoreactivity of human ck8/18 was seen only in human hepatocytes . in all the 10 hcv - infected livers but not the livers of three hcv noninfected mice , ish demonstrated diffuse cytoplasmic signals in the human hepatocytes . in figure 1 employing consecutive sections , hcv - rna was visualized with the ish technique in human ck8/18-positive chimeric hepatocytes from chimeric mice infected hcv genotypes 1a , 1b , and 2a ( in figure 1 , dotted lines encircle the mouse hepatocyte area ) . it is of note that hcv - rna was demonstrated in the cytoplasm of almost all chimeric human hepatocytes . the positive signals remained after dnase treatment but were completely abolished after rnase treatment . figure 2 illustrates hcv genotype 1b - infected chimeric mouse liver , showing he histology and positive cytoplasmic signals , which were resistant to dnase but sensitive to rnase . most of the hcv - positive patients showed high levels of hcv - rna in the serum . exceptionally , a surgical case of hcv - related hcc showed a low level of hcv - rna in the serum . nested rt - pcr genotyping for hcv using total rna extracted from ffpe sections was successful in 8 ( 73% ) of 11 biopsied chronic hepatitis c lesions , and in all the 8 cases , the genotypes were comparable with the serum analysis . lna - based ish detected signals in 9 ( 82% ) of 11 chronic hepatitis c lesions , including three showing diffuse cytoplasmic positivity , and six showing focal or partial positivity . in three nested rt - pcr negative lesions , ish revealed negativity ( 1 lesion ) or focal / partial positivity ( 2 lesions ) . no hcv - rna was detected in hbv - related lesions , fatty liver , autoimmune hepatitis , and in hcc lesions . nonspecific binding of the csaii reagent to kupffer cells or lymphoid cells was occasionally observed in both the hcv - infected and hcv - unrelated lesions , as arrows indicate . we showed herein that lna - based ish for hcv - rna yielded clear cytoplasmic reactivity in infected hepatocytes in ffpe sections . the lna - modified oligonucleotide probe we employed represents one of the most sensitive probes for ish [ 2529 ] . lna - based ish has been applied to detecting cellular microrna in ffpe tumor tissues [ 3032 ] . the 45-mer lna probe was targeted at the hcv-5utr containing a sequence common to the hcv subtypes . positive signals for hcv - rna were clearly demonstrated in hcv 1a- , 1b- , and 2a - infected humanized livers of chimeric mice . hcv - infected chimeric mice with humanized liver thus functioned as a useful model for histochemically demonstrating the hcv genome , particularly when immunostaining for human - specific ck8/18 was combined . in needle - biopsied human liver specimens , hcv - rna was detected in 8 of 11 hcv - infected samples with the nested rt - pcr analysis and in 9 of 11 with the ish technique . in nested rt - pcr negative cases , ish was negative or partially positive , suggesting that the detection threshold may depend on the virus load . hcv - rna was undetectable in the hepatocytes in hbv - infected livers , nonviral liver disorders ( fatty liver and autoimmune hepatitis ) , and in hcv - related hcc . with ish using a digoxygenin - labeled cdna probe , tang et al . reported positivity in 8 ( 67% ) of 12 hcv - related hcc . it has been documented with an in situ rt - pcr technique that hcv - rna decreases along with the progression from liver cirrhosis to hcc . more cases of hcc need to be analyzed , in relation to the distribution of the hcv genome in cancerous lesions . revie and salahuddin reviewed that hcv replicated in macrophages , b and t lymphocytes , and other nonhepatocellular components . however , we judged the nonhepatocellular bindings as nonspecific reactions , primarily because of the staining in non - hcv cases . under our present condition , nonspecific binding of the reagents , particularly csaii , to kupffer cells and lymphoid cells further technical improvement is requested for reliable and reproducible hcv - rna detection in ffpe specimens . in conclusion , the ish technique using the lna - modified oligonucleotide probe and csaii can be applied to detecting hcv - rna in liver biopsy specimens , the sensitivity probably being comparable to in situ rt - pcr . for detecting the hcv genome in routinely prepared liver biopsy specimens , our ish sequence is relatively simple and requires no special equipment to perform . at the moment , this method seems to be suitable for demonstrating hcv genome in ffpe sections and will become a valuable tool for routine histopathological diagnosis of hcv infection using ffpe liver biopsy specimens .

background . in situ hybridization ( ish ) with high sensitivity has been requested to demonstrate hepatitis c virus ( hcv ) rna in formalin - fixed , paraffin - embedded ( ffpe ) sections of the liver . methods . ish employing a locked - nucleic - acid- ( lna-)modified oligonucleotide probe and biotin - free catalyzed signal amplification system ( csaii ) was applied to hcv - rna detection in the liver tissue . nested reverse - transcription polymerase chain reaction ( rt - pcr ) was performed for hcv genotyping using total rna extracted from ffpe sections . the target tissues included ffpe tissue sections of humanized livers in hcv - infected chimeric mice ( hcv genotypes 1a , 1b , and 2a and noninfected ) and of needle - biopsied livers from hcv - infected patients . results . hcv - rna was demonstrated with the ish technique in hcv - infected liver tissues from both chimeric mice and 9 ( 82% ) of 11 patients with hcv infection . the hcv signals were sensitive to rnase . nested rt - pcr confirmed the genotype in 8 ( 73% ) of 11 livers ( type 1b : 6 lesions and type 2a : 2 lesions ) . hcv - rna was not identified in chronic hepatitis b lesions , fatty liver , autoimmune hepatitis , and hepatocellular carcinoma . conclusion . ish using the lna - modified oligonucleotide probe and csaii was applicable to detecting hcv - rna in routinely prepared ffpe liver specimens .

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there are many concerns about endocrine - disrupting chemical effects on wild animals as well as humans . considerable numbers of chemicals have been shown to alter the thyroid system in vertebrates , and disruption of the thyroid axis has been identified as an important consideration for the regulation of chemicals . thus , focusing on thyroid toxicity , the oecd and epa established guidelines for investigation of frog metamorphosis . because amphibian tadpoles have a thin , permeable skin and inhabit aquatic environments , they are exposed to toxicants through both dermal and dietary routes and appear to be particularly susceptible to a number of contaminants in wastewater discharged from agricultural fields and from industrial and household areas . in addition , the fact that frog metamorphosis is regulated by thyroid hormones ( ths ) , promoting the remodeling of the aquatic larvae into an adult tetrapod , means that the dramatic structural and functional changes of larval tissues can be readily applied as parameters reflecting endocrine disruption . 1 ) , show various morphological changes during metamorphosis and under conditions of exposure to certain compounds . here , the regulation by ths , focusing on affecting factors in addition to the metamorphosis assay , is reviewed . the thyroid gland secretes thyroxine ( t4 ) , which is converted to a more biologically active form , 3,3,5-triiodothyronine ( t3 ) , mainly in the peripheral target tissues . transport of ths in the blood is accomplished through binding to transport proteins , predominantly transthyretin ( ttr ) , with conjugation and degradation processes occurring in the liver and subsequent secretion in bile . some ths are coupled with bacterial deconjugation and undergo reabsorption in the intestine . th structures are conserved between vertebrates , and their production and secretion are essentially the same in amphibians and mammals . there are several processes that influence the thyroid hormone balance , such as sulfation , deiodination and glucuronidation ( see fig . 2 ) . both inhibition of thyroidal iodide uptake and suppression of thyroidal peroxidase activity can disrupt th synthesis and secretion . in addition , inhibition of 5-deiodinase in the peripheral tissue is related to reduction of conversion from t4 to t3 , while induction of hepatic microsomal enzymes such as phenol sulfotransferase and udp - glucuronyl transferase leads to increase in excretion of both t4 and t3 into bile . furthermore , competitive binding to thyroid transport proteins results in reduced levels of total and free ths in serum . therefore , there are many potential targets of environmental contaminants that could be involved in disruption of th metabolism . rahman and yamauchi found th sulfating activity to be present in the liver cytosol in frog tadpoles and that the t3:t4 sulfating activity ratio varies during developmental stages , sulfation being inhibited by chemical compounds such as halogenated phenol and phenolic compounds , p - nitrophenol , dopamine , 17beta - estradiol ( e2 ) and dihydroxyepiandrosterone . compared with sulfation , glucuronidation has been poorly studied in amphibians , in line with its character as a relatively minor pathway . however , a subset of the responsible enzymes , glucuronidases ( ugts ) , can reduce circulating levels of ths through biliary elimination as in mammals . regarding phase i species , hepatic microsomal cytochrome p450 2b1 was found to be induced by pentobarbital in adults of the semiaquatic frog , rana pipiens , but not in frog tadpoles and adults of the aquatic frog xenopus laevis . deiodinase enzymes , type 2 and 3 iodothyronine deiodinase , are found in several peripheral target tissues in amphibians , and the level of expression is closely connected with metamorphosis . the most characteristic feature of ttr from nonmammalian vertebrates , such as amphibians , is their higher affinity for t3 than for t4 . binding of chemicals to ttr decreases their effective free concentrations in plasma and changes the apparent affinity for ths , which can diminish cellular uptake and biological responses and would alter plasma th homeostasis . therefore , chemicals interfering with t3 binding to ttr may directly affect the free concentration of plasma t3 and its plasma clearance rate . chemical compounds including diethylstilbestrol ( des ) , phenolic and phenol compounds and pentachlorophenol and ioxynil have the potential to bind to ttr , and yamauchi et al . reported that with amphibian ttr , des possesses similar affinity to t3 , which is the natural ligand . the premetamorphic stage is the period until the appearance of the hind limbs , and the prometamorphic period is from their appearance to that of the forelimbs . during the period of metamorphic climax , resorption of the tail and gills and development of lungs amphibian metamorphosis is divided roughly into 3 stages , premetamorphosis , prometamorphosis and climax ( see fig . the premetamorphic stage is the period until the appearance of the hind limbs , and the prometamorphic period is from their appearance to that of the forelimbs . subsequently , during the period of metamorphic climax , resorption of tail and gills and development of lungs occur . for example , treatment of tadpoles with synthetic thyroid hormone receptor antagonists ( e.g. , nh-3 ) leads to retardation or even complete blockade of xenopus laevis ( x. laevis ) metamorphic development . in turn , exposure of x. laevis tadpoles to low nanomolar concentrations of t3 or synthetic thyroid hormone receptor agonists ( e.g. , gc-1 ) causes precocious induction of metamorphosis . the sensitivity to th varies according to the developmental stage , and the amount of spontaneously released thyroid stimulating hormone ( tsh ) is higher in late prometamorphic and climactic tadpoles than in early prometamorphic larvae and juvenile and adult frogs . low levels of circulating thyroglobulin are found in tadpoles at the early stages , but extremely high levels are evident at the metamorphic climax . in amphibians , both corticotrophin - releasing hormone ( crh ) and gonadotropin - releasing hormone ( gnrh ) , in addition to trh , have been considered as candidates for the tsh - releasing factor . in the bullfrog , crh was found to be the most potent tsh - releasing substance in both adult and larval pituitary cells , while both trh and gnrh moderately stimulated the release of tsh from the adult pituitary but not larval cells . kaneko et al . demonstrated that th suppressed the crh - induced release of tsh , but not the basal release , from larval , juvenile and adult bullfrog pituitaries in vitro . in addition , jacobs et al . reported that intravenously injected mammalian luteinizing hormone - releasing hormone ( lhrh ) was able to raise circulating levels of t4 as well as testosterone ( t ) in three frog species , rana ridibunda , rana temporaria and rana esculenta . regarding prolactin ( prl ) , inhibitory effects may be exerted on metamorphosis , but this is controversial . huang and brown concluded that prl does not play a role as a juvenile hormone in x. laevis , but overexpression of prl does specifically inhibit some but not all programs of tail resorption . research has indicated concomitant elevation of ths and corticoids as metamorphosis progresses . according to kikuyama et al . , the aldosterone plasma level is low prior to the onset of climax , but then there is a sharp rise , and combined adrenocorticotropin ( acth ) and t treatment causes a marked increase in concentration . gray and janssens observed that testosterone and e2 inhibited t3-induced metamorphosis in x. laevis tadpoles , while hogan et al . demonstrated a delay in the time for rana pipiens tadpoles to undergo metamorphosis when exposed to ethinylestradiol during either mid - metamorphosis or throughout the entire larval period . in the past few decades , several thyroid disrupting substances have been tested for toxicity using amphibians . xenopus and other anurans have been generally applied to assess the developmental effects of a variety of xenobiotics . there have been a considerable number of reports regarding inhibitors of t4 synthesis that inactivate peroxidases . after exposure to propylthiouracil ( ptu ) , also known to inhibit deiodinase , x. tropicalis tadpoles showed no signs of decline in body length or body weight but a considerable reduction in the developmental stage and hind limb length . reported effects of exposure to ptu and another peroxidase inhibitor ethylenthiourea ( etu ) , in x. laevis . metamorphic retardation caused by etu was associated with concentration - dependent histological changes in the thyroid gland and increased mrna expression of tsh - beta in the pituitary . exposed pre- and pro - metamorphic larvae to methimazole , ptu and t4 and induced changes in a concentration - dependent manner . methimazole and ptu caused a delay in larval development and morphological changes in the thyroid gland , which were characterized as reduced colloid , glandular hypertrophy and cellular hyperplasia and hypertrophy . on the other hand , t4 treatment resulted in a concentration - dependent increase in the developmental rate . one other potent inhibitor of t4 synthesis , perchlorate , disturbs iodide uptake by the follicular cells of the thyroid gland . ammonium perchlorate was found to inhibit forelimb emergence , hindlimb development and tail resorption , linked to significant hypertrophy of the thyroid follicular epithelium at concentrations below those reported in contaminated surface waters . showed that sodium perchlorate inhibits th synthesis via effects on the sodium - iodide symporter , resulting in retarded metamorphosis and histological effects on thyroid as the most sensitive endpoint . hu et al . reported the results of thyroid immunohistochemistry for t4 in x. laevis exposed to perchlorate ; t4 immunoreactivity was concentrated in a ring of colloid adjacent to follicle cells , independent of the developmental stage . theirs is the only report regarding this point of view , but they suggested the utility of this immunohistochemical biomarker , because the intensity of the colloidal t4 ring is more sensitive than any other morphological changes ( such as hind limb length , forelimb emergence , tail resorption , thyrocyte hypertrophy or colloid depletion ) . found that time until metamorphic transformation was prolonged , the body weights of froglets were increased after exposure to polychlorinated biphenyls ( pcb ) and t4 levels were lowered , although not significantly . established that bisphenol a ( bpa ) induced deceleration of both spontaneous and t4-induced metamorphic changes , with suppression of thyroid hormone receptor ( tr ) beta gene expression . polybrominated diphenyl ether ( bde-47 ) and a commercial pentabromodiphenyl ether mixture ( de-71 ) caused delay in tail regression in x. laevis . according to carlsson et al . , autoradiograms revealed high concentrations and long - term retention of 2,2,4,4,5-pentabromodiphenyl ether ( bde-99 ) in adipose tissue and melanin in frogs exposed as both tadpoles and juveniles , with lower developmental stages suggesting possible thyroid hormone disruption . recorded delayed metamorphosis and enlarged thyroid glands with follicular hyperplasia in x. tropicalis and x. laevis tadpoles exposed to the insecticide methoxychlor . regarding actual environmental contamination , sowers et al . reported that municipal wastewater effluent containing measurable amounts of a variety of pharmaceuticals , personal care products , industrial compounds and natural and synthetic hormones could alter the timing of the metamorphic process in frogs . in their latest report of a field study of the native wood frog in the athabasca oil sands region of northern alberta , canada , metamorphosis was delayed or not complete at the end of the 75 day study period . showed that the herbicide acetochlor accelerated forelimb emergence and the beginning of metamorphic climax , apparently by interacting with t3 , but also possibly by weakly opposing the inhibitory actions of corticosterone on development of premetamorphic tadpoles . the doses used in most of these studies were greater than the levels to which anurans are exposed in the natural environment , so further investigations are required to determine whether exposure to chemicals at environmentally relevant concentrations can affect frog metamorphosis . in contrast to the data showing a clear effect on metamorphosis of chemical compounds , some negative data have been reported . uv filters such as 4-methylbenzylidene camphor ( 4-mbc ) and 3-benzylidene camphor ( 3-bc ) have been found to not affect the thyroid system and the rate of metamorphosis at environmental concentrations , and thus , they seem not to exhibit thyroid - mimicking or antithyroid activity . carr et al . reported exposure to one herbicide , atrazine , did not exert any effects on metamorphosis . there are several reports concerning effects on amphibian larvae of triclosan , which is a bacteriocide used in personal care and consumer products such as shampoo , soaps , deodorants , toothpaste and plastic ware , and so on . the release of triclosan into the environment is of particular concern as it is structurally similar to th . conducted a glp study in compliance with the guidelines mentioned below , and indicated that environmentally relevant concentrations do not alter the normal course of thyroid - mediated metamorphosis in this standard anuran model . the amphibian hypothalamic - pituitary - thyroid ( hpt ) axis controls th levels in the same way as it does in mammals , although crh also plays a role . therefore , the amphibian metamorphosis assay ( ama ) represents a generalized vertebrate model to the extent that it is based on the functions of the hpt axis . on september 7 , 2009 , the organization for economic co - operation and development ( oecd ) adopted the ama using x. laevis as a guideline for testing of chemicals . the us environmental protection agency ( epa ) office of prevention , pesticides , and toxic substances ( oppts ) also includes the ama for detection of thyroid - active chemicals in tier 1 testing of their endocrine screening program . the epa developed the oecd test guidelines through a process of harmonization in october 2009 . previously , in 1998 , the oecd initiated a high - priority activity to revise existing guidelines and develop new guidelines for the screening and testing of potential endocrine disrupters . one element was a test guideline for the screening of substances active on the thyroid system of vertebrate species . the 1st oect expert consultation on endocrine disrupter testing in amphibians was held in 2001 . subsequently , the ama underwent extensive validation programs consisting of 3 phases , which included intra- and interlaboratory studies demonstrating the relevance and reliability of the assay . mainly the u.s . the primary objective was a comparative evaluation of the utility and sensitivity of two proposed exposure scenarios for detection of stimulating and inhibiting effects of thyroid system - disrupting substances . the united kingdom and switzerland also participated in this phase in addition to germany , japan and the united states . three model substances representing different modes of action on the thyroid system were used : sodium perchlorate , t4 and iopanoic acid ( iop ) . phase 3 studies were conducted to assess the utility of the ama protocol to detect weakly active thyroid system - disrupting substances and to distinguish thyroid system - related changes from activity resulting from mechanisms not directly related to thyroid system function . benzophenone-2 ( bp-2 ) and e2 were selected as weakly active and non- or indirectly related substances . as a result , the effect of a weakly antithyroid substance , bp-2 , was detected , while the endpoints with exposure to e2 did not indicate any effect on the thyroid system . subsequently , validation of the assay was subjected to peer review by a panel of independent experts . the primary endpoints and observation time points are mortality daily , developmental stage , hind limb length , snout - vent length and wet body weight on days 7 and 21 and thyroid gland histology on day 21 . the most prominent morphological staging landmark is hind limb morphology , which is positively associated with agonistic effects . regarding the histopathology of the thyroid gland , the guidelines mention core criteria and additional qualitative criteria along with grading , as shown in table 3 . for a more detailed understanding , information is available in amphibian metamorphosis assay : part 1 technical guidance for morphologic sampling and histological preparation and amphibian metamorphosis assay : part 2 approach to reading studies , diagnostic criteria , severity grading and atlas . in addition , in support of the oecd ama test guideline , a document was developed that provides a standardized approach for evaluating the histology / histopathology of thyroid glands , including an atlas of the normal architecture of amphibian thyroid glands over the course of metamorphosis . one article regarding an examination conducted in compliance with these guidelines supported the use of the ama as a tier 1 endocrine screen for detection of potential thyroid pathway activity , but the lack of a true negative response ( no effect ) during the validation process prevents full evaluation of this assay s specificity at this time . thyroid glands after exposure for 21 days beginning at stage 51 . left : control . each of the mechanisms mentioned above can alter concentrations of circulating ths . under conditions of th decrease , a result of feedback of circulating ths on the hpt axis , there is increased secretion of tsh from the pituitary gland . such excessive stimulation would be expected to result in an increased thyroid gland size , increased degree of follicular cell hyperplasia and/or increased degree of follicular cell hypertrophy , as is well documented for the rodent thyroid gland . stated that histopathological change of the thyroid gland is the most valuable information regarding thyroid toxicants , and histological examination of the amphibian thyroid gland has been shown to be a very sensitive approach in several studies . although the developmental stage and hind limb length are important endpoints of antithyroid activity , developmental delay can not , by itself , be considered a diagnostic indicator of antithyroidal activity . a series of validation assays verified that thyroid histopathology is sensitive and reliable for antithyroidal activity resulting from either inhibition of thyroidal iodide uptake or iodide organification . on the other hand , according to the phase 1 validation study , histopathological analyses of thyroid glands of t4-treated tadpoles were less consistent between the laboratories and more difficult to interpret compared with the effects seen in the ptu studies . furthermore , the phase 2 validation report mentioned that while thyroid histopathology was sensitive for antithyroidal activities , weak agonistic activity could not be reliably detected . depletion of colloid stores and increases in epithelial cell height are known to occur at climax stages during normal development when tsh synthesis and release by the pituitary and t4 synthesis and secretion by the thyroid gland reach maximum levels . therefore , there may be difficulty in distinguishing whether histological changes occur in response to alterations of the hpt axis or alternatively merely reflect advanced stages in tadpoles . in fact , the guidelines insist that the most appropriate sampling approach for histological analyses is to use stage - matched individuals whenever possible . in order to select stage - matched individuals , all larvae should first be staged prior to selection and subsequent processing for data collection and preservation . this is necessary because normal divergence in development will result in differential stage distributions within each replicate tank . there is one example showing that amphibian thyroid histopathology is extremely complex , and when misinterpretation occurs , it leads to the opposite conclusion . in a controversial issue regarding the effect of triclosan , fort and pawlowski reported in regard to the histopathological changes that an increase in the occurrence of minimal thyroid gland hypertrophy was not accompanied by follicular hypertrophy or hyperplasia . the lack of follicular hypertrophy or hyperplasia was suggested to be the result of both the minimal nature of the response and the increased body size of the treated specimens . because disruption of the thyroid axis has been identified as an important consideration for regulation of chemicals , the oecd and epa have established guidelines that make for use of larval african clawed frogs ( x. laevis ) and frog metamorphosis for screening and testing of potential endocrine disrupters . in the test guidelines , thyroid gland histology is one of the primary endpoints , along with mortality , developmental stage , hind limb length , snout - vent length and wet body weight . a series of validation studies and many reports have revealed that the histopathology of the thyroid gland is very sensitive and reliable for detection of antithyroidal activity but , on the other hand , is less useful for weak agonistic effects . since the morphology of the amphibian thyroid gland changes continuously throughout metamorphosis with the fluctuation in th levels , the guidelines stress the need for well experienced toxicologic pathologists who are familiar with normal x. laevis thyroid histology , thyroid gland physiology and general responses of the thyroid gland to agonists or antagonists .

there are continued concerns about endocrine - disrupting chemical effects , and appropriate vertebrate models for assessment of risk are a high priority . frog tadpoles are very sensitive to environmental substances because of their habitat and the complex processes of metamorphosis regulated by the endocrine system , mainly thyroid hormones . during metamorphosis , marked alteration in hormonal factors occurs , as well as dramatic structural and functional changes in larval tissues . there are a variety of mechanisms determining thyroid hormone balance or disruption directly or indirectly . direct - acting agents can cause changes in thyroxine synthesis and/or secretion in thyroid through effects on peroxidases , thyroidal iodide uptake , deiodinase , and proteolysis . at the same time , indirect action may result from biochemical processes such as sulfation , deiodination and glucuronidation . because their potential to disrupt thyroid hormones has been identified as an important consideration for the regulation of chemicals , the oecd and the epa have each established guidelines that make use of larval african clawed frogs ( xenopus laevis ) and frog metamorphosis for screening and testing of potential endocrine disrupters . the guidelines are based on evaluation of alteration in the hypothalamic - pituitary - thyroid axis . one of the primary endpoints is thyroid gland histopathology . others are mortality , developmental stage , hind limb length , snout - vent length and wet body weight . regarding histopathological features , the guidelines include core criteria and additional qualitative parameters along with grading . taking into account the difficulties in evaluating amphibian thyroid glands , which change continuously throughout metamorphosis , histopathological examination has been shown to be a very sensitive approach .

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cesarean sections ( cs ) performed following a medical indication is necessary for life - saving . it is a surgical intervention to prevent maternal or perinatal complications and the appropriate rate of use should be one associated with the lowest rate of maternal and perinatal morbidity and mortality . however , women are increasingly undergoing cs without any medical indication , which may contribute to the world - wide higher rates of cs . there has been a debate about the appropriateness of cs performed due to maternal request or what physicians suggest to their patients . result of the world health organization ( who ) global survey in a large cross - sectional study demonstrated severe maternal morbidity in planned cesarean delivery are higher than planned vaginal delivery ( vd ) . who recommended 10 - 15% rate of cesarean section . this recommendation was based on the cesarean section rates of the countries with the lowest maternal and neonatal mortality rates in the world at that moment . many studies have shown that the actual rate of cs in numerous countries is far higher than the recommended range . the rate of cesarean section out of all deliveries in iran increased by six - fold over the past three decades . these results are in accordance with previous reports that confirm the growing rate of cs in iran . however , the debate on the best practices ( vd vs. cs ) to minimize postnatal morbidity still is a matter of controversy both from professionals perspectives and from women 's perceptions of the childbirth experience . the postpartum period are critical life events for women leading to physiological , emotional , and social changes . postpartum mothers experience certain physical health conditions that may affect their quality - of - life ( qol ) , future health , and health of their children . several studies confirm that socio - economic deficiencies and medical problem are risk factors for decreased qol and depressive symptoms in women during the postnatal period . mode of delivery and childbirth experience may have a long - term effect on self - rated health . several studies have investigated the association between type of delivery and health related quality - of - life ( hrqol ) , but there is conflicting , with some studies reporting decreased hrqol in cesarean delivery . whereas , other studies did not confirm any relations between cs and hrqol . in a study it was found that patients after vd had higher mean physical hrqol scores than after cs while mean mental hrqol were similar among vd and cs groups . in another study , however , careful assessment of the predictor variables of poorer physical and mental health after childbirth may improve the quality of postpartum care . the aim of this study was to explore the impact of delivery mode on women 's postpartum physical and mental health related qol . a prospective study was performed in the period august 2007 to october 2008 of pregnant women who admitted for prenatal care in the health center . a total of 10 health center were selected randomly in kashan city in iran . after applying inclusion and exclusion criteria , there were 365 consecutive women that agreed to take part in the study and all women gave their informed consent before entering the study . of whom 342 women entering the study after delivery consist of 175 ( % 51.2 ) mothers after vd and 167 ( % 48.8 ) mothers after cs ( elective or emergency cs ) , in follow - up 2 months after delivery ( time 1 assessment ) 162 women after vd and 159 after cs completed qol questionnaire . of whom 150 women in each type of delivery completed qol questionnaire 4 months after delivery again ( time 2 assessment ) in health centers . in these analyses , we have included all women with singleton , live born infant , term pregnancy , prenatal care started before 20 weeks gestation , having uncomplicated pregnancies , parity 1 - 3 and the study population was ethnically iranian , not having : others pre - existing diseases , history of infertility , and divorce . excluding criteria were instrumental delivery , birth weight less than 2,500 g , child or fetal dead , child abnormality , not breast feeding and medical problems in child and mother . all patients were interviewed by the trained midwives and a structured questionnaire was used to evaluate women 's socio - demographic characteristics and obstetric variables . also we used a generic hrqol instrument to measure qol ( categorized into physical and mental health ) . qol was assessed using the iranian version of short form-36 health survey social functioning ( sf-36 ) . sf-36 is a standard and well - known generic health related qol instrument and proved to be highly feasible , reliable and is a good choice to measure health related qol after delivery . it consists of 36 items , organized into eight scales : physical functioning ( pf ) , role limitation due to physical problems or role physical ( rp ) , bodily pain ( bp ) , general health ( gh ) , vitality ( vt ) , social functioning ( sf ) , role limitation due to emotional problems or role emotional ( re ) , and emotional well - being ( ew ) . the scores on each subscale range from 0 to 100 with higher scores indicating a better condition , the sf-36 furthermore provides physical health ( based on the pf , rp , bp , and gh scales ) and a mental health ( relating to vt , sf , re , and ew ) . we compared women 's socio - demographic characteristics and physical and mental health scores between women with vd and cs that completed qol questionnaire at 2 and 4 months after delivery ( 150 vd and 150 cs ) . furthermore score differences within each mode of delivery between time 2 and time 1assessment were evaluated . differences in means were analyzed using the student 's t - test and the mann the study protocol was approved by the local research committee in kashan university of medical sciences . there were no significant differences between vd and cesarean groups with regard to these variables . maternal age was between 20 and 40 and vd was performed up to % 80 with episiotomy or laceration . the physical and mental health parameters related qol at 2 months follow - up has shown in table 2 ; total physical health score in vd women was higher than cs group . mean scores was 60.52 13.28 and 57.37 12.20 ( p = 0.034 ) respectively . on performing a detailed analysis , statistically significant differences in 2 parameters ; pf ( p = 0.001 ) and rp ( p = 0.026 ) were found . total mental health score in cs women was generally higher than vd , but differences were not significant . comparing physical and mental health parameters at 4 months postpartum results showed that the vd group in compared cs group had a higher score in total physical health ( 61.37 13.05 and 58.36 14.09 p = 0.05 ) and total mental health was ( 64.99 12.44 and 61.83 13.58 p = 0.036 ) respectively . in comparing subscales ; differences statistically significant were found for 1 parameter in physical health ( physical function p = 0.01 ) and for 2 parameter in mental health ( sf p = 0.036 and ew p = 0.042 ) . to compare the findings within each group results demonstrated that the vd group showed more improvements on total mental hrqol from time 1 to time 2 assessment [ table 4 ] . the mean score differences ( 4 months scores minus 2 months scores ) in vd and cs groups were 4.70 16.29 and 0.3 13.81 ( p = 0.01 ) respectively . in comparing subscales differences statistically significant were found for 2 parameter ( sf p = 0.03 and ew p = 0.01 ) . general characteristics of patients according to mode of delivery distribution of postpartum physical and mental health according to mode of delivery at 2 months after delivery distribution of postpartum physical and mental health according to mode of delivery at 4 months after delivery the mean score differences within each mode of delivery ( 4 months scores 2 months scores ) * our study showed that physical hrqol at 2 and 4 months after delivery is better in women with vd . there were significant differences in the pf and rp parameters in time 1 and pf in time 2 assessments . also mental hrqol at 4 months after delivery and improvement on mental hrqol from time 1 to time 2 assessments was better in vd group ; differences were significant in sf and ew . mental hrqol at 2 months after delivery was better in cs group but differences were not significant in each of parameters ; a reason for this occurred is mothers after cs supported better due to surgical intervention . also , in our study , nearly % 80 of vd was performed with episiotomy or laceration . in a study , the median time to restart intercourse in the normal vd with episiotomy was 40 days and in the cs group was 10 days postpartum and there was significant decreases in the sexual functioning scores after vd when compared to cs group . concerning its effects on postpartum sexual functioning . sexual health is perceived as an integral part of gh as it can affect postpartum depression . there are many studies that assess morbidity resulting from vd and cs , but a few studies have focused on women 's postpartum hrqol . however , our results are consistent with several studies but differ from some of the previous studies ; in a prospective study 100 women were interviewed ( 50 with normal delivery and 50 with cs ) . qol was measured using the sf-36 at two points ( time 1 : 6 - 8 weeks after delivery and time 2 : 12 - 14 weeks after delivery ; result showed vd group had a better qol for almost all subscales in both assessment times , the differences were significant for vt , mental health and pf . another study was performed to investigated fatigue and hrqol in women after vd , elective cs and emergency cs . a total of 141 women ( 71 after vd , 36 after elective cs and 34 after emergency cs ) completed the hrqolsf-36 questionnaires ; result showed patients after vd had higher mean physical hrqol scores than after cs , but the mean mental hrqol scores of the study groups were similar . in contrast these results a cross - sectional study evaluated the effect of delivery mode on women 's postpartum qol in rural china . in this study , none of six dimensions and total score of qol displayed significant difference between women with normal delivery and cs . in this study , cs rate was 70.0% , and most of them ( 59.7% ) were selected by maternal request . the most important in this data being a high frequency of cs due to maternal request also other factors such as lower education level and male gender of infant were associated with poor qol and whereas , in our study , these factors were similar in two groups . however , socio - cultural determinants may contribute in influencing postnatal qol . safarinejad et al . in a cohort study showed the qol parameters by using sf-36 questionnaire in planned cs women were generally higher than vd . there were differences significant for two physical health domains ( gh and rp ) and all of the mental health domains . in this study planned cs compared to vd and all of the women were parity 1 and younger age . one might argue the findings were influenced by the fact that women with elective or emergency cesarean may experience rather different qol during the postnatal period . furthermore in primipara women other reasons are associated with poor qol . in our study , cs consist of elective or emergency and parity was 1 - 3 . in another study dunn and oherlihy compared satisfaction levels by early postnatal questionnaire among 140 women who had a vaginal birth after delivery ( vbac ) with women who had cesarean section after vaginal delivery . the vbac group experienced minimal pain after delivery and had felt better prepared for delivery . in our study , subscale pain at 2 month after delivery was similar in two groups ; one reason was high frequency of episiotomy in vd group , it is important to pay attention to this risk factor to be able to offer good care and treatment to women during postpartum where necessary . in a prospective study in mothers who requested cesarean section in the absence of medical indication , their reason for the request , self - estimated health and experience of delivery were investigated . results showed women requesting cesarean section experienced their health as less good and were more often planning for one child only . they more often reported anxiety for lack of support during the labor , for loss of control and concern for fetal injury or death . after planned cesarean section women in this group reported a better birth experience compared to women planning a vaginal birth . there were no differences in signs of postpartum depression between the groups 3 months after birth . however intended caesarean delivery was found to be an independent risk factor for complications and is associated with an increased risk of maternal rehospitalization . furthermore mean duration of sickness and mean days required for returning to normal activities were also higher in case of caesarian section also it is expensive than normal vaginal delivery . our research was a prospective study , data were obtained from the participants by trained questionnaires and study population was ethnically uniform . unfortunately , we only used a general instrument and this might be considered as a limitation . another limitation of the study was that the type of cesarean section was not considered , women with elective or emergency cesarean may experience rather different qol during postpartum . the findings suggest that vd lead to a better physical health at 2 months after delivery and physical and mental health at 4 months after delivery . furthermore , we recommend the future studies include both general and specific questionnaire in assessing postpartum qol among women with vd , elective cs and emergency cs , in addition other studies needed for recognizing social and cultural factors that are related to physical and mental hrqol after delivery .

background : the postpartum period is a critical life event for women leading physical , emotional , and social changes . postpartum quality - of - life may be affected by delivery mode . the purpose of this study was to determine the association between postpartum health related quality - of - life ( hrqol ) and mode of delivery.materials and methods : in a prospective study , 300 women consisting of 150 vaginal deliveries ( vd ) and 150 cesarean sections ( cs ) were recruited between august 2007 and october 2008 from health centers . stratified random sampling was performed to select 10 health centers in kashan city , iran . physical and mental hrqol was measured using the sf-36 questionnaire and compared between vd and cs groups 2 and 4 months after delivery . data were analyzed by using the student 's t - test , mann whitney u - test , and chi - square test.results:results showed physical hrqol at 2 months after vd was better than cs significantly ; there were significant differences in the physical functioning and role physical subscales . furthermore mental hrqol at 4 months after vd was better than cs significantly ; there were significant difference in the social function and emotional health subscales.conclusion:the findings demonstrated that vd leads to a better physical health at 2 months after delivery and mental health at 4 months after delivery . efforts should be made to reduce cs .

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maslow 's hierarchy of needs has been a well - known theory since abraham maslow proposed it in 1943 and in his book motivation and personality in 1954 , in which he proposed that human needs can be portrayed in the shape of a pyramid , with the most fundamental levels of needs at the bottom . from bottom to the top are the needs : physiological needs , safety , belongingness and love , esteem , and self - actualization . physiological needs are the physical requirements for the survival of individual and the animal kind , such as food and sex , and safety needs are personal security , financial security , and health and well - being . maslow proposed that if the physiological needs are relatively satisfied , there then emerges a new set of needs , which may be categorized roughly as the safety needs . the organism may be wholly dominated by them , which may serve as the almost exclusive organizers of behavior , recruiting all the capacities of the organism in their service , and we may then fairly describe the whole organism as a safety - seeking mechanism . but maslow also found that practically everything looks less important than safety , even sometimes the physiological needs which being satisfied , are now underestimated . safety needs are personal security , financial security , and health and well - being , which are more fundamental than physiological needs ( figure 1 ) . for example , the deer can not eat ( physiological needs ) on the wild prairie when the wolves chase them ( safety ) . let me take one example from cosmides and tooby ( 2000 ) , who propose that the emotions serve to regulate behavior . he wrote about fear like this : imagine walking alone at night and hearing some rustling in the brush . your energies are aroused to be ready for action , you become acutely aware of sounds that could indicate that you are being stalked , the threshold for detecting movements is lowered , you no longer feel pangs of hunger , attracting a mate is the farthest thing from your mind . . whether physiological needs or safety needs are more important will affect our opinions about mental disorders , for example , freud proposed libido ( physiological needs ) more important . safety needs can block physiological needs ; the classical example is miller 's avoidance and approach experiments ( figure 2 ) . in 1961 , neal miller used behavioral measures to assess motivational disposition in rodents ( figure 2 ) . as the animal moves closer to the potential reward ( e.g. , food ) , the force exerted to obtain the reward increases . similarly illustrated is the avoidance : the force the animal exerted to avoid the aversive stimulus ( a shock ) also increases as the animal comes closer , and furthermore , the slope of the avoidance gradient tended to be steeper than that of approach . later experiments by ito found that the organisms tend to be more sensitive to the threatening information and generally process such information faster than the rewarding information . he called this phenomenon negativity bias and attributed it as a protective strategy through evolution , since even a single failure to respond adaptively to a survival threat may preclude passing on genetic information . he found that as a potential treat looms , the adaptive response of the brain is to amplify these threats and initiate appropriate behavioral responses , such as fleeing , freezing , or attacking . and he found that the negativity bias can be seen across all levels of the neuraxial organization . these data support the notion that safety needs are faster and more fundamental than hedonic needs . maslow thought adults usually inhibit the reaction for safety needs , so he used infants as an example and found that the child 's need for safety is his preference for some kind of undisrupted routine or rhythm . maslow mentioned , he seems to want a predictable , orderly world . for instance , injustice , or inconsistency in the parents seems to make a child feel anxious and unsafe . this attitude may be not so much because of the injustices per se or any particular pains involved , but rather because this treatment threatens to make the world look unreliable , or unsafe , or unpredictable . . maslow also mentioned , confronting the average child with new , unfamiliar , strange , unmanageable stimuli or situations will too frequently elicit the danger or terror reactions . this is consistent with the behaviorists , who propose that behavior is a process - form stimulus - reaction , while cognition scientists extend it to stimulus - opinion - reaction . is stimulus that has the hedonic value , which fits into our personal physiological needs . and it also has another feature : happening in an expected way , or unexpected way , which is related to threat and can be called safety value . we draw these two features of stressful events in two dimensions : hedonic value and safety value ( figure 1 ) . the safety value has nothing to do with the hedonic value , for both the liked things and disliked things can induce unsafety . for example , we worry about losing the liked things and also worry about getting the disliked things ; and we also will not be angry if we lose the good thing as expected and also get the disliked thing as expected . even though it is something you liked , if it is unexpected , you still feel afraid and angry . therefore the safety is related to unexpectancy . one feature of the safety need induced emotion is the rapid detection of potential threats and can initiate appropriate approach / avoidance behaviors . prediction error . the studies were done from electrophysiological recordings of dopamine neurons in awake , behaving monkey in schultz 's lab . these phased activation does not discriminate different types of rewarding stimuli . and it is quite unexpected that the reward delivery will not elicit dopamine neuron firing , once the animal has learned the stimulus and reward association . therefore , dopamine neurons are related to expectation about external stimulus rewarding , especially when it is uncertain or prediction error [ 6 , 7 ] . the studies of emotions have been expanded exponentially by two prominent researchers : magda arnold and richard lazarus , who proposed appraisal theory . appraisal theory states that emotions result from people 's interpretations and explanation of their circumstances . in the structural model of appraisal theory , lazarus borrowed the concept of appraisal from arnold and elaborated the concept as a key factor for emotions : emotional processes depend on the predictability of the stressful events . he distinguishes two basic forms of appraisal , primary and secondary appraisal , and he proposed that the primary appraisal and its induced emotions are a faster activating , automatic process , which is similar to the safety need . indeed , lazarus distinguishes three types of stressful events : harm , threat , and challenge , which are related to primary appraisal . the secondary appraisal is concerned with coping options , which include blame or credit , coping potential , and future expectations . it seems that the primary appraisal is related to fear and the secondary is related to anger ( figure 3 ) . lazarus mentioned that if a person appraises a situation as motivationally relevant , motivationally incongruent and also holds a person other than himself accountable , the individual would most likely experience anger in response to the situation . process model of the appraisal theory is more accurate to explain the safety needs , for personal safety , which are related to the unexpected ways of stimulus occurring . the process model proposed two main appraisal processes : perceptual stimuli and associative processing and reasoning . perceptual stimuli are what the individual picks up from his surroundings , such as sensation of pain or pleasure . then , the individual performs two main appraisal processes : associative processing , which is memory based , and reasoning , which is a slower and more deliberate process that involves logical thinking about the stimulus . for example , when you meet a car that quickly passes and stops before you , you will be firstly scared and then blame the car . let us take another example from izard 's paper , when rafe was hit from the back by a wheel chair , the first reaction of him was scared and angry , and showed angry expression and clenched fist . but after he turned back to see rebecca , a person with hemiplegia whose wheelchair had gone out of control and cause her to crash into rafe . so when something unexpected occurs , you will first evaluate its threat ( fear / anger ) and next evaluate its hedonic value ( happy / sad ) ( figure 3 ) . similar emotional flow happens in our lives all the time : everything in our lives is normally calm as expected ; but you will first feel scared ( fear ) when something unexpected occurs , and then you will blame ( anger ) the un - expectancy after fear is gone . and afterwards you might feel happy after successfully coping with the stressful events or feel sad if you failed to cope with them . finally , the stressful events go away , and people calm down . this kind of emotional flow , big or small , long or short , constitutes our everyday emotions . so fear - anger - happiness - sadness - calm might constitute the rainbow of emotions or emotional flow in our everyday life . the amygdala has been proved to be the neural basis for fear , and it is also recognized as the neural basis for stress elicited fear and anxiety [ 12 , 13 ] . in addition , electrical stimulation of the amygdala promotes autonomous reactions and stress - like behavioral , whereas amygdala ablation induced a marked tameness increase , motivation loss , and fear decrease to aversive stimuli [ 14 , 15 ] . amygdala is one of the most important limbic structures that link to fear , which was first suggested by klver & bucy in 1937 , who demonstrated that the lesion of the medial temporal lobe resulted in a wide range of odd behaviors , such as approaching normally to fearful objects . and about 20 years later , weiskrantz ( 1956 ) found that it is the amygdala whose impairment resulted in the odd behaviors , which are called klver and bucy syndrome . ledoux puts amygdala as the emotional computer to work out the emotional significance of stimuli . he demonstrated two neural pathways of sensory information from the thalamus to the cortex : ( 1 ) a slow - acting thalamus - to - cortex circuit , whose function is to analyze sensory information in detail , and ( 2 ) a fast - acting thalamus - amygdala circuit , whose major function is to analyze simple stimulus features , which bypass the cortex . these two pathways possibly underlie the two evaluation systems : the fast one for the fear / anger and the slow one for hedonic . other reports , such as findings from ohman and soares ( 1994 ) also support a fast - acting system for threat detection that involves only minimal cortical processing [ 19 , 20 ] . ( 2001 ) also reported a patient whose primary visual cortex was impaired and therefore showed no conscious visual perception but showed significant fearful reports . in all , the fast - acting thalamus - amygdala circuit is important for our ancient ancestors to rapidly recognize dangers to help with survival . it is an evolutionary adaptation for our ancestors to better cope with the unexpected environment with the fast activing thalamus - amygdala circuit . in addition to amygdala , the ne / lc system is important to direct behaviors of the animal to cope with the dangerous environment , and the well - known function of ne / lc system is to induce fight or flight behaviors . take a deer in the wild as an example . when a deer meets a lion , the reaction of the deer is flight ( fear ) , while the reaction of a lion is fight ( anger ) . so the emotion fear and anger might also be derived from the same neurotransmitter ne and the same stressful event ( figure 4 ) . ne is released from the locus coeruleus ( lc ) in the brain to keep the brain alert , which has been described as increasing the signal to noise of the sensory inputs . lc has been reported to be activated at stressful events : increased lc neuron firings were observed at visual threat . and lc sends projections to amygdala , which is the most important limbic structure that links to fear . therefore , amygdala and ne / lc system might constitute the neural structure for safety needs . our ancestors navigating rich environments had to face very complicated environments , with many forms of uncertainties . so they evolved an adaptive mechanism to first do a safety check for everything around them . if it would happen in an anticipated way , they will feel calm ; instead if something happened surprisingly , they will be scared and angry . therefore , fear and anger are due to things happening in an unexpected way ( figure 4 ) . of note , the unexpectancy will also increase the tension of hedonic emotions . with the same kind of hedonic stimulus , if it comes in an unexpected way , people will feel excited ; instead , people will feel happy . one reason for the clearer appearance of the threat or danger reaction in infants , is that they do not inhibit this reaction at all , whereas adults in our society have been taught to inhibit it at all costs . thus even when adults do feel their safety to be threatened we may not be able to see this on the surface . infants will react in a total fashion in infants we can also see a much more direct reaction to bodily illnesses of various kinds for instance , vomiting , colic or other sharp pains . . even the adults can inhibit our reactions , they still can react in some mental disorders . maslow wrote , some neurotic adults in our society are , in many ways , like the unsafe child in their desire for safety , although in the former it takes on a somewhat special appearance . their reaction is often to unknown , psychological dangers in a world that is perceived to be hostile , overwhelming and threatening . the neurotic individual may be described in a slightly different way with some usefulness as a grown - up person who retains his childish attitude toward the world . this is to say , a neurotic adult may be said to behave as if he were actually afraid of a spanking , or of his mother 's disapproval , or of being abandoned by his parents , or having his food taken away from him . it is as if his childish attitude of fear and threat reaction to a dangerous world had gone underground , and untouched by the growing up and learning processes , were now ready to be called out by any stimulus that would make a child feel endangered and threatened . fear - phobia . if something happens surprisingly , people will be scared and angry ; and if it would happen in an anticipated way , they will feel calm . so for the phobia patients , their problems might be that they can not successfully accomplish the emotional flow ( fear - anger - happiness - sadness - calm ) . the best way to remove fear is anger , these patients are too timid to show anger , so their emotions are checked at emotional flowing from fear to anger . therefore , anger might be the best treatment for these patients and ne is the neural substrate for them . depression is characterized by unrelenting sadness accompanied by an inability to derive pleasure from positively hedonic situations . therefore , depression might be related to the primary appraisal , the worrying about safety instead of physiological satisfaction . indeed , excessive self - blame and feeling worthless are symptoms of major depression episodes across cultures , which is similar to lazarus 's secondary appraisal . so the depressed patients have problems with anger or with coping appraisal or their problem is due to inability to cope with the unsafe stressful situation and showed inward anger . the difference between fear and anger is the direction of the behavior : fear is to throw oneself away from the stimulus and anger is to throw the stimulus away . for these patients , they do not have the ability to throw the outside stimulus due to repeated helplessness , they want to kill themselves . safety needs are the most fundamental needs for the human kind , which include personal security , financial security , and health and well - being . safety is the major reason for mental disorders , such as anxiety , phobia , depression , and ptsd . the neural basis for safety is amygdala and lc / ne system , which can be regarded as a safety circuitry , whose major behavior function is fight or flight and fear and anger emotions , or conditioned learning for these emotions . fear and anger are due to the safety needs , while joy and sadness are due to the physiological needs , which should come after safety needs in maslow 's hierarchy of needs . fear and anger are two sides of one sword , for they will act in different directions : fear is to flight away from the danger and anger is to fight the danger away . they are all due to the stressful events : normally everything is as expected , and life is calm . when something unexpected happens , the individuals first feel scared and then blame the unexpectancy ; this is the first safety check . afterwards the individual will have a hedonic need to see if it fits their personal needs and get the happy or sad emotions . finally , everything comes to an end , and people return to calmness or miss the lost things and worry for the uncertain bad things . so the emotional rainbow ( or emotion flow ) fear - anger - happiness - sadness - missing constitutes our emotions in everyday life .

safety first , we say these words almost every day , but we all take this for granted for what maslow proposed in his famous theory of hierarchy of needs : safety needs come second to physiological needs . here we propose that safety needs come before physiological needs . safety needs are personal security , financial security , and health and well - being , which are more fundamental than physiological needs . safety worrying is the major reason for mental disorders , such as anxiety , phobia , depression , and ptsd . the neural basis for safety is amygdala , lc / ne system , and corticotrophin - releasing hormone system , which can be regarded as a safety circuitry , whose major behavior function is fight or flight and fear and anger emotions . this is similar to the appraisal theory for emotions : fear is due to the primary appraisal , which is related to safety of individual , while anger is due to secondary appraisal , which is related to coping with the unsafe situations . if coping is good , the individual will be happy ; if coping failed , the individual will be sad or depressed .

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an imposing body of studies , spanning more than three decades , has cogently elucidated the involvement of epoxyeicosatrienoic acids ( eets ) in a wide array of critical physiological functions , inter alia , blood pressure regulation , nociception , adipogenesis , anti - inflammatory activity , organ regeneration , insulin potentiation , podocyte integrity , and cellular responses to bacterial infection . the most prominent regioisomer , 14,15-epoxyeicosa-5(z),8(z),11(z)-trienoic acid ( 14,15-eet ) , along with other members of this autacoid family , arise via metabolism of arachidonic acid by cytochromes p450 , especially members of the 2c and 2j families . the ratio of eet isomers and their stereocomposition are cyp p450 isoform - dependent and , thus , species- and tissue - specific . numerous chemical and metabolic factors conspire to complicate investigations into the roles of 14,15-eet and restrain its potential therapeutic utility ( figure 1 ) . its susceptibility toward aerial oxidation or auto - oxidation , i.e. , a nonenzymatic , free radical process involving the 1,4-dienyl substructures characteristic of polyunsaturated fatty acids , necessitates handling and/or storage under strict conditions that minimize exposure to oxygen and trace transition metals . a precis of inactivating enzymatic processes includes esterification , metabolism by other pathways of the arachidonate cascade , conjugation , -oxidation , chain elongation , and hydrolysis of the epoxide . in biological milieu , hydration of the epoxide by soluble epoxide hydrolase ( seh ) is a major determinant in maintaining the steady state levels of 14,15-eet , whose half - life has been estimated at no more than seconds to minutes . the pioneering studies of hammock and others confirmed inhibition of seh can elevate eet levels both in vitro and in vivo , thus offering an indirect means for pharmacologic intervention in eet - mediated processes . however , this strategy might prove less efficacious than an agonist replacement therapy whenever endogenous eet biosynthesis is compromised , for instance , as a consequence of disease , inflammation , radiotherapy , aging , and/or exposure to xenobiotics and drugs that inhibit cytochromes p450 . to address some of the stability limitations of natural eets , our laboratories previously prepared several iterations of eet surrogates with improved stability . advanced versions were evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of recombinant human seh . depending upon the bioisostere and its position along the carbon chain , varying levels of vascular relaxation and/or seh inhibition were observed . generally , oxamides and n - ipr - amides displayed useful 14,15-eet agonist activities but were modest to poor seh inhibitors . the in vitro success of this generation of analogues prompted us to consider further structural iterations . the surrogates described herein modify the free carboxylic acid of the previous generation of 14,15-eet mimics while , for the most part , retaining some key structural features of the pharmacophore identified earlier ( i.e. , cis--olefin and an epoxide bioisostere ) . they were evaluated as described before in precontracted bovine coronary artery rings for ( i ) % vasorelaxation at 10 m relative to a 14,15-eet control , ( ii ) ec50 for vasorelaxation , and ( iii ) ic50 for seh inhibition ( table 1 ) . interestingly , the simple expedient of conjugating the carboxylate with a short poly(ethylene oxide ) ( peg ) unit improved both % vasorelaxation and the ec50 somewhat compared with the parent free acids regardless the type of epoxide bioisostere , viz . , n - ipr - amide 1 , urea 2 , and oxamide 3 ; on the other hand , the soluble epoxide hydrolase ( seh ) inhibitory activity of urea 2 was significantly compromised versus the parent free acid ( ec50 7.5 m , ic50 46 nm ) . linkage of the carboxylate to the nitrogen of glycine ( 4 and 5 ) or aspartic acid ( 6 ) resulted in a modest boost to the vasoactivities while the seh ic50 of 6 dropped by an order of magnitude compared to the parent free acid . conversion to the n - phenyl and n - methylsulfonimides 7 and 8 , respectively , left the biological activities virtually unchanged in all categories while the simplified phenylsulfonamide 9 led to an improvement in the ec50 by a factor of 2 and smaller improvements in the other parameters . at 10 m , 14,15-eet induces 85% of maximum vasorelaxation and its ed50 is 2.2 m . for recombinant human seh , given the generally lackluster behavior of the esters and amides , our focus changed to replacement of the carboxylate with a variety of heterocyclic bioisosteres identified from literature sources . for phosphonate 10 and sulfonate 11 , the increase in polarity did not improve potency as seen with the peg esters . interestingly , sulfonate 11 retained its ability to suppress seh , in contrast to phosphonate 10 and peg ester 2 . vasorelaxation by s - alkylthiocatechol 12 was poor , but its ic50 for seh was pushed down into the single digits . the sulfone variant 13 regained some vasopotency , probably due to an increase in the acidity of the phenol but not sufficiently to be viable . replacement of the phenol moiety in 12 with a tetrazole ring produced 14 and was encouraging , but this trend was not continued in triazole 15 . sequential oxidation of the sulfur to sulfoxamide 16 and sulfonamide 17 , as seen for 12 13 , incrementally improved the ec50 . the ed50 jumped in the tetrazole bioisostere series 1821 , achieving a submicromolar value for urea 19 ( 12-fold better than 14,15-eet ) , while the % vasorelaxation versus 14,15-eet peaked . factoring in a very respectable low nanomolar ic50 , 19 is the best dual activity analogue in the study and a leading candidate for further development . notably , all three parameters began to erode , albeit minimally , in the one - carbon homologue 20 and further in oxamide 21 . the performance of another five - membered heterocycle , oxadiazol-5-one 22 , while acceptable , was not comparable to urea 19 . the closely related oxathiadiazole-2-oxides23 and 24 , on the other hand , demonstrated good vasorelaxant activities ; seh inhibition potency was consistent with our previous observations that ureas n - ipr - amides . the isomeric thioxo-1,2,4-oxadiazol25 registered a 6-fold improvement in ed50 over 14,15-eet yet was a comparatively poor seh inhibitor and , thus , is a good option whenever an eet agonist with minimal impact on seh function is required . the corresponding urea thioxo - oxadiazol,26 , could not sustain the vasopotency ; its ed50 sagged by almost an order of magnitude versus 25 while at the same time its seh ic50 improved by more than an order of magnitude . a final variant on this heterocyclic theme , 5-thioxothiadiazol27 , fell short of expectations on all levels and further study of this particular heterocycle was terminated . despite its success in the glitazone series of antidiabetic drugs , the 2,4-thiazolidinedione modification proved disappointing when incorporated into 28 . given the larger size of the n-(4-hydroxy-2-benzothiazolyl)acetamide bioisostere , the carbon chain utilized in most of the analogues ( figure 1 ) was trimmed by four carbons before attachment via the phenolic oxygen to generate 29 . following inspection of molecular models , the carbon chain was shortened even further and the olefin was deleted , leading to n - ipr - amide 30 , urea 31 , and oxamide 32 , whose total chain length more closely resembled that of 14,15-eet . as hoped , the ec50 values increased relative to 29 , although the % vasorelaxation was variable . contrary to expectations , there was no evident correlation between the pka of the eet analogues and the ed50 for vasorelaxation ( figure 2 ) , suggesting factors other than ionic interactions are involved in binding the carboxylate at the putative eet binding site . plot of calculated pka vs ed50 ( m ) of analogues in table 1 . the protonated form of analogues 6 , 11 , and 12 was used for the calculation . the syntheses of tetrazole 19 and thioxo-1,2,4-oxadiazol 25 are summarized in scheme 1 and illustrate the approach used to prepare the other analogues . semihydrogenation of the known acetylene 34 using p-2 nickel and h2 led to cis - olefin 35 that was subjected to azidation using diphenylphosphoryl azide ( dppa ) under mitsunobu conditions . staudinger reduction of the product , azide 36 , led to primary amine 37 that was reacted without purification with n - pentyl isocyanate to furnish urea 38 . an uneventful series of functional group interchanges proceeding through alcohol 39 , bromide 40 , and ending with nitrile 41 set the stage for the zinc bromide mediated annulation with sodium azide that delivered tetrazole 19 . reagents and conditions : ( a ) p2-ni/(h2nch2)2 , h2 ( 1 atm ) , etoh , rt , 1 h ( 96% ) ; ( b ) diad / ph3p / ph2p(o)n3 , thf , 20 to 23 c , 4 h ( 72% ) ; ( c ) ph3p , h2o / thf , rt , 12 h ( 76% ) ; ( d ) c5h11nco , thf , rt , 3 h ( 76% ) ; ( e ) nbu4nf , thf , rt , 12 h ( 8289% ) ; ( f ) cbr4/ph3p , ch2cl2 , rt , 2 h ( 8384% ) ; ( g ) kcn , dmso , rt , 12 h ( 7881% ) ; ( h ) nan3/znbr2 , iproh / h2o ( 1:3 ) , 110 c , 18 h ( 76% ) ; ( i ) ho(co)2nh(ch2)3ch3 , edci , dmf , rt , 12 h ( 73% ) ; ( j ) h2noh / na2co3 , meoh / h2o ( 4:1 ) , 60 c , 18 h ( 62% ) ; ( k ) im2c(s ) , thf , rt , 45 min ( 63% ) . following the same sequence of transformations as described above , 42 was converted into nitrile 45 . condensation of hydroxyimine 46 , obtained by addition of hydroxylamine to 45 , with 1,1-(thiocarbonyl)diimidazole yielded thioxo-1,2,4-oxadiazol 25 as a crystalline solid . final compounds were judged 95% pure by hplc using a zorbax eclipse c18 column ( 250 mm 4.6 mm ; agilent ) connected to an agilent 1200 api / lc - ms using acetonitrile / water combinations as eluent unless otherwise noted . nuclear magnetic resonance ( nmr ) spectra were recorded on varian 300 , 400 , or 500 spectrometers at operating frequencies of 300/400/500 mhz ( h ) or 75/100/125 mhz ( c ) in cdcl3 with tms as internal standard , unless otherwise stated . h nmr data are reported as follows : chemical shift ( ppm ) , multiplicity ( s = singlet , br s = broad singlet , d = doublet , t = triplet , q = quartet , app q = apparent quartet , qn = quintet , app qn = apparent quintet , m = multiplet ) , and coupling constant ( hz ) . high resolution mass spectra ( hrms ) were obtained at ut - arlington using a shimadzu it - tof mass spectrometer or at the medical college of wisconsin by prof . infrared ( ir ) spectra were obtained using a perkinelmer spectrum 1000 fourier transform spectrometer . melting points were measured using an optimelt from stanford research systems and are uncorrected . analytical thin layer chromatography ( tlc ) used emd chemicals tlc silica gel 60 f254 plates ( 0.0400.063 mm ) with visualization by uv light and/or kmno4 or phosphomolybdic acid ( pma ) solution followed by heating . all oxygen and/or moisture sensitive reactions were performed under an argon atmosphere using oven - dried glassware and anhydrous solvents . extracts were dried over anhydrous na2so4 and filtered prior to removal of all volatiles under reduced pressure . chromatographic purifications utilized preparative tlc or flash chromatography using prepacked sio2 columns on a combiflash rf200 chromatograph ( teledyne isco ) . unless otherwise noted , yields refer to isolated , purified material with spectral data consistent with assigned structures or , if known , were in agreement with published data . reagents were purchased at the highest commercial quality available and used without further purification , unless otherwise noted . anhydrous solvents were dried using a glass contours solvent system by passage through columns of activated packing material under argon immediately prior to use . a mixture of 1-(12-cyanododec-5(z)-enyl)-3-n - pentylurea ( 41 ) . ( 500 mg , 1.55 mmol ) , sodium azide ( 100 mg , 1.55 mmol ) , and zinc bromide ( 335 mg , 1.48 mmol ) was heated at 110 c in 2-propanol / h2o ( 1:3 , 8 ml ) while stirring vigorously in a sealed tube . after 18 h , the mixture was cooled to room temperature and the ph was adjusted to 1 using aq hcl ( 3 n , 4 ml ) . ethyl acetate ( 10 ml ) was added , and the stirring was continued until no solid was present . the organic layer was isolated and the aqueous layer extracted with etoac ( 2 25 ml ) . the combined organic fractions were washed with water ( 3 25 ml ) , dried , and concentrated in vacuo . the residue was purified by silica gel column chromatography to give the tetrazole 19 ( 431 mg , 76% ) as a colorless solid , mp 205.6205.8 c . h nmr ( cd3od , 300 mhz ) 5.405.30 ( m , 2h ) , 3.063.11 ( m , 4h ) , 2.93 ( t , j = 8.0 hz , 2h ) , 1.982.10 ( m , 4h ) , 1.701.82 ( m , 2h ) , 1.241.50 ( m , 16h ) , 0.90 ( t , j = 7.6 hz , 3h ) . c nmr ( cd3od , 75 mhz ) 160.16 , 156.81 , 129.77 , 129.47 , 39.81 , 39.68 , 29.88 , 29.80 , 29.35 , 28.99 , 28.69 , 28.55 , 27.48 , 26.85 , 26.81 , 26.68 , 22.96 , 22.31 , 13.22 . hrms calcd for c19h37n6o [ m + 1 ] 365.3029 , found 365.3030 . a mixture of n-(13-amino-13-(hydroxyimino)tridec-5(z)-enyl)-n - n - butyloxalamide ( 46 ) ( 100 mg , 0.27 mmol ) and 1,1-(thiocarbonyl)diimidazole ( 57 mg , 0.32 mmol ) in dry thf ( 5 ml ) was stirred at room temperature for 45 min . the mixture was diluted with water ( 20 ml ) and extracted with ethyl acetate ( 3 10 ml ) . the combined organic extracts were washed with water and dried , and the solvent was evaporated in vacuo . the residue was dissolved in acetonitrile ( 5 ml ) to which was then added dbu ( 61 mg , 0.40 mmol ) . after stirring at room temperature for 1 h , the mixture was diluted with water ( 10 ml ) , adjusted ph 4 with 1 n hcl , and extracted with ethyl acetate ( 3 10 ml ) to give thioxo-1,2,4-oxadiazol 25 ( 71 mg , 63% ) as a white solid , mp 110.6110.8 c . h nmr ( 400 mhz ) 8.90 ( br s , nh , 1h ) , 7.52 ( br s , nh , 2h ) , 5.285.40 ( m , 2h ) , 3.203.40 ( m , 4h ) , 2.59 ( t , j = 7.5 hz , 2h ) , 1.982.10 ( m , 4h ) , 1.211.70 ( m , 16h ) , 0.92 ( t , j = 7.3 hz , 3h ) . c nmr ( 100 mhz ) 160.12 , 160.08 , 153.31 , 130.62 , 129.46 , 39.93 , 39.85 , 31.35 , 29.33 , 28.94 , 28.89 , 28.68 , 27.02 , 26.84 , 26.69 , 23.96 , 20.23 , 13.90 . nabh4 ( 82 mg , 2.28 mmol ) was added in portions with vigorous stirring to a room temperature solution of ni(oac)24h2o ( 567 mg , 2.28 mmol ) in absolute ethanol ( 20 ml ) under a hydrogen atmosphere ( 1 atm ) . after 15 min , freshly distilled ethylenediamine ( 0.30 ml , 4.56 mmol ) was added to the black suspension , followed after a further 15 min by a solution of 12-(tert - butyldiphenylsilyloxy)dodec-5-yn-1-ol ( 34 ) ( 4.0 g , 9.16 mmol ) in absolute etoh ( 10 ml ) . after 1 h , the reaction mixture was diluted with et2o ( 20 ml ) and passed through a small bed of silica gel . the combined ethereal filtrates were concentrated under reduced pressure to afford alcohol 35 ( 3.85 g , 96% ) as a colorless oil sufficiently pure to be used directly in the next step . h nmr ( 300 mhz ) 7.647.68 ( m , 4h ) , 7.347.42 ( m , 6h ) , 5.425.28 ( m , 2h ) , 3.63 ( t , j = 6.4 hz , 4h ) , 2.081.96 ( m , 4h ) , 1.501.60 ( m , 4h ) , 1.401.24 ( m , 10h ) , 1.04 ( s , 9h ) . c nmr ( 100 mhz ) 135.81 , 134.40 , 130.61 , 129.71 , 129.60 , 127.80 , 64.21 , 63.14 , 32.78 , 32.60 , 29.98 , 29.27 , 27.42 , 27.14 , 27.10 , 26.08 , 25.92 , 19.48 . diisopropyl azodicarboxylate ( diad ; 1.46 ml , 7.35 mmol ) was added dropwise to a 20 c solution of pph3 ( 2.10 g , 8.0 mmol ) in dry thf ( 45 ml ) under an argon atmosphere . after 10 min , a solution of 12-(tert - butyldiphenylsilyloxy)dodec-5(z)-en-1-ol ( 35 ) ( 3.20 g , 7.35 mmol ) in dry thf ( 10 ml ) after 30 min , the mixture was warmed to 0 c and diphenylphosphoryl azide ( 1.58 ml , 7.35 mmol ) was added dropwise . after stirring 4 h at rt , the reaction mixture was quenched with water ( 150 ml ) and extracted with etoac ( 2 100 ml ) . the combined organic extracts were washed with brine ( 100 ml ) , dried ( na2so4 ) , and concentrated under reduced pressure . the residue was purified by sio2 column chromatography eluting with 4% etoac / hexane to afford azide 36 ( 2.45 g , 72% ) . h nmr ( 400 mhz ) 7.647.68 ( m , 4h ) , 7.347.42 ( m , 6h ) , 5.285.42 ( m , 2h ) , 3.70 ( t , j = 5.8 hz , 2h ) , 3.27 ( t , j = 6.3 hz , 2h ) , 1.962.10 ( m , 4h ) , 1.241.64 ( m , 12h ) , 1.04 ( s , 9h ) . c nmr ( 100 mhz ) 135.84 , 134.41 , 130.93 , 129.75 , 129.12 , 127.83 , 64.22 , 51.62 , 32.81 , 29.93 , 29.30 , 28.68 , 27.46 , 27.14 , 27.02 , 26.90 , 25.96 , 19.49 . ir ( neat ) 2930 , 2783 , 2331 , 2097 , 1106 cm . triphenylphosphine ( 1.18 g , 4.50 mmol ) was added to a stirring solution of 1-tert - butyldiphenylsilyloxy-12-azidododec-7(z)-ene ( 36 ) ( 1.90 g , 4.10 mmol ) in thf ( 12 ml ) containing 10 drops of deionized water . after 12 h , the reaction mixture was diluted with ch2cl2 ( 10 ml ) , dried , and concentrated in vacuo to give amine 37 ( 1.36 g , 76% ) as a viscous , colorless oil that was used directly in the next reaction without further purification . h nmr ( 400 mhz ) 7.627.68 ( m , 4h ) , 7.327.40 ( m , 6h ) , 5.305.40 ( m , 2h ) , 3.63 ( t , j = 5.2 hz , 2h ) , 2.62 ( t , j = 4.8 hz , 2h ) , 1.922.06 ( m , 4h ) , 1.401.58 ( m , 4h ) , 1.201.40 ( m , 8h ) , 1.03 ( s , 9h ) . c nmr ( 100 mhz ) 135.79 , 134.37 , 130.42 , 129.70 , 127.78 , 64.19 , 42.28 , 33.44 , 32.77 , 29.93 , 29.28 , 27.40 , 27.21 , 27.10 , 25.92 , 19.44 . a solution of 1-tert - butyldiphenylsilyloxy-12-aminododec-7(z)-ene ( 37 ) ( 1.32 g , 3.0 mmol ) in thf ( 5 ml ) was added dropwise to a stirring solution of n - pentyl isocyanate ( 0.386 ml , 3.0 mmol ) in thf ( 10 ml ) . after 3 h stirring at room temperature , all volatiles were removed under reduced pressure and the residue was purified by sio2 column chromatography eluting with 20% etoac / hexane to afford urea 38 ( 1.26 g , 76% ) as a viscous oil . h nmr ( 300 mhz ) 7.607.70 ( m , 4h ) , 7.357.42 ( m , 6h ) , 5.285.42 ( m , 2h ) , 5.16 ( br s , nh , 2h ) , 3.65 ( t , j = 6.5 hz , 2h ) , 3.083.20 ( m , 4h ) , 1.962.08 ( m , 4h ) , 1.221.60 ( m , 18h ) , 1.02 ( s , 9h ) , 0.89 ( t , j = 7.3 hz , 3h ) . c nmr ( 100 mhz ) 159.23 , 135.80 , 134.24 , 130.52 , 129.74 , 129.49 , 127.82 , 64.22 , 40.62 , 40.54 , 32.80 , 30.33 , 29.95 , 29.37 , 29.32 , 27.46 , 27.34 , 27.18 , 27.11 , 25.97 , 22.71 , 19.46 , 14.29 . hrms calcd for c34h55n2o2si [ m + 1 ] 551.4033 , found 551.4032 . a mixture of 1-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)-3-n - pentylurea ( 38 ) ( 1.12 g , 2.0 mmol ) and tetra - n - butylammonium fluoride ( 2.20 ml of 1 m soln in thf , 2.2 mmol ) in dry thf ( 10 ml ) was stirred at room temperature under an argon atmosphere for 12 h and then evaporated to dryness in vacuo . the residue was dissolved in etoac ( 50 ml ) and washed with water ( 30 ml ) , brine ( 30 ml ) , dried , and evaporated in vacuo . purification of the residue via sio2 column chromatography gave alcohol 39 ( 0.56 g , 89% ) as a colorless solid , mp 63.763.8 c . h nmr ( 300 mhz ) 5.255.42 ( m , 2h ) , 4.48 ( br s , nh , 2h ) , 3.64 ( d , j = 6.5 hz , 2h ) , 3.083.20 ( m , 4h ) , 1.962.14 ( m , 4h ) , 1.221.60 ( m , 18h ) , 0.88 ( t , j = 7.0 hz , 3h ) . c nmr ( 125 mhz ) 159.26 , 130.23 , 129.62 , 63.72 , 40.33 , 40.29 , 32.92 , 30.30 , 30.26 , 29.74 , 29.35 , 29.13 , 27.26 , 27.20 , 27.13 , 25.82 , 22.69 , 14.27 . cbr4 ( 0.55 g , 1.66 mmol ) and pph3 ( 0.43 g , 1.66 mmol ) were added to a 0 c solution of 1-(12-hydroxydodec-5(z)-enyl)-3-n - pentylurea ( 39 ) ( 0.43 g , 1.38 mmol ) in ch2cl2 ( 20 ml ) . after 2 h at room temperature , the reaction mixture was concentrated in vacuo and the residue was purified via sio2 column chromatography to give 1-(12-bromododec-5(z)-enyl)-3-n - pentylurea ( 40 ) ( 0.43 g , 83% ) as a solid , mp 46.746.8 c . h nmr ( 300 mhz ) 5.225.42 ( m , 2h ) , 4.40 ( br s , 2h ) , 3.42 ( t , j = 9.3 hz , 2h ) , 3.103.20 ( m , 4h ) , 1.982.10 ( m , 4h ) , 1.801.90 ( m , 2h ) , 1.251.55 ( m , 16h ) , 0.92 ( t , j = 7.2 hz , 3h ) . c nmr ( 100 mhz ) 159.51 , 130.14 , 129.69 , 40.48 , 40.39 , 34.20 , 32.96 , 30.34 , 29.67 , 29.36 , 28.58 , 28.25 , 27.31 , 27.27 , 27.17 , 22.68 , 14.26 . hrms calcd for c18h36brn2o [ m + 1 ] 375.2011 , found 375.2014 . a mixture of potassium cyanide ( 0.23 g , 3.54 mmol ) and 1-(12-bromododec-5(z)-enyl)-3-n - pentylurea ( 40 ) ( 0.90 g , 2.40 mmol ) was stirred in dmso ( 5 ml ) at room temperature . after 12 h , the reaction mixture was diluted with water ( 20 ml ) and extracted with ethyl acetate ( 2 50 ml ) . the combined organic extracts were washed with water ( 2 25 ml ) , brine ( 25 ml ) , dried ( na2so4 ) , and passed through a silica gel column to give nitrile 41 ( 0.62 g , 81% ) as a colorless solid , mp 5657 c . h nmr ( 300 mhz ) 5.295.40 ( m , 2h ) , 4.27 ( br s , nh , 2h ) , 3.103.20 ( m , 4h ) , 2.34 ( t , j = 7.0 hz , 2h ) , 1.982.08 ( m , 4h ) 1.241.70 ( m , 18h ) , 0.89 ( t , j = 7.0 hz , 3h ) . c nmr ( 125 mhz ) 159.41 , 129.94 , 129.86 , 120.14 , 40.45 , 40.35 , 30.30 , 29.50 , 29.33 , 28.70 , 28.51 , 27.26 , 27.16 , 25.47 , 22.66 , 17.28 , 14.24 . ir ( neat ) 2930 , 2281 , 2184 , 2042 , 1936 , 1613 , 1197 , 1042 cm . hrms calcd for c19h36n3o [ m + 1 ] 322.2858 , found 322.2867 . a mixture of 2-(n - butylamino)-2-oxoacetic acid ( 0.40 g , 2.70 mmol ) , 1-tert - butyldiphenylsilyloxy-12-aminododec-7(z)-ene ( 37 ) ( 1.20 g , 2.70 mmol ) , 1-hydroxybenzotriazole ( hobt ; 0.44 g , 3.30 mmol ) , and [ 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride ] ( edci : 0.63 g , 3.30 mmol ) in dry dmf ( 5 ml ) was stirred at room temperature overnight . the reaction mixture was quenched with water ( 30 ml ) and extracted into ethyl acetate ( 3 20 ml ) . the combined organic extracts were washed with water ( 2 10 ml ) and brine ( 10 ml ) , dried , and concentrated in vacuo . the residue was purified by sio2 column chromatography to give n - n - butyl - n-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)oxalamide ( 42 ) ( 1.10 g , 73% ) . h nmr ( 400 mhz ) 8.05 ( br s , nh , 2h ) , 7.667.74 ( m , 4h ) , 7.327.42 ( m , 6h ) , 5.305.42 ( m , 2h ) , 3.67 ( t , j = 3.9 hz , 2h ) , 3.31 ( q , j = 5.2 hz , 4h ) , 1.962.10 ( m , 4h ) , 1.501.64 ( m , 6h ) , 1.221.44 ( m , 10h ) , 1.06 ( s , 9h ) , 0.92 ( t , j = 7.8 hz , 3h ) . c nmr ( 100 mhz ) 160.33 , 135.80 , 134.35 , 130.73 , 129.74 , 129.20 , 127.83 , 64.17 , 39.89 , 39.69 , 32.79 , 31.48 , 29.94 , 29.29 , 29.07 , 27.46 , 27.23 , 27.14 , 27.0 , 25.96 , 20.29 , 19.46 , 13.96 . hrms calcd for c34h53n2o3si [ m + 1 ] 565.3826 , found 565.3824 . n - n - butyl - n-(12-(tert - butyldiphenylsilyloxy)dodec-5(z)-enyl)oxalamide ( 42 ) ( 1.20 g , 2.12 mmol ) was desilylated as described above for 39 to give n - n - butyl - n-(12-hydroxydodec-5(z)-enyl)oxalamide ( 43 ) ( 0.568 g , 82% ) as a colorless solid , mp 102.8102.9 c . h nmr ( 400 mhz ) 7.69 ( br s , 2h ) , 5.205.35 ( m , 2h ) , 3.56 ( t , j = 4.2 hz , 2h ) , 3.26 ( q , j = 5.6 hz , 4h ) , 2.17 ( br s , 1h ) , 1.952.02 ( m , 4h ) , 1.441.56 ( m , 6h ) , 1.201.40 ( m , 10h ) , 0.87 ( t , j = 7.2 hz , 3h ) . c nmr ( 100 mhz ) 160.15 , 130.66 , 129.21 , 62.98 , 39.80 , 39.63 , 32.93 , 31.39 , 29.77 , 29.18 , 28.95 , 27.26 , 27.0 , 26.88 , 25.80 , 20.18 , 13.85 . n - n - butyl - n-(12-hydroxydodec-5(z)-enyl)oxalamide ( 43 ) ( 330 mg , 1.0 mmol ) was brominated as described above for 40 to give n-(12-bromododec-5(z)-enyl)-n - n - butyloxalamide ( 44 ) ( 330 mg , 84% ) as a white solid , mp 46.046.3 c . h nmr ( 400 mhz ) 7.79 ( br s , nh , 1h ) , 7.77 ( br s , nh , 1h ) , 5.205.32 ( m , 2h ) , 3.32 ( t , j = 6.4 hz , 2h ) , 3.22 ( q , j = 7.2 hz , 4h ) , 1.902.00 ( m , 4h ) , 1.721.82 ( m , 2h ) , 1.421.56 ( m , 4h ) , 1.201.40 ( m , 10h ) , 0.85 ( t , j = 7.3 hz , 3h ) . c nmr ( 100 mhz ) 160.17 , 160.15 , 130.40 , 129.34 , 39.77 , 39.59 , 34.12 , 32.93 , 31.40 , 29.62 , 29.0 , 28.54 , 27.25 , 27.24 , 27.0 , 26.91 , 20.18 , 13.85 . n-(12-bromododec-5(z)-enyl)-n - n - butyloxalamide ( 44 ) ( 250 mg , 0.642 mmol ) was treated with potassium cyanide as described above for 41 to give n - n - butyl - n-(12-cyanododec-5(z)-enyl)oxalamide ( 45 ) ( 168 mg , 78% ) as a colorless solid , mp 83.083.3 c . h nmr ( 400 mhz ) 7.45 ( br s , nh , 2h ) , 5.305.40 ( m , 2h ) , 3.34 ( q , j = 8.6 hz , 4h ) , 2.32 ( t , j = 7.6 hz , 2h ) , 1.982.08 ( m , 4h ) , 1.301.68 ( m , 16h ) , 0.92 ( t , j = 7.2 hz , 3h ) . c nmr ( 100 mhz ) 160.03 ( 2c ) , 130.03 , 129.08 , 120.10 , 39.88 , 39.42 , 31.22 , 29.40 , 28.82 , 28.60 , 28.42 , 27.07 , 27.06 , 26.82 , 25.54 , 20.06 , 17.01 , 13.80 . hrms calcd for c19h34n3o2 [ m + 1 ] 336.2651 , found 336.2650 . to a suspension of n - n - butyl - n-(12-cyanododec-5(z)-enyl)oxalamide ( 45 ) ( 420 mg , 1.29 mmol ) in meoh / h2o ( 4:1 ; 12 ml ) was added h2nohhcl ( 228 mg , 3.28 mmol ) and na2co3 ( 344 mg , 3.25 mmol ) . the reaction mixture was heated at 60 c for 18 h then cooled to room temperature , and all volatiles were removed in vacuo . the residue was diluted with water ( 30 ml ) and extracted into ethyl acetate ( 2 25 ml ) . the combined organic extracts were washed with water ( 2 10 ml ) and brine ( 10 ml ) , dried , and purified via silica gel column chromatography to give n-(13-amino-13-(hydroxyimino)tridec-5(z)-enyl)-n - n - butyloxalamide ( 46 ) ( 287 mg , 62% ) as a colorless solid , 116.3116.4 c . h nmr ( cd3od , 400 mhz ) 5.285.40 ( m , 2h ) , 3.24 ( t , j = 6.4 hz , 4h ) , 1.982.00 ( m , 6h ) , 1.501.60 ( m , 6h ) , 1.261.40 ( m , 10h ) , 0.92 ( t , j = 7.3 hz , 3h ) . c nmr ( cd3od , 100 mhz ) 160.55 ( 2c ) , 156.31 , 130.05 , 129.18 , 39.23 , 39.09 , 31.18 , 30.63 , 29.51 , 28.83 , 28.69 , 27.10 , 26.87 , 26.59 , 19.88 , 12.88 . the influence of eicosanoids and analogues on coronary vascular tone was measured by the induced changes in isometric tension of bovine coronary artery rings precontracted with the thromboxane - mimetic , u46619 , as previously described . all assays were conducted in triplicate or greater and are means 10% sd of the reported value . recombinant human seh was produced in a baculovirus expression system and was purified by affinity chromatography . human seh ( 1 nm ) was incubated with inhibitors ( 0.4 < [ i]final < 100000 nm ) for 5 min in 25 mm bis - tris / hcl buffer ( 200 ml , ph 7.0 ) at 30 c before the substrate , cyano(2-methoxynaphthalen-6-yl)methyl trans-(3-phenyl - oxyyran-2-yl]methyl carbonate ( cmnpc ; [ s]final = 5 mm ) , was added . activity was assessed by measuring the appearance of the fluorescent 6-methoxynaphthaldehyde product ( em = 330 nm , ex = 465 nm ) at 30 c during a 10 min incubation ( spectramax m2 , molecular device , inc . , ic50s refer to the concentrations of inhibitor that reduced activity by 50% and are the averages of three replicates .

the cytochrome p450 eicosanoid 14,15-epoxyeicosa-5,8,11-trienoic acid ( 14,15-eet ) is a powerful endogenous autacoid that has been ascribed an impressive array of physiologic functions including regulation of blood pressure . because 14,15-eet is chemically and metabolically labile , structurally related surrogates containing epoxide bioisosteres were introduced and have become useful in vitro pharmacologic tools but are not suitable for in vivo applications . a new generation of eet mimics incorporating modifications to the carboxylate were prepared and evaluated for vasorelaxation and inhibition of soluble epoxide hydrolase ( seh ) . tetrazole 19 ( ed50 0.18 m ) and oxadiazole-5-thione 25 ( ed50 0.36 m ) were 12- and 6-fold more potent , respectively , than 14,15-eet as vasorelaxants ; on the other hand , their ability to block seh differed substantially , i.e. , 11 vs > 500 nm . these data will expedite the development of potent and specific in vivo drug candidates .

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in response to the increasing demand for an improved healthcare system in the united states , the american academy of family physicians , the american academy of pediatrics , the american college of physicians , and the american osteopathic association developed the joint principles of the patient - centered medical home ( table 1 ) . the patient - centered medical home ( pcmh ) is an extension of internationally employed edward wagner 's chronic care model ( ccm ) . the ccm was developed to address the increasing rate of patients with chronic conditions in the united states using team - based care . the rate of chronic conditions is currently estimated to be 2.2 conditions for individuals having 60 years old and up , on average . in its implementation , the ccm has proven to reduce patients ' healthcare costs and improve patient care quality , two elements directly aligned with the goals of the pcmh . the pcmh model strives to provide quality , coordinated , and cost - effective care to patients and to increase access to services . in addition , it aims to increase practice efficiency and subsequently provider and patient satisfaction . within this paper , we follow the process of implementing the pcmh model within primary care practices and discuss the difficulties these practices have encountered in the transition as well as potential solutions . our goal is to provide future pcmhs with an insight into the transition process and to remove the likelihood of these problems reoccurring . in spite of difficulty with the transition process , over 1,500 sites and 7,700 clinicians across the united states have successfully completed the national committee for quality assurance ( ncqa ) recognition process and are functioning as patient - centered medical homes . they see pcmh as a way to better serve their patients , to address the crisis in primary care , and to seize evolving payment opportunities . encouraged by the expansion of the pcmh across the united states , in june 2010 , medical practitioners and healthcare administrators ( military providers , civilian doctors and nurses , researchers , hospital staff , and administration ) met in alexandria , virginia , to discuss their experiences with the transition . from the conference , we were able to collect detailed experiences that will provide a unique insight and data from the transition process . from the panel of attendees , two specific healthcare providers carillion clinic and the air force contributed the vast majority of the examples and experiences discussed here . carillion clinic is a large healthcare organization located in southwest virginia , providing healthcare to individuals in both urban and rural settings . their organization comprises over 600 physicians in multicare - specialty group practices and eight not - for - profit hospitals . the examples in this paper are primarily taken from their experiences transitioning their urban primary care practices into pcmhs . more specifically , their experiences were directly pulled from practices with multiple physicians ( carilion clinic does group certifications of pcmhs by region ) . the air force has employed their version of the pcmh , termed the air force patient - centered medical home ( previously the family health initiative ) , at several of their bases within the united states . these air force pcmh practices are within the air force bases themselves , and each of them has a patient panel of military beneficiaries ( active duty members , retirees , and families ) creating a unique healthcare environment because the air force operates under a complex healthcare and insurance system with both military care and purchased care ( our focus in this paper is on the onbase care provided by the air force and does not pertain to care purchased offbase through the air force 's tricare program ) . from these healthcare organizations ' experiences , combined with a literature review of empirical work , this paper addresses the challenges and successes encountered in the transition to pcmh . the purpose of this paper is to realize the difficulties that arise in the transition into a pcmh across various settings and to provide solutions for practices to follow in their transition given their particular needs . this way these practices can provide better quality of care to patients , decrease patient 's health care costs , and improve the system as a whole . lessons learned discusses the most common concerns among primary care practices who have initiated the transition to a pcmh , which are promoting physician buy - in , changing office culture , care coordination , staffing and space allocations , and leveraging electronic medical records ( emrs ) . under each of these topics , difficulties and successes are examined using specific examples provided by the pcmh workshop and previous studies . the first step in the transition process is to understand what procedures and standards a primary care practice must follow in order to obtain recognition . recognition as a pcmh increases the likelihood of reimbursement for the pioneering pcmh procedures that are currently undercompensated . as more studies discuss the positive results of pcmh , new reimbursement methods become more of an obtainable goal . for a practice to become recognized as a pcmh by the ncqa , the practice must provide documentation of the practice 's guidelines for implementation ( table 2 , ) . however , ncqa standards do not offer instructions for practices to follow in making the transition . this flexibility in the ncqa standards is necessary due to the uniqueness of each practice as it would be difficult to create one that addresses the implementation of all the standards of the pcmh for each practice type . consequently , each practice must create its own written policies that adhere to pcmh principles and fit the specific practice structure . practices must account for their patient panels , location , and financial resources when creating policies . the practices must then decide whether it would better suit their needs to attempt the transformation incrementally or all at once . once these policies have been written , implemented , and then the outcomes documented , the practice can apply for ncqa recognition through an online survey that collects information regarding its guidelines about administration ( appointments , access , telephone calls ) , clinical services ( patient satisfaction , tracking critically important conditions ) , and performance tracking [ 8 , 9 ] . once these steps are completed successfully as determined by the ncqa review process , the practice is awarded one of three tiers of recognition by the ncqa ( the ncqa is currently debating reframing the recognition system to a two - tiered recognition system . some supporters of the pcmh feel that the implementation of the model should be to the fullest extent possible and only acknowledged in by the ncqa in these cases . however , as discussed within this paper , there are difficulties that arise when attempting to administer all of the changes necessary and not all practices can achieve the standards required for these recognition levels ) . each tier reflects how many performance elements under each ncqa standard the practice has satisfied . as with the introduction of any innovative healthcare model , the transition and recognition of a pcmh can be time consuming and expensive , but rewards range from better patient care to a more coordinated practice structure and also potentially reducing the overall cost of care for the patient in their lifetime through preventative and care management services , the latter of which is becoming more and more of a concern for primary care practices due to the current debate over reimbursement schedules . primary care physicians typically leave the workforce earlier in their careers than specialists with complaints of being overworked and poorly compensated . therefore , selling the model to physicians , with their already hectic schedules , so that their participation and contribution to pcmh is pivotal in a successful transformation . however , many physicians involved in the transformation resist the change due to a lack of the appropriate pcmh training , misaligned financial incentives , underreimbursement , and time - consuming procedures . the pcmh model recommends that practice staff and providers ( nurses and physicians ) take time to analyze patients ' needs as a whole . the model encourages practice leaders to empower ancillary staff to enlist protocols when meeting chronic , acute , and health maintenance needs . this team - based care is delivered prior to or after the provider encounter during a visit . the air force gave the following example of team - based collaboration when addressing a patient with a sprained ankle . first , the technician evaluates the patient and then performs the ottawa ankle rules , which clearly delineate whether the patient requires an x - ray . next , nurses will order x - rays based upon the results of the ottawa ankle rules the technician provides . subsequently , the skilled nurses return to educate the patient on ankle exercises and necessary bracing . training a nurse to do this initial triage and intervention enables the provider to meet with the patient after x - rays are complete , which allows for a more efficient appointment . the team - based care creates a routine in which it is no longer necessary for a provider to address the patient each time they come to the practice . many physicians are reluctant to release these face - to - face encounters with patients to their staff . this can be explained by the lack of training specific to pcmh practices , as well as the current reimbursement schedule , which encourage physicians to treat patients based on volume of care , rather than quality . practices must emphasize that the reallocation of responsibilities across the practice 's medical team can create more time for them to complete other requirements and make the practice more efficient as seen in the above air force example . second , the current form of financial reimbursement creates misaligned incentives for the physicians with their new responsibilities . for instance , according to the medicare resource - based relative value scale ( rbrvs ) , physicians are only reimbursed for a patient visit if they can report a 15-minute office evaluation , discouraging the physicians from relinquishing any of their patients ' visits to their nurses . restructuring the reimbursement scheme so that such face - to - face visits between physicians and patients are not required would allow physicians to provide care within the best interest of the patient and their practice . however , this modification may involve many hours of lobbying and petitioning the federal government and insurance companies . with many successful pcmhs across the united states , in order to sustain the model payment schemes will eventually need to adjust to reimburse physicians for improving patients ' quality of care and overall health , rather than for just the volume of services they provide . aside from physicians ' reimbursement , other components of the pcmh policies , such as care coordination and team - based care , require the use of resources not currently reimbursed in most health insurance packages . in addition to the above - mentioned responsibilities of the physicians and their staff , the pcmh model introduces team - based care to primary care practices . team - based care requires staff to address patient treatment decisions collectively . in advance of each appointment , a previsit meeting is conducted to allocate resources , to ensure that adequate health maintenance is performed , and to see that chronic care management is addressed by the care team ( nurses , care coordinators , physicians , other providers in the practice , and front office staff ) . however , it is important to emphasize that previous studies show the use of previsit huddles has resulted in improved decisionmaking and care for patients , as well as patient satisfaction [ 15 , 16 ] . the studies also show that patients provided with team - based care had increases in their satisfaction levels from 5 to 10 points in five out of six satisfaction care scales . in addition , their research shows that once providers accepted the team - based care structure , their satisfaction levels increased as well . in spite of the current difficulty with physician buy - in to pcmh , we found that many physicians are inspired by their ability to provide patients with the quality care they deserve through pcmh . a lead primary care physician from a carillion clinic has seen an improvement in the health of patients due to the coordination and team - based care pcmh offersthe pcmh care model has helped us provide more comprehensive care to our patient population . the use of registries and care coordinators have enabled us to focus on patients that are not getting needed care or are not returning for follow - up appointments as they should . the pcmh care model has helped us provide more comprehensive care to our patient population . the use of registries and care coordinators have enabled us to focus on patients that are not getting needed care or are not returning for follow - up appointments as they should . the transformation of a primary care practice into a pcmh requires significant changes in office culture . in this section , we will discuss several areas that need to be addressed by the primary care practice , including nurse and patient experience . implementing the pcmh model is not as simple as assigning new staff roles and restructuring old staff with new titles . the healthcare organization must understand how changing positions within the practice brings new experiences to all of their current members and how these experiences can potentially affect their capability of delivering care . for instance , in the air force , many nurses in the pcmh transition shift from positions where they have not been exposed to patients to being required to evaluate patients face to face regularly ; this new interaction created anxiety among these nurses . the air force has addressed this issue through additional training programs so nurses can better understand their new relationship with patients . in addition to providing new training for nurses , it is important also to train front office staff in their new roles as they play a part in carrying out several pcmh principles , such as ensuring greater access and increased communication . both clerical and clinical staff members are encouraged to develop a more sophisticated level of decision - making skills , which may require additional training on the pcmh principles , new workflow processes in the office , and increasingly integrated responsibilities to patients and each other . every healthcare model must take into account patients ' response to the new healthcare structure . patients value the respect , quality , and concern of their healthcare provider in addition to accessibility ; their reaction to the change in structure significantly affects the ability of a team to provide care . it is equally important that patients understand that the ultimate goal of pcmh is to improve their quality of life and participate in achieving this objective . pcmh focuses not only on the manner in which care is administered , but also educating patients regarding their health and ensuring they have the knowledge necessary to improve it . national pcmh demonstration projects have shown patient education has increased their participation in their own care and subsequently their quality of life . patients are encouraged to interact with practice staff and their providers regularly during appointments and through new means of communication , such as online patient portals , and to take advantage of health education opportunities within the community to which they are referred by staff . in pcmh , practices are tasked with improving care coordination and documenting patient engagement in self - management . this requirement has prompted many pcmhs to create a care manager or care coordinator position . this staff member ( e.g. , a nurse or nonclinical staff member ) performs tasks that were previously delivered inconsistently or not at all , or that were formerly the responsibility of the physician , such as patient followups and patient education ( when introducing the care coordinator to the patients , we encourage caution to avoid dramatization of past inadequacies in care delivery during patient and physician interactions , which could undermine their relationship with the physician . ) throughout the pcmh conference , discussants emphasized their success with care coordinators in their practices , many of whom have received positive feedback from their patients . for instance , a diabetic patient from one of carilion clinic 's roanoke , virginia practices reportedi have recently been diagnosed with diabetes . while this was initially very upsetting for me , i was able to receive immediate counseling about my diabetes from the care coordinator . we reviewed some initial diet and exercise information , and then she set me up to see a dietitian in the diabetes management program.this patient 's improvement is unquestionable and shows the benefits this new position can provide . however , problems arise with the adoption of multidisciplinary team members in the education and support of patients , their time , salary , responsibilities , and workload are not defined in the cost or structure of a typical practice . additionally , many practices that hire care coordinators face the challenge of reimbursing their work ; this is because their work is typically not covered by health insurance at all or only to a limited extent . while this was initially very upsetting for me , i was able to receive immediate counseling about my diabetes from the care coordinator . we reviewed some initial diet and exercise information , and then she set me up to see a dietitian in the diabetes management program . in addition to self - management coaching of those patients with chronic conditions , the care coordinators at carilion clinic have been assigned the task of population management . the care coordinators use registries ( registries are a list of patients with a chronic disease displayed with many clinical features to stratify level of disease control ) , to focus on specific chronic diseases , such as high blood pressure and diabetes . care coordinators use predetermined protocols to contact those patients in poor disease control or those who are due for a follow - up visit . once these patients come back into the office , a number of things happen as they reengage with their care team . the nurse updates the health maintenance care in the chart , the provider focuses on the acute or chronic disease issues , and the care coordinator offers to meet with the patient , further identifying barriers they face that may interfere with disease self - management . a sophisticated emr or free - standing disease registry facilitates this task . these technologies are used to organize and target the entire patient panel to track health indicators and in turn , isolate the patients of interest . this disciplined approach to population management is not typical in most primary care offices ; therefore , this activity tends not to be included in the budget , and time is not allocated for it . if the transitioning pcmh practice is committed to carrying out population management work , there needs to be added space for this individual to work along with a private consultation area to perform the self - management coaching . along with refocusing the office culture to adhere to the new comprehensive level of care , pcmh connects patients with resources in the healthcare system and the community . primary care practices need to take time to build collaborative relationships with other professionals , such as social workers , nutritionists , and health educators , to provide needed services . it is not feasible for most pcmh practice sites to have space on hand for these professionals to work side by side at all times . the pcmh model does not require that a specific staff member document the services offered , ordered , or rendered , and neither does ncqa . rather , what is important is that there is a workflow process in place for such documentation in patients ' medical records . for instance , at carilion , ambulatory nurses have taken on roles in both chronic disease management and health maintenance . in some practices , nurses have taken on the documentation of all health maintenance needs of patients , while in others , this responsibility is shared among team members . no matter who plays what role , they need to be clearly within individual practice workflows and protocols . there may be a period when nurses will need to familiarize themselves with the new structure . during this time for example , the air force experienced a backlog problem with the transition of nurses from floor nurses to disease managers . the disease manager started as one of the nurses in the clinic but was taken out of patient treatment and put into this new position . the problem is that this created backfill , so the disease management nurses were not able to concentrate on the task at hand but instead needed to address the problems that arose in the practice during their training . once the air force 's disease management nurses were able to focus on disease management , there was significant improvement in the quality of care for patients . it was also found in an outside study that patients who participated in chronic disease management had less frequent hospitalizations and on average spent 0.8 fewer nights in the hospital once admitted . carilion clinic has also implemented disease management and has found that scanning the patient registry on a regular basis uncovers patients who would benefit from intensive disease management . in particular , one patient under carilion 's care , a 70-year - old woman with diabetes , coronary artery d status after coronary artery bypass grafting , and congestive heart failure , was oxygen dependent and wheelchair bound . she was contacted after the care coordinator saw in her emr that she had visited the emergency room three times within the past eight months and one visit resulted in a prolonged hospital and intensive care unit stay . following the practice 's protocol , her care team referred her to cardiac rehab . subsequently , she has not visited the emergency room in over a year , has lost 30 pounds , no longer needs oxygen , and walks without assistance . the size of the primary care practice can be an obstacle in implementing the pcmh model . here , the term small is used to refer to primary care practices with only one or two physicians and an average - sized patient panel ( it is not uncommon for a primary care practice with one physician to have a couple thousand patients ; however , practices discussed within this paper do not have over 2,000 patients in their panel ) . approximately 32% of healthcare physicians practice in either an individual or a partnership practice , and 60% of practices function with 50 physicians or fewer . smaller practices typically can not employ the same resources as larger facilities due to budget constraints , and therefore , they may have a harder time making the changes necessary to become a pcmh and have to find ways around their limited resources . for instance , rather than full - time coordinators in small practices , carilion employs shared coordinators in sites where there are only one or two full - time providers ; as such these smaller practices can not rationalize adding a full - time staff member . national pcmh demonstrations have shown that these smaller practices with such resource constraints have a more difficult time transforming to a high - functioning pcmh ; they may qualify as level 1 , rather than level 3 , which means they only meet the minimum requirements of the ncqa [ 1719 ] . an emr is not required by the ncqa for a practice to apply for pcmh recognition , but having one may facilitate the practice 's goals for care coordination . implementing an emr is not particularly difficult ; instead , the concern is who will service , update , and sustain it . regardless of the size of practices , pcmh principles encourage the use of electronic medical records to increase the efficiency of care coordination , although it is not required . this change in record keeping removes the need for medical record storage space within the practice and in turn allows for clinical space to be optimized . prior to fully transitioning to the pcmh model , healthcare organizations must address the issue of space . for instance , if a practice employs a care coordinator , they must be provided with space to communicate with patients in private , to perform their health assessments and individual disease coaching on the phone or in person , and to prepare plans for care , such as materials for the pre - visit huddle . it is uncommon for the traditional primary care practices to have a location for these types of private conversations and activities . empty exam rooms or doctors ' offices may just be sufficient , especially since these conversations taking place in waiting rooms or more public areas can easily violate confidentiality standards and the health insurance portability and accountability act of 1996 . a fully integrated emr helps pcmhs conduct team - based care for chronic conditions by allowing members to visualize the patient instantly , through the use of problem lists , immunization history , and medication lists . however , creating an emr that administers the principles of pcmh has been a difficult task for primary care practices in transition . in a 2006 study , it was reported that only 42% of primary care practices found it easy to generate a disease registry , while only 18% felt it was easy to create special follow - up appointments based on high - risk medications patients were taking . many of the pcmh conference attendees had complaints about their difficulty in creating an emr that suited their specific practice 's needs . for practices transitioning from a paper - based system to an emr , it takes staff members a great deal of time to input all of the information into a computerized system . practices sometimes find the sheer volume of information needing to be recorded overwhelming and difficult to decipher , because there may not have been a pcmh template in paper charts . if the practice is unable to employ the emr to its advantage , many of the goals of pcmh are very difficult to satisfy . for instance , care coordination and disease management are very difficult for large practices to handle using a paper - based system . once employed , emrs can be used to improve patient care and therefore patient health by monitoring their health status to anticipate necessary treatments . using the emr , care coordinators and nurses are able to more efficiently search for specific patient types by viewing the patient 's medical record on the computerized database and to acknowledge those who require immediate attention and contact them in a timely fashion . staff then uses population management tools in the emr to contact these individuals and encourage them to make an appointment . in a review of the pcmh model , the authors were encouraged by pcmh physicians who are using proactive methods to assist unhealthy patients , rather than waiting for patients to seek help . carilion clinic has created a structured method of administering disease management resulting in the optimization of their emr . their approach is to first search and then contact their patients who have a priority one rating . their searches are focused on patients with a chronic disease , which can be sorted in the emr based on their conditions . thus far , in their transition to a fully integrated emr , carillion clinic has formed registries for asthma , congestive heart failure ( chf ) , diabetes ( dm ) , and high blood pressure ( htn ) . within each of the registries , the patient 's clinical information , such as blood pressure , their registries can be sorted by the necessity of care or the priority as determined by carillion clinic . priority one patients are those who have at least one of three predefined health conditions determined by carilion clinic ( carillion developed these predefined health conditions from nationally recognized medical guidelines ) . an example of a priority one patient , as defined by carilion clinic , would be a diabetic ( dm ) with an a1c over 10 . another example of a priority one patient would be a patient who is hypertensive , or a patient with a systolic bp over 160 . the carillion staff contacts patients with these conditions as soon as possible . between the months of march and may 2010 , of the diabetic patients and hypertensive patients contacted , 43% committed to an appointment with carillion clinic and their care team to address their current health status ( as of june 11 , 2010 , 63% of the appointments made have been kept ) . without a fully functioning emr with capabilities that allow carilion clinic and other health care providers to search for these patients efficiently , they would remain invisible to the practice and may not receive the care they need . this paper has examined lessons learned from the implementation of pcmh at several sites , both military and civilian . pcmh demonstrations show there is still much work to be done , particularly through educating the medical community about the benefits of the model . current pcmh demonstrations have shown that this method of care can improve the quality of life for patients , such as reducing the patient 's frequency of er visits and hospital admissions , as well as reducing health care costs [ 25 , 26 ] . overall , the results illustrate that pcmh decreases the gap between what providers say they want to do in order to improve patient care and what they can actually do with their current resources . as with any new healthcare structure , current pcmh practices are still developing methods to improve the quality of care provided to their patients , such as care coordination , team - based care , and the use of emr to promote care coordination . there continues to be a struggle for transitioning pcmhs in determining how to implement the guidelines that the ncqa provides , which is a concern because ncqa is the most widely recognized organization in the united states for certifying practices as pcmhs . ncqa recognition of pcmh does not require primary care practices to excel at every standard , but rather it requires a system of documentation about the quality of care being provided , and a plan for continuous improvement that follows each of the principles . however , this does not guarantee a successful practice ; a successful practice should be defined as one that realizes pcmh recognition is not an end at all , but there is always room for improvement and maturation . with a great deal of enthusiasm from adopters thus far , pcmh can certainly have a permanent place in the future us healthcare system . healthcare professionals and organizations will be under pressure to prove the value of their services . in implementing pcmh , one will track hba1c , bmi , and/or other parameters as indicators of quality , but at what point would a pcmh be qualified to receive additional payment for their improvement in the quality of patient care ? without funding for additional resources , practices will continue with the discrepancy between the current volume - based healthcare system and a system that focuses on the quality of care . the question remains of whether pcmh is the direction healthcare should go in , and many studies show that preventative care improves the quality of life . pcmh holds a great deal of potential in addressing the broken healthcare system in the united states , but at the same time , it also faces many obstacles in terms of payment reform , professional support , and patient participation .

the patient - centered medical home ( pcmh ) is a primary care model that provides coordinated and comprehensive care to patients to improve health outcomes . this paper addresses practical issues that arise when transitioning a traditional primary care practice into a pcmh recognized by the national committee for quality assurance ( ncqa ) . individual organizations ' experiences with this transition were gathered at a pcmh workshop in alexandria , virginia in june 2010 . an analysis of their experiences has been used along with a literature review to reveal common challenges that must be addressed in ways that are responsive to the practice and patients ' needs . these are : ncqa guidance , promoting provider buy - in , leveraging electronic medical records , changing office culture , and realigning workspace in the practice to accommodate services needed to carry out the intent of pcmh . the ncqa provides a set of standards for implementing the pcmh model , but these standards lack many specifics that will be relied on in location situations . while many researchers and providers have made critiques , we see this vagueness as allowing for greater flexibility in how a practice implements pcmh .

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incremental improvements in the survivorship from cpr occurred as more and more people were trained in cpr and as defibrillators became portable and were deployed in more locations . finally , most people who had suffered from cardiac arrest did not survive to leave the hospital or did so in a neurological devastated state . in the early of 21 century , the survival rate of cardiac arrest outside of the hospital remained 78% . about one - quarter of patients regained pulses after cpr , and about one - third of the patients with those initial successes survived hospitalization . the severe brain defect after cpr stimulated many investigations into the pathophysiology of , and treatments for , global brain ischemia . for the protection of patient 's brain , mild hypothermia therapy has been proposed . in many animal experiments for example , it can increase the survival rate and improve neurological function of animal . besides , it reduces metabolic rate and the expression of some pro - apoptosis proteins . in clinical trials , the effectiveness of hypothermia also has been proved . hence , 2010 american heart association guidelines for cardiopulmonary resuscitation and emergency cardiovascular care recommended , comatose patients with out - of - hospital ventricular fibrillation cardiac arrest were cooled to 3234c for 12 or 24 h. however , recent years some other clinical trials showed that mild hypothermia can not improve the prognosis of patients after cpr . therefore , we conducted a meta - analysis of all relevant published studies to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest . studies were considered for inclusion if they met the following criteria : ( 1 ) all published randomized controlled trials ( rcts ) . ( 2 ) we included studies in adult patients who suffered from cardiac arrest and were successfully resuscitated . ( 3 ) the intervention was therapeutic mild hypothermia , no matter how to lower the body temperature . the temperature should remain between 32c and 34c . and the treatment for the control group was according to the standard treatment after cardiac arrest without hypothermia . the secondary outcomes were adverse events , such as rearrest , renal failure , pulmonary edema and so on . ( 5 ) the relative risks ( rrs ) with their corresponding 95% confidence intervals ( cis ) were reported . studies were considered for exclusion if they met the following criteria : ( 1 ) we excluded studies on children and pregnant women . ( 2 ) cardiac arrest patients under the treatment of mild hypothermia received other drugs in combination therapy . ( 3 ) there was a history of central nervous system depressant drug medication prior to cardiac arrest . an extensive literature search was conducted using electronic databases , manual searching , and correspondence with authors of included studies . the cochrane library , pubmed , web of science , embase , cnki , and wan fang data were searched from the date of their establishment to october 2014 . search terms used were cooling , hypothermia , cpr , cardiac arrest and rct . two reviewers independently reviewed the citations , abstracts , and full - text articles and determined the eligibility of all the studies identified in the initial search . when the entire process was completed , the two cross - checked with each other . in cases of disagreements , a third reviewer was consulted . the following details were extracted : authors , year of publication , sample size , interventions , and outcomes . assessment of the quality of the included studies was performed using the methodology recommended by cochrane collaboration . this method comprised assessments of the risk of potential bias in six domains : random sequence generation ( correct , incorrect or unclear ) , allocation concealment ( correct , incorrect or unclear ) , blinding of outcome assessment ( correct , incorrect or unclear ) , incomplete outcome data ( complete , incomplete or unclear ) , selective reporting ( yes , no or unclear ) , other bias ( yes , no or unclear ) , such as the baseline , source of funding , and academic biases . if all quality criteria were met , the trial was considered to have a low risk of bias ( score : a ) . if one or more of the quality criteria were only partially met , the trial was considered to have moderate risk of bias ( score : b ) , and if one or more criteria not met , the trial was considered to have high risk of bias ( score : c ) . statistical analysis was performed using review manager version 5.2 ( cochrane collaboration , oxford , uk ) software . the heterogeneity of the qualitative analysis was assessed by chi - square test , and the significant level was set to p = 0.1 . if p > 0.1 , i < 50% , the different rcts can be regarded as homogeneous . if p < 0.1 , i 50% , the different rcts can be regarded as heterogeneity . we used weighted mean deviation ( wmd ) and 95% ci to represent the continuous data . and studies were considered for inclusion if they met the following criteria : ( 1 ) all published randomized controlled trials ( rcts ) . ( 2 ) we included studies in adult patients who suffered from cardiac arrest and were successfully resuscitated . ( 3 ) the intervention was therapeutic mild hypothermia , no matter how to lower the body temperature . the temperature should remain between 32c and 34c . and the treatment for the control group was according to the standard treatment after cardiac arrest without hypothermia . the secondary outcomes were adverse events , such as rearrest , renal failure , pulmonary edema and so on . ( 5 ) the relative risks ( rrs ) with their corresponding 95% confidence intervals ( cis ) were reported . studies were considered for exclusion if they met the following criteria : ( 1 ) we excluded studies on children and pregnant women . ( 2 ) cardiac arrest patients under the treatment of mild hypothermia received other drugs in combination therapy . ( 3 ) there was a history of central nervous system depressant drug medication prior to cardiac arrest . an extensive literature search was conducted using electronic databases , manual searching , and correspondence with authors of included studies . the cochrane library , pubmed , web of science , embase , cnki , and wan fang data were searched from the date of their establishment to october 2014 . search terms used were cooling , hypothermia , cpr , cardiac arrest and rct . two reviewers independently reviewed the citations , abstracts , and full - text articles and determined the eligibility of all the studies identified in the initial search . when the entire process was completed , the two cross - checked with each other . in cases of disagreements , a third reviewer was consulted . the following details were extracted : authors , year of publication , sample size , interventions , and outcomes . assessment of the quality of the included studies was performed using the methodology recommended by cochrane collaboration . this method comprised assessments of the risk of potential bias in six domains : random sequence generation ( correct , incorrect or unclear ) , allocation concealment ( correct , incorrect or unclear ) , blinding of outcome assessment ( correct , incorrect or unclear ) , incomplete outcome data ( complete , incomplete or unclear ) , selective reporting ( yes , no or unclear ) , other bias ( yes , no or unclear ) , such as the baseline , source of funding , and academic biases . if all quality criteria were met , the trial was considered to have a low risk of bias ( score : a ) . if one or more of the quality criteria were only partially met , the trial was considered to have moderate risk of bias ( score : b ) , and if one or more criteria not met , the trial was considered to have high risk of bias ( score : c ) . statistical analysis was performed using review manager version 5.2 ( cochrane collaboration , oxford , uk ) software . the heterogeneity of the qualitative analysis was assessed by chi - square test , and the significant level was set to p = 0.1 . if p > 0.1 , i < 50% , the different rcts can be regarded as homogeneous . if p < 0.1 , i 50% , the different rcts can be regarded as heterogeneity . we used weighted mean deviation ( wmd ) and 95% ci to represent the continuous data . and a total of 1613 potentially relevant studies were identified from the following databases in our initial articles search : two hundred and twenty - six from pubmed , 412 from embase , 161 from cochrane library , 292 from web of science , 245 from cnki , 237 from wan fang data . after screening the titles or abstracts , the full text of the remaining 60 studies was retrieved and assessed for eligibility . of the retrieved studies , 54 studies were excluded because of other type of publications ( review , case report , retrospective studies , prospective studies ) . finally , a total of six studies met the inclusion criteria [ figure 1 ] . the six rcts included 531 adult patients who suffered from cardiac arrest and were successfully resuscitated . the original articles compared the therapeutic effect under different conditions ( hypothermia or normothermia ) . characteristics of the rcts included in the meta - analysis rcts : randomized controlled trials , vf : ventricular fibrillation , ca : cardiac arrest . ( 1 ) all of the six studies mentioned random , but only three studies described the method of generating a random sequence correctly . ( 2 ) only two studies showed us they use a sealed envelope to conduct allocation concealment . ( 3 ) six studies mentioned blinding in which five studies were double - blind . ( 4 ) all of the six studies described the case of which quit or lost to follow - up . the number of quit or lost to follow - up of each study were < 20% of the total number . the methodological quality of the rcts included in the meta - analysis rcts : randomized controlled trials . the survival rate of patients who suffered from cardiac arrest and were successfully resuscitated is one of the primary outcomes which we concerned . all of the six studies reported the survival rate in which three studies reported survival rate at hospital discharge ( subgroup 1 ) , the other three studies reported survival rate after 6 months ( subgroup 2 ) . there was no heterogeneity from the outcome in subgroup 1 ( p = 0.50 , i = 0 ) and subgroup 2 ( p = 0.32 , i = 12% ) . the pooled results showed [ figure 2 ] no significant difference in the survival rate at hospital discharge between treatment group and control group ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) . however , there was significant difference in the survival rate after 6 months ( rr = 1.23 , 95% ci : 1.021.48 , p = 0.03 ) . summarized the above six studies , no heterogeneity among them ( p = 0.60 , i = 0 ) , and the pooled results showed hypothermia does improve the survival rate ( rr = 1.25 , 95% ci : 1.051.49 , p = 0.01 ) . the neurological function of patients who suffered from cardiac arrest and were successfully resuscitated is another primary outcome which we concerned . in clinical , cerebral performance category ( cpc ) the cpc scale ranges from 1 to 5 , with 1 representing good cerebral performance ( conscious , alert , capable of normal life ) or minor disability which do not significantly compromise cerebral or physical function , 2 moderate cerebral disability ( function is sufficient for independent activities of daily life ) , 3 severe cerebral disability , dependent on others for daily life support , 4 coma or vegetative state ( not conscious , unaware of surroundings , no cognition ) , and 5 brain death . included studies considered cpc 12 as good neurological recovery and cpc 35 as bad neurological recovery . hence , these data can be converted to dichotomous data . all of the six studies reported the neurological function in which three studies reported neurological function at hospital discharge ( subgroup 3 ) , the other three studies reported neurological function after 6 months ( subgroup 4 ) . there was no heterogeneity from the outcome in subgroup 3 ( p = 0.30 , i = 18% ) and subgroup 4 ( p = 0.23 , the pooled results showed [ figure 3 ] no significant difference in the neurological function at hospital discharge between treatment group and control group ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) . however , there was significant difference in the neurological function after 6 months ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) . summarized the above six studies , no heterogeneity among them ( p = 0.38 , i = 6% ) , the pooled results showed hypothermia does improve the neurological function ( rr = 1.37 , 95% ci : 1.131.66 , p = 0.001 ) . summary of data on neurological function for mild hypothermia versus normothermia . in the included studies , only four studies mentioned rearrest . meta - analysis [ figure 4 ] demonstrated mild hypothermia does not influence the incidence of rearrest ( rr = 1.19 , 95% ci : 0.871.61 , p = 0.27 ) . the result [ figure 5 ] showed no significant difference between treatment group and control group ( rr = 0.88 , 95% ci : 0.481.61 , p = 0.68 ) . other adverse events such as pulmonary edema , pneumonia , bleeding , showed the same results ( data not shown ) . because only a single article mentioned the above adverse events , we did not put them into meta - analysis . most studies at the top of the funnel plot , it meant the studies ci is narrow and their precision is high . to evaluate the influence of any single study on the pooled rr and ci we omitted two high - risk studies from the overall analysis , the pooled rr ( 95% ci ) ranged from 1.25 ( 1.051.49 ) to 1.21 ( 1.011.45 ) for studies reporting the rrs by survival rate and from 1.37 ( 1.131.66 ) to 1.30 ( 1.061.59 ) for studies reporting the rrs by neurological function . funnel plot compares log relative risk ( rr ) versus the standard error of log rr for survival rate and neurological function ( : at hospital discharge ; : at 6 months ) . a total of 1613 potentially relevant studies were identified from the following databases in our initial articles search : two hundred and twenty - six from pubmed , 412 from embase , 161 from cochrane library , 292 from web of science , 245 from cnki , 237 from wan fang data . after screening the titles or abstracts , the full text of the remaining 60 studies was retrieved and assessed for eligibility . of the retrieved studies , 54 studies were excluded because of other type of publications ( review , case report , retrospective studies , prospective studies ) . finally , a total of six studies met the inclusion criteria [ figure 1 ] . the six rcts included 531 adult patients who suffered from cardiac arrest and were successfully resuscitated . the original articles compared the therapeutic effect under different conditions ( hypothermia or normothermia ) . characteristics of the rcts included in the meta - analysis rcts : randomized controlled trials , vf : ventricular fibrillation , ca : cardiac arrest . ( 1 ) all of the six studies mentioned random , but only three studies described the method of generating a random sequence correctly . ( 2 ) only two studies showed us they use a sealed envelope to conduct allocation concealment . ( 3 ) six studies mentioned blinding in which five studies were double - blind . ( 4 ) all of the six studies described the case of which quit or lost to follow - up . the number of quit or lost to follow - up of each study were < 20% of the total number . the methodological quality of the rcts included in the meta - analysis rcts : randomized controlled trials . the survival rate of patients who suffered from cardiac arrest and were successfully resuscitated is one of the primary outcomes which we concerned . all of the six studies reported the survival rate in which three studies reported survival rate at hospital discharge ( subgroup 1 ) , the other three studies reported survival rate after 6 months ( subgroup 2 ) . there was no heterogeneity from the outcome in subgroup 1 ( p = 0.50 , i = 0 ) and subgroup 2 ( p = 0.32 , i = 12% ) . the pooled results showed [ figure 2 ] no significant difference in the survival rate at hospital discharge between treatment group and control group ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) . however , there was significant difference in the survival rate after 6 months ( rr = 1.23 , 95% ci : 1.021.48 , p = 0.03 ) . summarized the above six studies , no heterogeneity among them ( p = 0.60 , i = 0 ) , and the pooled results showed hypothermia does improve the survival rate ( rr = 1.25 , 95% ci : 1.051.49 , p = 0.01 ) . the neurological function of patients who suffered from cardiac arrest and were successfully resuscitated is another primary outcome which we concerned . in clinical , cerebral performance category ( cpc ) the cpc scale ranges from 1 to 5 , with 1 representing good cerebral performance ( conscious , alert , capable of normal life ) or minor disability which do not significantly compromise cerebral or physical function , 2 moderate cerebral disability ( function is sufficient for independent activities of daily life ) , 3 severe cerebral disability , dependent on others for daily life support , 4 coma or vegetative state ( not conscious , unaware of surroundings , no cognition ) , and 5 brain death . included studies considered cpc 12 as good neurological recovery and cpc 35 as bad neurological recovery . all of the six studies reported the neurological function in which three studies reported neurological function at hospital discharge ( subgroup 3 ) , the other three studies reported neurological function after 6 months ( subgroup 4 ) . there was no heterogeneity from the outcome in subgroup 3 ( p = 0.30 , i = 18% ) and subgroup 4 ( p = 0.23 , i = 31% ) . the pooled results showed [ figure 3 ] no significant difference in the neurological function at hospital discharge between treatment group and control group ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) . however , there was significant difference in the neurological function after 6 months ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) . summarized the above six studies , no heterogeneity among them ( p = 0.38 , i = 6% ) , the pooled results showed hypothermia does improve the neurological function ( rr = 1.37 , 95% ci : 1.131.66 , p = 0.001 ) . meta - analysis [ figure 4 ] demonstrated mild hypothermia does not influence the incidence of rearrest ( rr = 1.19 , 95% ci : 0.871.61 , p = 0.27 ) . the result [ figure 5 ] showed no significant difference between treatment group and control group ( rr = 0.88 , 95% ci : 0.481.61 , p = 0.68 ) . other adverse events such as pulmonary edema , pneumonia , bleeding , showed the same results ( data not shown ) . because only a single article mentioned the above adverse events , we did not put them into meta - analysis . most studies at the top of the funnel plot , it meant the studies ci is narrow and their precision is high . to evaluate the influence of any single study on the pooled rr and ci , we performed a sensitivity analysis . we omitted two high - risk studies from the overall analysis , the pooled rr ( 95% ci ) ranged from 1.25 ( 1.051.49 ) to 1.21 ( 1.011.45 ) for studies reporting the rrs by survival rate and from 1.37 ( 1.131.66 ) to 1.30 ( 1.061.59 ) for studies reporting the rrs by neurological function . funnel plot compares log relative risk ( rr ) versus the standard error of log rr for survival rate and neurological function ( : at hospital discharge ; : at 6 months ) . as the cpr technology became more and more mature , a growing number of patients with cardiac arrest were saved . in the half past century especially the last 10 years , hypothermia is the only method that approved by a large amount of clinical trials which can improve the prognosis of patients . meta - analysis can evaluate the effectiveness and safety of hypothermia which results are quite credible . in this study , we used meta - analysis to integrate these different independent researches , in order to get more reliable analysis about the efficacy of mild hypothermia for the treatment of patients with cardiac arrest . for patients rescued from cardiac arrest , we pay most attention to their prognosis . the data of three studies were measured at hospital discharge , the other three were measured after 6 months . in order to avoid the heterogeneity first we analyzed the data by subgroups . the mild hypothermia therapy can not improve survival rate and neurological function at hospital discharge . we speculated the possible reasons are as follows : ( 1 ) whole - body hypothermia influences all organ systems , and any potential benefit should be balanced against possible side effects ; ( 2 ) in clinical treatment , combination hypothermia with many other drugs may bring about some unknown influences , for example it will reduce the rate of drug metabolism and so on ; ( 3 ) hypothermia revives neurons is a long and slow process , for some other reasons patients die before neurons recovery . the pooled results showed us that mild hypothermia does improve survival rate and neurological function of patients with cardiac arrest . for example : hypothermic blanket , ice packs , cold saline intravenous infusion and so on . however , some other studies or meta - analysis hold the view that early initiation of rapid cooling can not improve the prognosis . the possible mechanisms include : ( 1 ) hypothermia can reduce cerebral oxygen consumption and energy metabolism ; ( 2 ) it can reduce reactive oxygen species generation and release of excitatory amino acids ; ( 3 ) it also can preserve the integrity of the blood - brain barrier , regulate the gene expression of inflammatory protein and apoptotic proteins and reduce cell death . although there are many advantages of mild hypothermia , we should not ignore the side effects . in the included six studies , the main adverse events were re - arrest , renal failure , pulmonary edema and so on . hence , we drew a conclusion mild hypothermia therapy for cardiac arrest patients is safe . however , the study also suffers from several limitations : ( 1 ) the number of trial and the study size may be inadequate . therefore , the precision of the outcome parameters obtained is generally low ; ( 2 ) another potential limitation of this meta - analysis is clinical and methodological heterogeneity . according to the six studies , we knew cardiac arrest patients with different first recorded cardiac rhythm are included . besides , the time from start of cooling to target temperature was different , the duration of keeping hypothermia was different and the methods of cooling body temperature were also different in these studies . all of the above factors would lead to inaccurate results . in this meta - analysis , we conducted a subgroup analysis according to different situations , although there was almost no heterogeneity . but in order to get more accurate results , further research should include high - quality studies to analyze the impact of different cooling measures or different hypothermia duration on the survival rate and neurological function . in conclusions , this meta - analysis demonstrated mild hypothermia can not improve survival rate and neurological function of patients at hospital discharge . the pooled results showed mild hypothermia does improve prognosis of patients . and it does not influence the incidence of adverse events compared with the control group .

background : therapeutic hypothermia has been recommended for the treatment of cardiac arrest patients who remain comatose after the return of spontaneous circulation . the aim of this study was to evaluate the effectiveness and safety of mild hypothermia on patients with cardiac arrest by conducting a meta-analysis.methods:the relevant trials were searched in cochrane library , pubmed , web of science , embase , cnki and wan fang data from the date of their establishment to october 2014 . thereafter , the studies retrieved were screened based on predefined inclusion and exclusion criteria . data were extracted , and the quality of the included studies was evaluated . a meta - analysis was conducted using the cochrane collaboration review manager 5.2 software.results:six randomized controlled trials involving 531 cases were included , among which 273 cases were assigned to the treatment group and the other 258 cases to the control group . the meta - analysis indicated that mild hypothermia therapy after cardiac arrest produced significant differences in survival rate ( relative risk [ rr ] = 1.23 , 95% confidence interval [ ci ] : 1.021.48 , p = 0.03 ) and neurological function ( rr = 1.33 , 95% ci : 1.081.65 , p = 0.007 ) after 6 months compared with normothermia therapy . however , no significant differences were observed in the survival to the hospital discharge ( rr = 1.35 , 95% ci : 0.872.10 , p = 0.18 ) , favorable neurological outcome at hospital discharge ( rr = 1.53 , 95% ci : 0.952.45 , p = 0.08 ) and adverse events.conclusions:the meta - analysis demonstrated that mild hypothermia can improve the survival rate and neurological function of patients with cardiac arrest after 6 months . on the other hand , regarding the survival to hospital discharge , favorable neurological outcome at hospital discharge , and adverse events , our meta - analysis produced nonsignificant results .

pubmed

for several years , a male factor has been recognized in the diagnosis and treatment of all infertility ; approximately 40% of marital infertility is due to a male factor . it is a condition characterized by persistent enlargement and elongation of the pampiniform plexus , venous convolution , and sinus formation with transient or permanent backflow . the etiopathogenesis of varicocele remains unclear and , at present , there is still no unequivocal explanation of their formation and effect on the excretory and secretory functions of the testis . the left testicular vein runs vertically and joins the left renal vein opposite the origin of the lower adrenal vein . thus , blood turbulence occurs in the vicinity of the opening of the testicular vein and , in consequence , retrograde flow of renal metabolites and adrenal hormones to the testis is observed . the elevated hydrostatic pressure within the testicular vein leads to venous stasis within the testis resulting in testicular hyperemia and compromised testicular oxygen concentration [ 1 , 2 ] . one of the hypotheses of spermatogenesis impairment is the concept of atrophic changes within convoluted tubules due to increased scrotal temperature or tissue hypoxia ; in consequence , testicular growth is arrested in children and adolescents on the affected side [ 3 , 4 ] . a high grade varicocele may be detected during a meticulous andrologic examination involving inspection and palpation of the genital organs while standing up . grading the varicocele is usually based on the dubin amelar 1 to 3 scale . we believe that a diagnosis based on inspection and palpation should subsequently be confirmed by accessory ultrasound . doppler examination allows the assessment of pampiniform plexus diameters , presence and character of backflow , as well as objective and comparative analysis of the size of both testes [ 1 , 5 , 6 ] . a palpable varicocele with testicular hypotrophy on the affected side , confirmed by color doppler , is an indication for surgical repair . from 1996 to 2011 , eighty two adolescents were operated on for idiopathic varicocele with testicular hypotrophy . sixty eight patients were subjected to the current analysis ( 14 men were lost in follow up ) . the age of the patients was 13 to 17 years ( mean 15.3 years ) . this is a retrospective study , where data from patients charts were analyzed . as the study is not an experimental one it did not need approval from the ethics committee . the clinical diagnosis was established on the basis of physical and andrologic examinations with an assessment of testicular size and varicocele severity on the dubin amelar 1 to 3 scale . color doppler ultrasound was performed in all patients ; testicular size was measured according to costabile with an orchidometer . in subjects with varicocele disease , a difference in testicular volume of 2 ml was used as the criterion of testis hypotrophy . laparoscopic repair was performed via a transperitoneal approach with division of the testicular vein only . the evaluation of surgical outcome was carried out at 1 to 9 years following laparoscopic varicocele repair , and was based on ultrasound evidence of varicocele subsidence and increase of testicular volume on the affected side . according to who directions , all subjects had their semen parameters checked twice after they had turned eighteen . the abnormal values of semen parameters were consistent with who 2010 guidelines also adopted in poland [ 12 , 13 ] . blood tests to determine levels of testosterone ( t ) , follicle stimulating hormone ( fsh ) , and lutenizing hormone ( lh ) were also performed and presented as qualitative variables ( normal / abnormal ) . abnormal adopted levels of the abovementioned hormones were : less than 300 ng / dl for testosterone and less than 1.5 iu / l for both lh and fsh . statistical analysis was carried out using the t student test ( statistica version 7.0 ) . for dependent variables , the paired test was applied and for independent variables , the unpaired t a grade 2 varicocele was found in 32 ( 47.1% ) subjects and a grade 3 was found in 36 ( 52.9% ) ( table 1 ) . before laparoscopic repair , mean left and right testicular volumes were 16.2 ( sd = 4.3 ) cm and 19.7 ( sd = 6.4 ) cm , respectively . thus , left testicular volume was significantly lower compared to that of the right testis ( p = 0.03 ) . an increase in left testicular volume when compared to the contralateral testis was found in 25 ( 78.1% ) subjects with clinical grade 2 varicocele , and in 32 ( 88.8% ) subjects with grade 3 abnormality . patients divided according to preoperative varicocele severity and postoperative improvement in testicular diameter and semen parameters number in brackets reflects % of the whole group in patients with unilateral grade 2 varicocele mean baseline left and right testicular volumes were 16.7 ( sd = 4.2 ) cm and 19.8 ( sd = 2.4 ) cm , respectively . after surgery the respective volumes were 20.9 ( sd = 3.7 ) cm and 21.4 ( sd = 1.6 ) cm ; the increase was statistically significant ( p = 0.01 ) . in patients with unilateral grade 3 varicocele , mean initial left and right testicular volumes were 16.2 ( sd = 2.3 ) cm and 18.7 ( sd = 3.9 ) cm , respectively . after surgery the respective volumes were 19.9 ( sd = 2.5 ) cm and 20.2 ( sd = 3.3 ) cm ; the increase was statistically significant ( p = 0.03 ) . an increase in left testicular volume was found in 46 ( 85.1% ) of 54 patients with unilateral varicocele and in 12 ( 85.7% ) of 14 subjects operated on for bilateral disease . in patients with bilateral varicocele mean baseline left and right testicular volumes were 15.5 ( sd = 6.6 ) cm and 17.9 ( sd = 3.2 ) cm , respectively . after surgery the respective volumes were 19 ( sd = 6.9 ) cm and 20.2 ( sd = 5.6 ) cm . the left testicular volume increases were comparable irrespective of whether unilateral or bilateral repair was performed . bilateral hypotrophy was seen in two ( 14.2% ) of fourteen subjects with bilateral disease ; they had no testicular volume increase at follow up . semen analysis was performed in all subjects who had undergone laparoscopic varicocele repair in adolescence . the check revealed normozoospermia in 58 ( 85.2% ) of our 68 patients while the remaining 10 ( 14.7% ) had oligozoospermia ( < 15 million sperm / ml ) or low percentage of motile spermatozoa , ie . , asthenozoospermia ( < 40% ) or low percentage of normally shaped sperm , ie . , teratozoospermia ( < 4% ) with persistent testicular hypotrophy . normozoospermic men also had normal fsh , lh , and t levels as opposed to 10 subjects with different variants of spermatogenesis abnormalities , who showed fsh and lh elevation with low t levels . varicocele recurrence was only observed in two ( 2.9% ) patients ; they underwent repeat repair . complications developed in six ( 8.8% ) of patients , and included a hydrocele testis five patients ( 7.3% ) , and epididymitis hospital stay was 1 to 3 days ( mean 2.1 days ) and overall convalescence was 7 to 12 days ( mean 8.2 days ) . the varicocele was described in antiquity ; since the end of the xix century it has been considered one of the causes of male factor infertility . although several convincing hypotheses exist to explain the etiology and pathomechanisms of varicoceles , there is no consensus about the advisability of early surgical repair and harmful effect of the vascular lesion on male fertility potential . we agree with greenfield , cayan , and other authors that in the case of suspicion or preliminary diagnosis of genital abnormality , the patient should be referred to a urologist or andrologist [ 2 , 6 , 14 , 15 , 16 ] . an increase in testicular volume on the affected side indicates the efficiency of surgical repair . we also agree that varicocele with testis hypotrophy on the affected side , confirmed by color doppler ultrasound , is an indication for surgical repair [ 2 , 9 , 15 , 19 , 20 , 21 ] . laparoscopic varicocele repair resulted in significant increase in hypotrophic testicular volume in 58 ( 83.8% ) of our subjects . numerous surgical techniques have been developed for treatment of varicocele , which are aimed at closing incompetent veins of the spermatic cord , thus preventing retrograde blood flow towards the testis and venous stasis in dilated vessels of the pampiniform plexus . surgical repair of varicocele can be achieved by conventional open varicocelectomy , laparoscopic , microsurgical intervention , or transdermal sclerotherapy and embolization of the testicular vein . technological progress of the last twenty years has allowed the introduction of a new minimally invasive surgical modality , ie . , we believe that laparoscopic varicocele repair as described by palomo ( ligation and division of both testicular vein and artery ) or bernardi ( testicular artery sparing ) should be recommended as the treatment of choice for the vascular lesion . however , opinions of experts on the efficacy of the above mentioned modalities are divided [ 2 , 9 , 17 , 18 ] . while recognizing the value of both procedures for varicocele correction , it is assumed that the laparoscopic bernardi technique should be given priority in boys and adolescents . axis occur earlier than their physical manifestation , ie . , decreased testicular volume and consistence especially on the varicocele affected side . concomitant damage to the right testis results when a immunological factor is present as it is in the case of unilateral cryptorchidism or unilateral testicular torsion . hormonal regulation of the reproductive system in adolescent boys and men of procreative age depends on sertoli and leydig cells stimulation in the testis by pituitary lh causes leydig cells to synthesize and , subsequently , secrete testosterone , which controls meiosis and spermatid transformation into a spermatozoon . fsh acts with testosterone to induce the secretory activity of sertoli cells ; they synthesize proteins indispensable for normal spermatogenesis . clinical observations and results of experimental research also suggest important roles of estradiol ( e2 ) and prolactin ( prl ) in the process [ 1 , 2 , 15 , 20 , 23 , 24 ] . postoperative follow up of young men after they turn 18 facilitates objective evaluation of the efficacy of surgical varicocele repair . our results , based on sperm examination at 1 to 9 years after laparoscopic intervention , mostly revealed normal sperm count , motility , and morphology . other authors also report a significant improvement of sperm parameters following surgical varicocele repair , thus confirming the advisability of surgical correction for the vascular lesion . normal fsh , lh , and t levels found in young men operated on in adolescence are also significant ; late diagnosis and treatment frequently result in spermatogenesis disturbances and abnormal concentrations of sex hormones [ 2 , 23 , 25 , 26 ] . laparoscopic varicocele repair resulted in significant increase of hypotrophic testicular volume in 83.8% of our subjects . assessment of the efficacy of surgical varicocele repair should include hormonal tests and semen analysis in procreative age group .

introductionfailure to perform surgical repair of varicocele before puberty is among the common causes of male infertility . the purpose of this study was to evaluate the testicular volume and fertility potential in men after laparoscopic varicocelectomy conducted in adolescence due to varicocele and concomitant testicular hypotrophy.material and methodsfrom 1996 through 2011 , eighty two adolescents were operated on for unilateral primary varicocele with testicular hypotrophy . sixty eight patients were subject to the current analysis . the age of the patients was 13 to 17 years ( mean 15.3 years ) . clinical diagnosis was established on the basis of andrologic examination and ultrasonography with an assessment of testicular size and varicocele severity . laparoscopic surgical repair was performed by a transperitoneal approach with division of testicular vein only.resultsan increase in left testicular volume when compared with the contralateral testis was found in 25 ( 78.1% ) young men with clinical grade 2 varicocele ( p = 0.02 ) and in 32 ( 88.8% ) subjects with grade 3 abnormality ( p = 0.04 ) . an increase in left testicular volume was found in 46 ( 85.1% ) of 54 patients with unilateral varicocele and in 12 ( 85.7% ) of 14 subjects operated on for bilateral disease . a left testicular volume increase was comparable independent of the use of uni or bilateral repair . fifty eight ( 85.2% ) of our 68 patients had normozoospermia.conclusionslaparoscopic varicocele repair resulted in a significant increase of hypotrophic testicular volume in 83.8% of our subjects .

pubmed

radical retropubic prostatectomy is the surgical treatment of choice for patients with localized prostate cancer . numerous studies have been published demonstrating excellent tumor control , good functional results , and low morbidity associated with the procedure ( 1 - 4 ) . however , the major long - term morbidity from radical retropubic prostatectomy that remains is erectile dysfunction , despite advances in surgical techniques . prior to studies done by walsh and donker ( 5 ) using male fetuses , the cause of erectile dysfunction after radical prostatectomy was not well understood . they proposed and eventually demonstrated that erectile dysfunction occurred secondary to the injury of cavernosal nerves . these nerves were identified branching from the pelvic plexus and running as a plexus of small nerves within a prominent neurovascular bundle on the posterolateral border of the prostate , terminating in the region of the membranous urethra . these nerves are mainly concentrated laterally to the prostate and these neural structures are referred to as neurovascular bundles . nerve sparing radical retropubic prostatectomy has led to improvements in potency rates , with studies reporting a 16 to 76% recovery of erectile function in men whose bilateral neurovascular bundles were preserved and 0 to 56% in those with unilateral neurovascular bundle preservation ( 6 , 7 ) . this variability can be attributed to the fact that there was no definite and exact anatomy of periprostatic nerve fibers , especially the cavernosal nerves . although some papers about the anatomy of periprostatic nerves were published recently , there are still controversies about detailed descriptions of the exact periprostatic nerve distribution in the literature . thus , in the present study , we described periprostatic nerve distribution and constructed a 3-dimensional model of nerve distribution . the prostate , together with seminal vesicle , bladder , and rectum , was removed en bloc from five adult male human cadavers ( age at death 43 - 72 yr ) after filling urinary bladder with 40% formaldehyde using urethral catheter . each specimen was sliced by 5 mm step sections from the base of the prostate to the apex , and a total of eight slices of whole - mount histologic sections were embedded in paraffin , and prepared , and then stained with anti - s-100 antibody neural stain to delineate the precise location of this neural tissue . immunostaining with anti - s-100 protein antibody was performed by dewaxing the slides in xylene three times for three minutes and then rehydrating in ethanol ( two changes of three minutes in 100% ethanol and two changes of three minutes in 90% ethanol ) and distilled water . the rabbit polyclonal primary antibody to s-100 protein ( immunon , pittsburgh , pa , usa ) was applied in a 1:500 dilution and incubated for one hour at room temperature . the samples were then washed in pbs , subjected to envision - hrp kit ( dako , carpinteria , ca , usa ) , and the reaction product was visualized with chromogen 3 , 3'-diaminobenzidine ( dab ) for three minutes . the stained whole - mount section slide was divided into four arbitrary sectors : two laterals , one ventral , and one dorsal ( rectal side ) according to one fourth the circumference of whole - mount specimens . whole - mount specimens were then divided by two axes , such as from 1.5 to 7.5 o'clock and 10.5 to 4.5 o'clock direction ( fig . each slide was taken from the base , mid - part , and apex of the prostate . nerve fibers 100 micrometer or greater in diameter were counted in 200 magnification microscopically ( olympus bx51 ; tokyo , japan ) , in addition to the number of nerve fibers , the distance from nerve fiber to the true prostate capsule , and nerve fiber diameter on each sector from representative slides . the three - dimensional periprostatic nerve distribution model was made by connecting the stained nerve fibers from the same location of the same sectors of the eight whole - mount slides from each prostate and reconstructing its image after taking a picture of the three - dimensional model ( fig . the difference in variables was assessed using anova and kruskal - wallis nonparametric anova . statistical significance in this study was considered at p<0.05 . the average number of nerve fibers located in the ventral part of the prostate was 5.751.20 , and comparatively , the average number in the dorsal ( rectal ) part was 28.502.17 . there were even fewer nerve fibers in the ventral part of the prostatic base area , numbering 3.751.38 . in the right lateral part the average number of nerve fibers was 25.259.92 , and in the left lateral part 25.7512.76 ; thus the number of nerve fibers in the dorsal ( rectal ) , right lateral , and left lateral parts of the prostate capsule showed no significant difference , and nerve fibers were distributed evenly around the prostatic capsule microscopically in the posterior , right lateral , and those in left lateral areas . however , nerve fibers in the ventral part were small in number compared to the dorsal , right lateral , and left lateral parts at all levels ( p=0.001 ) ( table 1 ) . moving from base to apex , the average number of nerve fibers increased in the ventral area , but this increase was not statistically significant ( p=0.386 ) . the average distance from prostatic capsule to nerve fibers was 1.730.43 mm in the ventral part , 1.800.16 mm in the dorsal ( rectal ) part , 1.580.26 mm in the right lateral part , and 1.230.29 mm in the left lateral part . there was no difference in the average distances when comparing the four different parts statistically ( p=0.266 ) . also there was no significant difference among the base , mid - part , and apex levels ( p=0.673 ) ( table 2 ) . the average thicknesses of nerve fibers in the ventral and dorsal parts were 0.190.02 mm and 0.300.03 mm , and those in the right and left lateral parts were 0.300.03 mm and 0.280.03 mm , respectively . there was no difference in nerve fiber thickness according to level such as base , midpart , and apex ( p=0.180 ) . however , the average thickness of the nerve fibers in the ventral part was significantly decreased as compared to the other parts ( p=0.005 ) ( table 3 ) . the three - dimensional model of the periprostatic nerve fibers revealed an almost even distribution and nearly similar thickness of periprostatic nerve fibers on dorsal and both lateral parts . furthermore , there was absence of a cord - like structure , such as neurovascular bundle , noted on the study . however , the nerve fibers in the ventral part were substantially fewer in number and decreased in thickness . traditionally , it was thought that cavernosal nerves branched from the pelvic plexus and ran as a plexus of small nerves within a prominent cord - like neurovascular bundle on the posterolateral border of the prostate ( 5 , 8) . new advancements in surgery , including the use of laparoscopic and robotic modalities and magnifying visual devices in open surgery , have enabled precise nerve dissection . however , despite all these advancements in nerve preservation or restoration , potency rates have remained unsatisfactory ( 9 - 11 ) . recently , several descriptions of the neurovascular bundle have been proposed : the neurovascular bundle , in contrast to the usual concept , appears to contain few pelvic splanchnic nerve components at the bladder - prostate junction . the pelvic splanchnic nerve branches demonstrate a spray - like distribution , and caudal branches reach the dorsolateral portion of the prostate at levels more than 20 mm inferior to the bladder - prostate junction . therefore , the cavernous nerves appear to be spread and located beyond the neurovascular bundle ( 12 ) . ( 13 ) found , in addition to the main neurovascular bundles , other nerve branches running between the prostatic capsule and endopelvic fascia , and called them distal accessory nerve pathway . according to these authors , this pathway could explain the unpredictable response to erection recovery and the lack of correlation between surgeon perception of the quality of neurovascular sparing and functional results after radical prostatectomy . recently , several descriptions of the neurovascular bundle have shown variations in the distributions of autonomic fibers between different individuals . about half of the patients have been reported as having a ' fan - like ' nerve fiber distribution on the lateral aspect of the prostate ( 14 ) . as mentioned above , the traditional knowledge of the neurovascular bundle has been challenged . therefore , we performed this study to distinguish microscopic features and anatomic navigation of periprostatic nerves using male adult cadavers . we found that the number of periprostatic nerve fibers was distributed evenly to dorsal and both lateral area of the prostate in our study . unlike the study of kiyoshima et al . ( 14 ) , no cord - like neurovascular bundle was observed in any cadaver case in our study . in addition , the distances from the prostatic capsule were almost the same in both the lateral and dorsal ( rectal ) parts of the base , middle , and apex levels of the prostate . these data , including numerical values about periprostatic nerve fibers , have not been reported and will serve as a guide for improved results for nerve sparing radical prostatectomy . several urologists reported surgical techniques according to the anatomy of the neurovascular bundle related to postoperative sexual dysfunction ( 14 , 15 ) . ( 16 ) suggested a modified technique of nerve sparing called " curtain dissection " , which involves incising the periprostatic fascia and dissecting the neurovascular bundle far more anteriorly than previously described . ( 2 ) reported the technique of open retropubic nerve - sparing radical prostatectomy . in their study , the parapelvic fascia of the prostate is incised at the lateroventral aspect of the prostate at 10 o'clock and 2 o'clock . they noted the importance of starting the incision high up on the ventral aspect of the prostate to preserve a maximum number of nerve fibers because a substantial number of nerve fibers are located at the ventral area . it has been shown that potency rates strongly correlate with the number of preserved nerve fibers . had introduced their modified technique of robotic radical prostatectomy , named vattikuti institute prostatectomy ( 17 ) . the features of their technique are early transaction of the bladder neck , preservation of the prostatic fascia ( veil of aphrodite ) , and control of the dorsal vein complex after dissection of the prostatic apex . they reported that the veil nerve sparing procedure offers superior erectile function compared with conventional nerve - sparing surgery without compromising cancer control ( 18 ) . ( 19 ) performed a histologic study of comparison between the veil of aphrodite technique and standard nerve - sparing technique during a robotic prostatectomy . they concluded that the veil of aphrodite technique is a safe procedure that effectively preserved the lateral prostatic fascia , which contains nerve bundles that run along the surface of the anterolateral zones . in their study , the nerve bundle counts of the anterolateral zones was different between the two techniques with statistical significance . our result means modified nerve - sparing techniques by incising the periprostatic fascia more anteriorly are feasible for saving periprostatic nerves . but an excessive high up incision ( over 2 , 10 o'clock ) would not be effective because the number of nerve fibers of both lateral parts is much more than that of the ventral part of the periprostatic area . these findings are differernt from eichelberg 's reports that the sum of the median percentage of nerves detected in anterior half of the prostate was more than 20% ( 20 ) . our result revealed that the mean percentage of nerves in ventral part was 6.7% ( range : 4.5 - 9.4% ) ( table 1 ) . according to costello et al . ( 21 ) , the majority of nerves which exist on the anterior aspect of the prostate laterally are not functionally significant parasympathetic nerves . so , there is little evidence to support higher incisions in the lateral prostatic fascia anatomically . based on this study , the number of periprostatic nerve fibers is evenly distributed on both lateral and dorsal ( rectal ) parts from base to apex of the prostate without forming any cord - like structures . because the number of nerve fibers on the ventral part is few in comparison , an excessive high up incision over 2 , 10 o'clock on the ventral part of the prostate is insignificant during nerve - sparing radical prostatectomy .

we investigated the distribution and navigation of periprostatic nerve fibers and constructed a 3-dimensional model of nerve distribution . a total of 5 cadaver specimens were serially sectioned in a transverse direction with 0.5 cm intervals . hematoxylineosin staining and immunohistochemical staining were then performed on whole - mount sections . three representative slides from the base , mid - part , and apex of each prostate were subsequently divided into 4 sectors : two lateral , one ventral , and one dorsal ( rectal ) part . the number of nerve fibers , the distance from nerve fiber to prostate capsule , and the nerve fiber diameters were analyzed on each sector from the representative slides by microscopy . periprostatic nerve fibers revealed a relatively even distribution in both lateral and dorsal parts of the prostate . there was no difference in the distances from the prostate capsule to nerve fibers . nerve fibers in the ventral area were also thinner as compared to other areas . in conclusion , periprostatic nerve fibers were observed to be distributed evenly in the periprostatic area , with the exception of the ventral area . as the number of nerve fibers on the ventral part is fewer in comparison , an excessive high up incision is insignificant during the nerve - sparing radical prostatectomy .

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rheumatoid arthritis ( ra ) is a progressive , autoimmune disease , characterized by chronic inflammation of synovial membrane and consistently degradation of articular cartilage and subchondral bone . serum amyloid a ( saa ) is a highly conserved acute - phase protein , released in response to inflammation or infection . production of acute - phase saa ( a - saa ) is stimulated by proinflammatory cytokines , such as interleukin-6 ( il-6 ) , il-1 , tumor necrosis factor ( tnf ) , interferon- , and transforming growth factor- ( tgf- ) . the concentration of a - saa increases dramatically during acute inflammation and injury , reaching within 5 - 6 hours levels that are 1000-fold greater than normal [ 13 ] . as with other acute - phase reactants , however , extrahepatic production of saa by several tissues and cell types has been described in patients with chronic diseases , for example , alzheimer 's disease , cancer , diabetes , obesity , insulin resistance , metabolic syndrome , and atherosclerosis [ 46 ] . it has been even suggested that saa may play an important , pathogenic role in the proinflammatory cascade in the course of ra . saa is not only an acute - phase protein but also a kind of apolipoprotein , relevant in cholesterol metabolism . normally , saa circulates in low levels bound to high - density lipoprotein ( hdl ) , but during inflammation saa can contribute up to 80% of hdl apolipoprotein ( apo ) composition , exceeding apo - a1 in quantity and impairing the protective function of hdl . saa may also increase the oxidation of low - density lipoprotein ( ldl ) and thus may be associated with cardiovascular ( cv ) disease ( cvd ) and atherogenesis [ 5 , 7 ] . saa is also the precursor of amyloid a protein , a fibrillar , insoluble product deposited in major organs , which carries a risk of organ failure and premature death in the course of secondary amyloidosis . aa amyloidosis is a late complication of ra , connected with active , long - standing , and disabling disease . the prevalence of aa amyloidosis in ra reported in different studies is highly variable because the complication frequently remains undetected due to lack of clinical manifestations in many patients . persistently high concentration of saa is a prerequisite for aa amyloidogenesis [ 1 , 3 ] . high levels of saa correlate with the progression of amyloidosis and low levels are associated with its regression [ 2 , 9 ] . it was reported that , in aa amyloidosis patients , serum saa concentration was the most important predictive parameter of the risk of death , associated with renal function . the aim of the study was to assess the concentration of saa in ra patients with different disease activity , with reference to other inflammatory , metabolic parameters , as well as cv and renal disease risk factors . the study group consisted of 140 ra patients admitted consecutively and treated in the department of rheumatology and connective tissue diseases , medical university of lublin . the patients were qualified for the treatment in order to make a decision whether to maintain or modify the ongoing therapy , depending on an assessment of the disease activity . the ethical committee of the medical university of lublin approved the design of the study . demographic and clinical information was obtained through structured interview , review of medical records , self - report questionnaires , physical examination , laboratory tests , and high - resolution b - mode ultrasonography . disease activity was measured using the disease activity score ( das ) based on evaluation of 28 joints ( das28 ) , calculated with the number of tender joint counts ( tjc ) and swollen joint counts ( sjc ) , erythrocyte sedimentation rate ( esr ) value , and patient 's global assessment ( pga ) of disease activity in visual analogue scale ( vas ) . ability to perform daily activities was measured using the modified health assessment questionnaire ( m - haq ) with the range 03 ( a score 0 representing no impairment of function ) . erosive form of ra was diagnosed in those patients , who presented erosions on joint surfaces of bones in radiograms of hands and/or feet , according to steinbrocker et al . extra - articular symptoms were noted , which occurred during the whole course of the disease . blood was collected after an overnight fasting to determine the complete blood cell count , erythrocyte sedimentation rate ( esr ) , serum concentration of crp , creatinine ( cr ) , total protein , albumin , fibrinogen , total cholesterol ( tc ) , hdl - cholesterol [ hdl - c ] , ldl - cholesterol [ ldl - c ] , and triglycerides ( tg ) at the university hospital central laboratory . serum level of crp was measured by immunoturbidimetric assay , with the upper limit of the normal range at 5 mg / l . concentrations of tc , hdl - c , and tg were measured using the standard enzymatic technique ( biomaxima ) ; ldl - c was calculated according to the friedewald formula . modification of diet in renal diseases ( mdrd ) was calculated for every patient to estimate the glomerular filtration rate ( gfr ) using serum cr concentration and demographic factors . blood samples were also stored at 80c for further assessment of cystatin - c ( cys - c ) and saa . cys - c is used as an endogenous marker for gfr , more precise than creatinine . serum cys - c was measured using quantitative enhanced immunonephelometry method ( with commercially available assay developed by dade behring ) . the producer recommended the normal range of cys - c between 0.53 and 0.95 mg / l independently of sex , age , and body mass . serum concentration of saa was determined by a commercial enzyme - linked immunosorbent assay ( elisa ) , with detection limit 0.005 mg / l ( human saa ; biosource europe s.a . igm - rheumatoid factor ( rf - igm ) was determined using the antirheumatoid factor elisa ( igm ) test ( euroimmun ) with the recommended upper limit of the normal range 20 units ( ru)/ml . anticitrullinated protein antibodies ( acpa ) were determined using the quanta lite ccp3.1 igg / iga elisa assay ( inova diagnostics ) with negative results < 20 u / ml . height and weight were measured barefoot wearing light clothes . body mass index ( bmi ) was calculated as the ratio of weight and squared height . during physical examination blood pressure ( bp ) was performed in every patient , with assessment of corrected qt interval ( qtc ) . the 10-year risk of fatal cvd using the systemic coronary risk evaluation ( score ) model according to the eular recommendations was estimated in every patient . carotid intima - media thickness ( cimt ) was measured using high - resolution b - mode ultrasound ( logiq 7 ge ) . in every subject imt was assessed bilaterally in the three regions : common carotid artery ( cca ) , carotid bulb ( bulb ) , and internal carotid artery ( ica ) . the average of the maximal imt from all 6 carotid segments ( defined as mean cimt ) was used in the analyses . it has been reported that cimt 0.6 mm is a marker of subclinical atherosclerosis . plaques were defined as a distinct protrusion , greater than 1.5 mm into the vessel lumen . results were expressed as mean ( standard deviation , sd ) or number ( % ) . group differences were tested using student 's t - test and mann - whitney u test for normally and nonnormally distributed parameters , respectively . spearman 's or pearson 's correlation test was used to determine the association between saa and clinical and laboratory variables . multivariable analysis ( multiple linear regression ) was performed according to a forward selection procedure , introducing those variables that showed a statistically significant association with saa . characteristics of patients with ra are presented in table 1 ; clinical and laboratory variables are presented in table 2 . most patients were positive for rf - igm and acpa and had erosive form of ra , of low or moderate activity ( l / mda ) ( das28 5.1 ) at the time of assessment . remission of ra according to das28 ( < 2.6 ) was observed in 8 patients ( 7 women and 1 men ) . extra - articular symptoms during the course of ra were noted in 58 patients ( 41.4% ) and included rheumatoid nodules ( 40 patients ) , sicca syndrome ( 16 patients ) , interstitial lung disease ( 7 patients ) , amyloidosis ( 1 patient ) , and vasculitis ( 1 patient ) . at the time of examination disease modifying antirheumatic drugs ( dmards ) were not used in 5 patients ( 3.6% ) . in the remaining 135 patients , treatment with at least 1 synthetic dmard was administered : methotrexate ( mtx ) ( 58.6% of all patients ) , leflunomide , sulfasalazine , chloroquine , and cyclosporine . biological dmards were used in 36 patients ( 25.7% ) ( adalimumab in 3 , etanercept in 15 , infliximab in 15 , and rituximab in 3 cases ) . simultaneously , low - dose prednisone ( 10 mg / day ) was used in 108 patients ( 77.1% ) . the mean saa concentration in the group of 140 patients was 327.0 ( 263.4 ) mg positive , significant ( p < 0.05 ) correlations were found between saa and das28 , tjc , sjc , pga of the disease activity , morning stiffness , m - haq and esr value , concentration of crp , fibrinogen , and cys - c , as well as with white blood cell count ( wbc ) , platelet count ( plt ) , and score value . negative , significant ( p < 0.05 ) correlations were found between saa and concentration of albumin , hemoglobin , and qtc value . all the above - mentioned variables were included in the multiple linear regression analysis , which confirmed significant associations for crp , wbc , and qtc ( table 3 ) . the mean saa concentration was significantly higher in men than in women ( p = 0.01 ) ( table 4 ) . the group of men , when compared with women , was characterized by higher inflammatory parameters [ crp 27.4 ( 23.3 ) versus 17.4 ( 21.9 ) mg / l , p = 0.005 ; fibrinogen 5.4 ( 1.2 ) versus 4.6 ( 1.3 ) g / l , p = 0.006 ] , unfavorable lipid parameters [ hdl 50.7 ( 11.6 ) versus 62.3 ( 15.0 ) mg / dl , p = 0.0001 ; tc / hdl index 3.8 ( 1.0 ) versus 3.4 ( 0.8 ) , p = 0.01 ] , higher cv risk markers [ cimt 0.86 ( 0.17 ) versus 0.77 ( 0.14 ) mm , p = 0.01 ; score 4.3 ( 3.9 ) versus 1.1 ( 1.5 ) , p < 0.0001 ] , and higher cys - c concentration [ 0.83 ( 0.24 ) versus 0.75 ( 0.21 ) mg / l , p = 0.04 ] . significantly higher concentrations of saa were found in patients with high disease activity ( hda ) versus l / mda ( p < 0.0001 ) and in patients currently treated versus not treated with glucocorticoids ( gcs ) ( p = 0.002 ) ( table 4 ) . significantly higher concentration of saa was also observed in patients with advanced atherosclerosis when compared with those without atherosclerotic plaques ( p = 0.04 ) and in patients with increased ( 1.0 mg / l ) versus normal cys - c level ( p = 0.004 ) ( table 4 ) . the mean saa concentration was significantly lower in women currently treated with biological dmards ( p = 0.01 ) ( table 4 ) . there was no such a correlation in men . among female patients treated with biological dmards , anti - tnf inhibitors were used in 32 ( 94% ) and rituximab was used in 2 cases . the normal saa level ( < 10 mg / l ) was observed in 11 patients ( 7.9% ) , 10 women , and 1 man . the mean concentration of saa in this group was 6.4 ( 4.4 ) mg / l ( range 09.9 ) . patients with normal versus increased saa concentration were characterized by lower concentration of crp [ 11.4 ( 18.2 ) mg / l versus 20.2 ( 22.7 ) , p = 0.03 ] and fibrinogen [ 4.1 ( 1.6 ) versus 4.8 ( 1.3 ) g / l , p = 0.05 ] , lower wbc [ 6.0 ( 1.2 ) versus 8.1 ( 2.5 ) 10/l , p = 0.003 ] , and higher qtc duration [ 373.8 ( 52.8 ) versus 332.2 ( 57.2 ) ms , p = 0.04 ] . there were no significant differences in methods of treatment between the two groups of patients . the major finding of the study was that serum saa concentration was high above normal in the vast majority of ra patients . the normal saa level was found only in < 10% of patients , characterized by lower inflammatory burden and normal qtc duration . elevated above normal saa levels were found not only in patients with hda , but also in those with l / mda assessed with das28 . this observation suggests that in spite of favorable clinical assessment , sustained inflammation persists in most of ra patients and may be connected with the disease complications . in this study , we demonstrated a direct relationship between saa and ra activity ( with both clinical and laboratory parameters of activity ) . significantly higher saa was associated with the current treatment with gcs , which are commonly used in patients with hda . women treated with biological dmards had significantly lower saa concentration than women treated only with synthetic dmards . these results confirm better therapeutic effects of biological dmards in comparison to synthetic dmards resulting in lower disease activity . the mean saa level was significantly higher in patients with plaques as a manifestation of advanced atherosclerosis . significantly higher mean saa concentration was noticed in patients with increased cys - c level as an early marker of chronic kidney disease ( ckd ) . the results of our study indicate that , in ra patients , saa could be considered as a biomarker associated with both the current inflammatory disease activity and chronic complications , the risk of cv and renal involvement in the course of ra . persistently elevated saa concentration increases the risk of amyloidosis development due to deposition of amyloid proteins in different tissues and organs . our results are consistent with data in literature , which reported a strong correlation between saa and ra activity . the saa level correlated with clinical disease activity ( 28-joint sjc ) and it has been demonstrated that saa may be also produced by rheumatoid synovial tissue ( but not in normal synovial membrane ) , inducing cell growth , angiogenesis , invasion and migration of cells , secretion of proinflammatory cytokines ( tnf , il-1 , il-6 , and il-8 ) , chemokines , reactive oxygen species , and matrix metalloproteinases ( mmp ) [ 2 , 6 , 20 , 21 ] . therefore , saa may play a pathogenic role locally in joint destruction and cartilage damage [ 2022 ] . the correlation between saa level and wbc may be associated with the reported function of saa as a priming agent , rendering neutrophils more responsive to opsonized particles , as well as inducing chemotaxis and adhesion to endothelial cells and cellular migration [ 23 , 24 ] . significantly higher saa levels were found in patients currently treated with gcs , which seems to be a paradoxical effect . however , gcs are commonly used in patients with hda , presenting increased inflammatory parameters . on the other hand , it was reported that gcs induce extrahepatic saa expression , which has been shown in tissue cultures and confirmed in patients with chronic obstructive pulmonary disease . further prospective determinations of saa are necessary to define an accurate answer . according to our knowledge , we also found significantly higher saa concentration in women not treated with biological dmards , which indicates more active disease in these cases . however , patients in our group received therapy with different biological dmards ( one of the three tnf inhibitors or rituximab ) , which hinders a reliable assessment of relationship between saa and biological treatment . in literature , there is no data on the association between saa and rituximab . there are few reports on the effect of anti - tnf [ 2731 ] and anti - il-6 [ 31 , 32 ] treatment on saa concentration , dealing mostly with patients with amyloidosis , secondary to ra . the blockade of tnf prevents high systemic saa concentrations that are largely driven by tnf . the effective and early dmards treatment works as the primary prevention of amyloidosis , as well as the secondary prevention in a subclinical phase of this complication . however , even during anti - tnf therapy , excessive , extrahepatic expression of saa , mediated by il-6 and gcs , could probably be maintained in rheumatoid synovial tissue . in this case , saa may upregulate tnf itself and may be a potent upstream mediator of tnf , influencing response to existing anti - tnf therapy . high level of saa during anti - tnf treatment might serve as a biomarker of persistent synovial inflammation , resistant to the therapy currently used . in our patients , the mean saa concentration was significantly higher in men than in women , which may be related to significantly higher parameters of inflammation and unfavorable cv risk factors in men . according to literature , women have higher saa levels than men , for various reasons , due to hormonal status , interaction with leptin , or different body composition [ 33 , 34 ] . men have larger intra - abdominal fat deposits , which produce significant amounts of cytokines ( tnf , il-6 ) , even in larger quantity than subcutaneous adipose tissue . we observed significantly higher saa levels in patients with atherosclerotic plaques and increased cys - c serum concentration , which indicates the risk of cv and renal complications . it has been demonstrated that cells within human atherosclerotic lesions ( foam and endothelial cells , smooth muscles ) could express saa and release to circulation at the site of plaque rapture [ 35 , 36 ] . no independent effect of saa on cimt value was observed in one study . in literature , saa is reported as a predictor of coronary artery disease and further coronary events , as well as biomarker of cerebrovascular disease [ 38 , 39 ] . cys - c has been reported as a prognostic marker of risk for death , cv , and kidney outcomes in elderly patients , with and without ckd . in the whole group of our patients , the mean qtc duration was below 350 ms and was inversely correlated with saa concentration . intriguingly , in patients with normal saa level , the mean qtc duration was normal . according to our knowledge , this is the first report on the association between saa and short qtc , which should be taken into account , considering a risk of life - threating arrhythmias , especially in patients with high saa concentration . cardiovascular mortality is significantly increased in ra , with a risk of sudden death about twofold higher than in non - ra subjects [ 41 , 42 ] . it has been suggested that this phenomenon may be associated with serious arrhythmias as a result of nonstructural cardiac alterations , such as reduced heart rate variability ( hrv ) or qt interval abnormalities . shortening of qtc , recognized with the value < 350 ms , is associated with increased risk of atrial fibrillation with rapid ventricular tachycardia . the study in patients with inflammatory arthritis demonstrated that high sensitivity crp ( hscrp ) was inversely correlated with hrv and directly correlated with qtc duration . the study population consisted mostly of patients with spondyloarthropathies and only 25% of ra patients of unknown disease activity . it is suggested that systemic inflammation may affect cardiac nervous system , irrespectively of other cv risk factors . it seems that a short qt interval does not by itself predict risk of life - threating arrhythmias but rather should be taken in context of each individual patient . the strengths are as follows : ( 1 ) relatively large sample of consecutive patients with ra , treated in our department , ( 2 ) the detailed characteristics of patients , who have been considered in all aspects of ra pathology including several inflammatory , cv , metabolic parameters , and assessment of renal function , ( 3 ) the design of the study which was based on clinical practice , and ( 4 ) according to our knowledge being the first study which evaluates the potential association of high saa level with qtc shortening , increasing the risk of serious arrhythmias and sudden death . there are also some limitations including the following : ( 1 ) our patients were treated with different biological dmards ; that is why it is difficult to determine the exact association between saa and effect of therapy ; ( 2 ) ecg recording was performed routinely in every patient and qtc duration was assessed automatically , with no special procedure , and hrv was not assessed ; and ( 3 ) we did not collect detailed information about the ongoing treatment , which may influence heart rate . as a consequence , further investigation is needed to confirm the observed relationship , in relation to concurrent treatment . in our study , in patients with ra , serum saa levels strongly correlated with the activity of the disease and were significantly higher in patients with high disease activity . however , high saa concentrations were observed also in patients with low or moderate disease activity , in spite of clinical improvement . concurrent treatment with gcs was associated with higher saa and therapy with biological dmards was associated with lower saa concentration . the mean saa level was inversely associated with qtc duration and markedly higher in patients with atherosclerotic plaques , emphasizing increased cv risk resulting from chronic inflammatory process . we also found significantly higher saa levels in patients with increased cys - c as a marker of ckd , emphasizing increased risk of renal disease . high saa concentration was strongly connected with the activity of the disease and the risk of cv and renal involvement in ra patients . prospectively , repeated assessment of saa may facilitate searching ra patients with persistent inflammation and increased risk of extra - articular complications , as well as amyloidosis development .

objectives . rheumatoid arthritis ( ra ) is a systemic , inflammatory disease . serum amyloid a ( saa ) is an acute - phase protein , involved in pathogenesis of atherosclerosis . the aim of the study was to assess serum concentration of saa in ra patients , with reference to other inflammatory parameters and markers of extra - articular involvement . methods . the study population consisted of 140 ra patients , low / moderate disease activity ( l / mda ) in 98 ( 70% ) patients and high disease activity ( hda ) in 42 ( 30% ) . comprehensive clinical and laboratory assessment was performed with evaluation of electrocardiogram and carotid intima - media thickness . results . the mean saa concentration [ 327.0 ( 263.4 ) mg / l ] was increased highly above the normal value , even in patients with l / mda . simultaneously , saa was significantly higher in patients with hda versus l / mda . the mean saa concentration was significantly higher in patients treated with glucocorticoids , was inversely associated with qtc duration , and was markedly higher in patients with atherosclerotic plaques , emphasizing increased cv risk . saa was significantly higher in patients with increased cystatin - c level . conclusions . in ra patients , high serum saa concentration was strongly associated with activity of the disease and risk of cv and renal involvement . recurrent assessment of saa may facilitate searching patients with persistent inflammation and risk of extra - articular complications .

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glucocorticoids have been extensively characterized as being anti - inflammatory and/or immunosuppressive in therapeutic settings and are generally discussed as opposed to the development of inflammation and limiting of production or maturation of some effector immune cell types , in a physiological context . nevertheless , there is a well - established association between stress and allergic diseases , including asthma [ 4 , 5 ] . glucocorticoids , which are an essential part of systemic stress responses , play coadjuvant roles in promoting inflammation and may promote th2-type immunity through differential effects on th1 th2 cytokine production [ 5 , 6 ] . chronically stimulated eosinophil production ( eosinopoiesis ) is an important feature of human asthma and of murine allergic asthma models . in both cases , allergen challenge of sensitized subjects increases eosinopoiesis in the bone - marrow [ 7 , 8 ] . this effect is antigen - specific and can be abolished by inducing oral tolerance to the allergen , which affects both eosinophils and neutrophils in bone - marrow . the effects of oral tolerization in bone - marrow neutrophil and eosinophil granulocytes can be duplicated by transfer of splenic t lymphocyte subpopulations from tolerized / sensitized / challenged donors to histocompatible naive recipients . these observations highlight the importance of acquired cellular immunity in regulating the hematological response to allergen sensitization and challenge . they further suggest the possibility that granulopoiesis , encompassing both eosinophil and neutrophil production , might be regulated by lymphocyte populations in nonsensitized subjects as well . in an allergic asthma model , we demonstrated a critical role for endogenous glucocorticoids in the hematological response to allergen challenge : challenge induces a corticosterone surge that is paralleled by increased eosinophilia of bone - marrow in vivo and by increased responsiveness to il-5 , the major eosinopoietic cytokine , ex vivo ; the bone - marrow response to challenge is abolished by blockade of glucocorticoid signaling or glucocorticoid production . on the other hand , in the absence of allergen sensitization and challenge , we have also obtained evidence of a link between the corticosterone surge induced by mild surgical trauma and short - term bone - marrow eosinophilia ; again , blockade of endogenous glucocorticoid production or action abolished the bone - marrow eosinophilia induced by trauma . these in vivo effects of corticosterone , an endogenous glucocorticoid released by the adrenal glands , are paralleled by those of exogenously provided corticosterone or dexamethasone on murine bone - marrow [ 12 , 13 ] and of other glucocorticoids on human hemopoietic cells . in balb / c mice , dexamethasone increases eosinophil production in murine bone - marrow culture [ 12 , 13 ] and primes bone - marrow cells in vivo for increased ex vivo responses to il-5 . during further screening of inbred mouse strains for differences in the granulopoietic responses to dexamethasone , we observed bone - marrow eosinophilia in mice of the c57bl/6 ( b6 ) background injected with dexamethasone , which was undetectable in perforin - deficient b6 mutants submitted to the same treatment . perforin is a major mediator of cellular immunity [ 1519 ] , expressed in lymphocyte populations which fight viral and bacterial pathogens [ 16 , 17 ] as well as malignant cells in the context of both innate and acquired immune responses . perforin is also expressed by murine bone - marrow neutrophils , which have a critical regulatory role in t cell - mediated contact hypersensitivity . perforin deficiency is known to induce complex changes in leukocyte populations in humans and mice [ 16 , 17 , 20 ] , including a classical presentation of familial hemophagocytic lymphohistiocytosis , characterized by early life onset , high mortality , and multiple immunological defects , including uncontrolled activation and proliferation of cd4 + and cd8 + t cells , cytokine storm , macrophage activation and proliferation , pancytopenia , and anemia . here we report that perforin deficiency also presents a selective defect in granulocyte production , which can be corrected by wild - type lymphocyte transfer . sh30088.03 ) , rpmi1640 ( sh30011.01 ) , and imdm ( sh30228.01 ) were from hyclone ( logan , ut , eua ) ; l - glutamine ( g7513 ) , penicillin - streptomycin solution ( p4333 ) , essential amino acids solution ( 50x ) ( m5550 ) , methylcellulose ( m0387 ) , dexamethasone ( 21-phosphate , disodium salt , d1159 ) , mifepristone ( ru486 , m8046 ) , and histopaque-1083 ( 10831 ) were from sigma - aldrich corporation ( st . louis , mo , eua ) ; agar noble ( 0142 - 15/21422 ) was from difco ( detroit , mi , eua ) ; nonessential amino acids solution ( 100x ) , ( 11140 - 050 ) and mem vitamin solution ( 100x ) ( 11120 - 052 ) were from gibco life technologies ( carlsbad , ca , usa ) ; recombinant murine il-5 ( 405-ml-025 ) was from r&d systems ( minneapolis , mn , usa ) ; recombinant murine gm - csf ( 315 - 03 ) was from preprotech ( rocky hill , nj , usa ) ; rat antimouse cd8b ( clone : ebioh35 - 17.2 , 12 - 0083 - 82 , 0.2 mg / ml ) was from ebioscience ( san diego , ca , usa ) ; magnetic microspheres conjugated to antimouse cd4 ( l3t4 , 130 - 049 - 20 ) and to goat antirat igg ( 130 - 048 - 501 , as secondary antibody to primary rat antimouse cd8b ) were from miltenyi biotec ( ambriex , sp , brazil ) . wild - type c57bl/6 , perforin - deficient ( pfp ) mutants of the b6 background , and wild - type b6.129 mice were bred by cecal - fiocruz , rio de janeiro , brazil . the pfp stock was derived from the original b6.129s6-pfp stock by backcrossing to c57bl/6 at fiocruz . unless otherwise indicated , the wild - type controls for the experiments shown were c57bl/6 ; in selected experiments , b6.129 wild - type controls were used and yielded the same results as c57bl/6 ( not shown ) . the animals were housed and handled following institutionally approved guidelines under license l-00209 from ceua - fiocruz . routinely , female mice were used for the experiments , since male mice characteristically fight for dominance in the same cage , and the stress associated with fighting may confound the interpretation of the results . we have no evidence , however , that the granulopoietic responses described here , including the strain differences , are restricted to females . dexamethasone in saline solution was injected i.p . ( 200 l , i.p . , amounting to 5 mg / kg ; ) . ru486 in 0.1% methylcellulose was given intragastrically 2 h before dexamethasone with a gavage needle ( 200 l , amounting to 100 mg / kg ) . for lymphocyte transfers , 10 nylon - wool purified cells from naive c57bl/6 donors in a 100 l volume of sterile saline were injected into the tail vein of pfp recipients , once . controls received an equal number of cells from pfp donors . where lymphocytes depleted of cd4 + or cd8 + cells were used , the amount of cells injected was that recovered from 10 initial unseparated lymphocytes , in 100 l sterile saline , i.v . dexamethasone was administered to the recipients , in both cases , 48 h after lymphocyte transfer . where indicated , bone - marrow , peripheral blood from the abdominal vena cava , spleens , and whole thymuses were collected , for enumeration of cells or determination of relative weight of the thymus ( mg / g body weight ) [ 21 , 23 ] . bone - marrow collected from both femurs of each mouse in 5 ml rpmi1640 medium/1% fbs with a 22-gauge needle and kept on ice was used for total and differential counts and cell culture ( see below ) . spleen mononuclear cells were the source for isolation of lymphocytes ( see below ) [ 24 , 25 ] . differential counts were carried out in cytocentrifugates after fixation in pbs-10% formaldehyde and staining for eosinophil peroxidase ( epo [ 26 , 27 ] ) followed by counterstaining with harris ' hematoxylin . liquid bone - marrow cultures were established at 37c , with 10 bone - marrow cells in 1 ml rpmi1640/10% fbs , in 5% co2/95% air , plated in 24-well plates ( cat . n : 142475 , nunc brand products ) with il-5 ( 1 ng / ml ; 7 days ) . where indicated , dexamethasone was added ( 10 l / well , to 10 m final concentration ) . absolute numbers were calculated from total cell counts in turk 's solution multiplied by the % of epo+ cells ( eosinophil - lineage cells , both mature and immature ) in cytocentrifuge smears . triplicate semisolid ( clonal ) bone - marrow cultures were established for 7 days with 2 10 cells adjusted to 1 ml of 1 : 1 imdm / agar mix , in 35 mm tissue culture plates ( nunc ) , in the presence of gm - csf ( 2 ng / ml final ) , with or without dexamethasone ( 10 m ) , at final 20% fbs and 0.3% agar concentrations . total colonies ( defined as aggregates > 50 cells derived from a single progenitor cell ) [ 8 , 12 ] were scored at day 7 under an inverted microscope ( 400x , phase contrast ) . for lymphocyte isolation , spleens were collected and minced in rpmi1640/1% fbs on tissue culture plates . spleen mononuclear cells ( 2 10 in 10 ml rpmi1640/1% sfb ) were isolated by centrifugation on a 1.083-density ficoll - hypaque cushion ( 3 ml , at 400 g , for 30 minutes , at room temperature , following manufacturer 's instructions ) [ 24 , 25 ] . cells recovered from the medium / ficoll - hypaque interface were collected , washed in serum - free medium , resuspended , counted , and further separated on nylon - wool columns [ 24 , 25 ] at 4 10 cells/2 ml / g nylon wool . cells eluted in a total 25 ml warm medium , dropwise , were washed and counted before cytocentrifugation / staining or incubation with antibodies . depletion of cd4 + cells was done with 10 l l3t4/10 nylon - wool purified lymphocytes in 100 l serum - free rpmi1640 , on ice , following manufacturers ' instructions . depletion of cd8 + cells was in two steps , with rat antimouse cd8b conjugated with pe , followed by goat antirat igg ( 20 l/10 lymphocytes in 80 l medium , on ice ) . columns were eluted with 9 ml medium , dropwise , over a 5-minute period . the numbers of experiments ( n ) are indicated in the caption of the figures , to avoid overcharging the figures and captions . for comparisons of two groups ( figures 1 and 2 ) , we used the two - tailed t - test with separate variances ( systat for windows , version 5 , systat inc . , evanston , il ) . for multiple comparisons ( figures 3 , 4 , 5 , and 6 ) , we used anova , with the tukey hsd correction for groups of equal size ( systat for windows ) or with bonferroni 's correction for groups of unequal size ( using prisma 5 for windows , graph pad , la jolla , ca ) , unless otherwise indicated in section 3 . following preliminary experiments ( not shown ) that evidenced a positive response to dexamethasone in b6 wild - type mice , as well as the absence of any response , positive or negative , in pfp mutants of the b6 background , we reviewed data on freshly harvested bone - marrow from a large number of mice of both strains ( n = 44 and n = 48 , resp . ) , to look for evidence of strain differences in bone - marrow steady - state parameters , in the absence of dexamethasone exposure ( figure 1 ) . a significant difference was observed in this large series , with pfp mice having lower bone - marrow cellularity than wild - type controls ( figure 1(a ) ; p < 0.001 ) . the data available for epo+ cells ( figure 1(b ) ; p = 0.033 ) and neutrophils ( figure 1(c ) ; p = 0.010 ) of the mice in the large series showed significant differences as well , with lower counts in pfp mutants . as the two groups were defined only on the basis of genetic differences ( presence or absence of functional perforin genes ) , these differences could still be accounted for , in principle , by variance due to nongenetic factors within these groups , especially those which influence growth and development . because bone - marrow cellularity is roughly proportional to the size of the animal , we next evaluated whether these differences would disappear in the comparison between groups of control and mutant mice matched by weight ( figures 1(d)1(f ) ) . significant differences were still observed for total cells ( figure 1(d ) ; p = 0.001 ) , eosinophils ( figure 1(e ) ; p = 0.004 ) , and neutrophils ( figure 1(f ) ; p = 0.011 ) , all three parameters being lower in the mutant mice . because age might affect bone - marrow function through mechanisms unrelated to body weight gain ( as senescence may have an earlier onset in some strains ; furthermore , age is a major determinant of incidence of many pathological processes , such as malignancies ) , we next examined whether matching by age ( all animals at 12 weeks ) would eliminate the differences . significant differences were still found for all three parameters ( for figures 1(g)1(i ) , resp . , p = 0.001 , p = 0.006 , p = 0.001 ) , which were lower in the mutant mice . even matching by both weight and age ( for figures 1(j)1(l ) , resp . , p = 0.022 , p = 0.032 , p = 0.001 ) failed to eliminate these significant differences between controls and mutants for any of the three parameters , which were all lower in the mutant mice . overall , this suggests that bone - marrow cellularity , as well as bone - marrow eosinophil and neutrophil counts , is significantly lower in pfp mutants than in b6 mice and that this difference can not be dismissed as created by undue comparisons of two groups differing in body weight , age , or both . we further examined ( figure 2 ) the counts of total cells , lymphocytes , neutrophils , and eosinophils in peripheral blood of weight - matched ( median 21 g , range 1923 g ) b6 and pfp mice . unlike bone - marrow , peripheral blood total nucleated cell counts did not differ significantly between these strains ( figure 2(a ) , p = 0.795 ) , nor did lymphocyte counts ( figure 2(b ) , p = 0.417 ) . by contrast , both neutrophil ( figure 2(c ) , p = 0.025 ) and eosinophil ( figure 2(d ) ; p = 0.030 ) counts were significantly different , and , like in bone - marrow , lower in pfp mice . together , the data in figures 1 and 2 suggest that , even in the absence of dexamethasone , pfp mice have reduced granulocyte numbers both inside and outside of bone - marrow , relative to wild - type controls of comparable body weight . our original observation of this strain difference was increased % eosinophils in cultured bone - marrow from b6 , but not pfp , mice when both il-5 and dexamethasone were present ( jones and cardoso de mendona , unpublished observations ) . this is , however , insufficient to characterize a strain difference in response to dexamethasone , because the % of a given cell type in a bone - marrow sample , which has a highly heterogeneous composition , can be increased artifactually by a corresponding decrease in another cell type , rather than by a positive effect of dexamethasone on the cell type of interest . to confirm a positive effect of il-5 and dexamethasone in stimulation of wild - type and mutant eosinopoiesis , liquid cultures were established from the same number of bone - marrow cells ( 10 ) from b6 or pfp donors , in the presence of il-5 , alone or associated with dexamethasone , and the absolute numbers of eosinophils in the culture were determined . as shown in figure 3 , eosinophils were produced in cultures of il-5-stimulated wild - type and mutant bone - marrow ( figure 3(a ) ) . control cultures lacking il-5 do not contain eosinophils at the end of the culture period ( not shown ; ) . counts of epo+ cells recovered at the end of the culture were increased in the presence of dexamethasone ( 10 m ) , relative to il-5 controls , in cultures from b6 controls , but not from pfp mutants ( figure 3(a ) , p < 0.001 ) . nevertheless , the il-5 present was sufficient to sustain eosinopoiesis in the absence of dexamethasone by pfp bone - marrow to the same level observed in b6 control cultures . on the other hand , data from semisolid cultures , which allowed us to examine the effects of dexamethasone on several classes of progenitors ( colony - forming cells ) , including the granulocyte ( g ) and granulocyte - macrophage ( gm ) progenitors , which produce neutrophils , are also shown in figure 3 . the total counts of colonies formed by gm - csf - stimulated pfp bone - marrow progenitors , in the absence of dexamethasone , also differed significantly from those of b6 controls ( p 0.001 ) : even though identical numbers of bone - marrow cells were plated , less colonies were made by perforin - deficient bone - marrow ( figure 3(b ) ) . furthermore , dexamethasone ( 10 m ) significantly stimulated colony formation by b6 bone - marrow ( p = 0.002 ) but failed to stimulate ( p = 0.558 ) pfp bone - marrow in the same conditions ( figure 3(b ) ) . these observations show that bone - marrow progenitors from pfp mice differ significantly from those of b6 controls , because they form less colonies and do not respond to dexamethasone , which significantly enhances colony formation in the wild - type cultures . we next examined the effect of dexamethasone ( 5 mg / kg injection ) on bone - marrow eosinophils and neutrophils , as well as on the relative weight of the thymus , which is significantly reduced by glucocorticoids . as shown in figure 4 , dexamethasone injection increased the numbers of bone - marrow eosinophils ( figure 4(a ) , p = 0.003 ) and neutrophils ( figure 4(b ) , p = 0.019 ) in vivo , relative to saline - injected controls , in wild - type b6 mice , but not in pfp mice . the effect of dexamethasone on eosinophil numbers in wild - type b6 mice was abolished by ru486 pretreatment , while ru486 had no effect of its own in the absence of dexamethasone , in either b6 or pfp mice ( not shown ) . the relative weight of the thymus was significantly different ( p = 0.010 ) between body weight - matched pfp mice and wild - type controls , suggesting a decreased thymic cellularity in mutant thymus , even without exogenous glucocorticoid administration ( figure 4(c ) ) . importantly , dexamethasone injection did reduce significantly the relative weight of the thymus in both wild - type ( p = 0.007 ) and mutant ( p = 0.001 ) mice ( figure 4(c ) ) . again , the effect of dexamethasone on the thymus was abolished in both cases by ru486 pretreatment ( not shown ) . together , these observations show that pfp bone - marrow is unresponsive to dexamethasone stimulation in vivo , although dexamethasone significantly increases eosinophil and neutrophil numbers in the bone - marrow of wild - type controls . they also show that lack of responsiveness to dexamethasone in pfp bone - marrow is not due to a general lack of response to glucocorticoids , because the thymus of pfp mice responds to dexamethasone injection as expected . because perforin is mainly expressed in various effector / regulatory lymphocyte subsets , it was important to test whether the perforin - related defect in bone - marrow response to dexamethasone could be corrected by introducing b6 lymphocytes in pfp mice . we have done so with splenic lymphocytes from naive b6 donors , because both the baseline granulopoiesis defect and the defective granulopoietic response to dexamethasone were observed in the absence of allergic sensitization . the number ( 10 ) of lymphocytes for transfer into individual mice was defined on the basis of similar reconstitution studies . to define whether elimination of a particular lymphocyte subpopulation defined by standard surface markers ( cd4/cd8 ) prevented reconstitution of the dexamethasone response , 10 total lymphocytes purified from spleen were submitted to alternative depletion protocols with marker - specific microbeads , and the depleted cells in the column effluent , corresponding to the number of cells negative for the selection marker present in the original lymphocyte sample , were injected . as shown in figure 5 , pfp recipients of wild - type lymphocytes ( unseparated ) show a significant in vivo response to dexamethasone injection , by increased total cell ( figure 5(a ) ; p = 0.032 , t - test ) , eosinophil ( figure 5(b ) ; p = 0.04 ) , and neutrophil ( figure 5(c ) ; p < 0.001 ) counts in the bone - marrow , sharply contrasting with observations in pfp mice in the absence of wild - type cell transfer ( compare figure 4 ) . on the other hand , depletion of cd4 + or cd8 + cells in the wild - type lymphocyte preparation gave distinct results in the reconstitution assay , depending on the granulocyte population examined : while depletion of either subset abolished the ability of splenic lymphocytes to reconstitute the eosinopoietic response to dexamethasone ( figure 5(b ) ) , depletion of cd4 + cells did not prevent reconstitution of the neutropoietic response ( figure 5(c ) ; p = 0.013 ) , while depletion of cd8 + cells prevented reconstitution . as a further control , we performed transfer of pfp lymphocytes into pfp recipients and obtained no reconstitution of dexamethasone responses , judged by any of these three parameters ( figure 6 ) . in these controls , dexamethasone reduced significantly the relative weight of the thymus , showing that dexamethasone was able to reach systemically active levels even if bone - marrow showed no evidence of responding to it . overall , these observations suggest that lymphocytes from the spleen of naive wild - type mice , but not pfp mice , are able to reconstitute in the short term the granulopoietic responses to dexamethasone , but different cells may be involved in reconstitution of lineage - specific ( eosinophilic versus neutrophilic ) responses . this is , to our knowledge [ 1520 ] , the first description of a selective defect in granulopoiesis associated with perforin deficiency in mice and of its correction by the intravenous transfer of wild - type lymphocytes . as such , it extends the range of manifestations associated with perforin deficiency and raises the issue of how perforin contributes to regulation of granulocyte lineages in vivo . because the wild - type lymphocyte populations interact with a drug ( dexamethasone ) which initiates signaling through the ru486-inhibitable glucocorticoid receptor , to stimulate in vivo eosinophil and neutrophil production , these observations may be relevant to processes in which bone - marrow is stimulated by immune responses or trauma with involvement of the same receptor and of endogenous glucocorticoids released by the adrenal glands . perforin is a well - characterized effector protein , mainly ( but not exclusively ) expressed in lymphocytes which share the ability to induce cell - mediated cytotoxicity [ 1520 ] . perforin deficiency was initially characterized by the loss of important cytotoxic lymphocyte functions [ 1520 ] . however , it was soon realized that perforin deficiency entails other , more complex , functional consequences , leading to the development of type ii familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 ] , which shares pathophysiological features with the macrophage activation syndrome , including interferon- ( ifn- ) overproduction . hence , perforin deficiency has broader consequences in addition to impairment of cytotoxic lymphocyte function , and ifn- , alone or in association with other cytokine storm components , may transduce its effects on other leukocyte lineages . one of the issues raised by our findings is whether the lower granulocyte numbers in blood and bone - marrow of pfp mice are due to the progression of the murine equivalent of the human familial hemophagocytic lymphohistiocytosis [ 16 , 17 , 20 , 28 ] . it should be noted that the human familial hemophagocytic lymphohistiocytosis is a severe condition , presenting early in life in most cases and associated with important mortality [ 20 , 28 ] . also , phenotypically distinct forms correspond to different mutations with perforin expressed at lower levels and/or with altered properties , a situation that is not applicable to the present study , which employed perforin null mice . the perforin - deficient mice in the original walsh et al . study ( from which the mice used in this study descend ) were described as healthy for at least 5 months . in our study , mice were certified spf and maintained in microisolator units , with no evidence of mortality , stunting , or signs of infection during the entire observation period of up to 3 months ( 12 weeks ) . on the other hand , pfp defect in response to dexamethasone could be corrected by transfer of as few as 10 lymphocytes 48 h before the stimulus . the prompt reconstitution of this defect argues against the lack of dexamethasone response in pfp animals being caused by a chronic inflammatory disease of the bone - marrow , such as familial hemophagocytic lymphohistiocytosis , which in humans is challenging for therapy . because correction was achieved by simple transfer of lymphocytes from naive donors , we think it is more likely that the transferred lymphocytes provided something that was lacking in the recipient . neutrophils were reduced in bone - marrow and blood of pfp mice relative to b6 controls . this not only shows that the neutrophil deficiency has peripheral expression but further argues against reduced numbers in bone - marrow being due to an increased export of neutrophils to the periphery . the evidence from colony - forming assays suggests , instead , that production is greatly reduced in pfp relative to b6 mice , as there were less gm - csf - responsive progenitors in an identical number of bone - marrow cells . reduced production would explain the decrease in granulocyte counts both outside and inside of bone - marrow . on the other hand , normal numbers of lymphocytes and total leukocyte counts show that the defect is selective for granulocytes , which are not the major circulating leukocyte subpopulation in mice , so that reduction in their numbers does not have a major impact on total circulating leukocyte counts . neutropenia is associated with increased susceptibility to bacterial and fungal infection , provided it is severe enough . this was not observed in our study , so presumably the pfp mice were capable of coping with the microorganisms present in a rather clean environment ( spf conditions , microisolator housing ) . it remains to be seen , however , whether following a more severe infectious exposure , such as that associated with sepsis induction , pfp mice would prove more vulnerable to bacterial dissemination . this will be addressed in future studies , since emergency granulopoiesis , as opposed to baseline granulopoiesis , is driven by gm - csf and related hemopoietic cytokines . the lack of appropriate response to gm - csf , both in the absence and in the presence of dexamethasone , would predict that emergency granulopoiesis would be defective in pfp mice and might therefore negatively influence the outcome of sepsis . the hypothalamus - pituitary - adrenal axis is activated in sepsis , and therefore it is possible that emergency granulopoiesis is driven by endogenous glucocorticoids , in a way consistent with our observations . the prediction , in this case , is that perforin would be required for normal host response to bacterial sepsis through an effect on glucocorticoid - mediated signaling in the bone - marrow . the ability of lymphocyte preparations to reconstitute responses to dexamethasone was abolished by depletion of cd4 + and/or cd8 + cells before transfer , depending on the granulocyte population . for neutrophils , cd8 while this suggests that different mechanisms are involved for eosinophils and neutrophils , it certainly raises the issue of the lymphocyte subpopulation involved . since naive lymphocytes are sufficient , it is not likely that conventional t cells , cd4 + or cd8 + , are responsible , for they would not be activated in this isolation protocol [ 24 , 25 , 31 ] . conventional nk cells would not be a strong candidate , since cd4 + or cd8 + lymphocyte depletion would probably not eliminate them [ 32 , 33 ] . innate lymphocytes other than conventional nk cells , however , would be expected to be present in naive mouse spleens [ 32 , 33 ] . natural killer t cells , which include subsets expressing cd4 and cd8 , are found in significant numbers in b6 spleen , present an activated memory phenotype without known activating exposure , and exert multiple regulatory functions through rapid secretion of large amounts of cytokines [ 32 , 33 ] , might perhaps account for some of our observations . regardless of innate or adaptive lymphocyte population involved , two questions which require further study are as follows : ( a ) is perforin the missing component that is brought by the transferred lymphocytes ? and ( b ) do these lymphocytes act by homing to the recipients ' bone - marrow and interacting locally with granulocyte progenitors / precursors ? both questions , however , require a more complex experimental design and resources to allow us to detect and modify perforin expression inside living lymphocytes , as well as monitor their homing to the bone - marrow compartment . for this reason , such experiments fall outside the scope of this paper , which is limited to the demonstration of the defect and of its correction . finally , it should be noted that lymphocyte transfer restored the short - term response to dexamethasone but did not bring the baseline counts of eosinophils and neutrophils to the same level of the b6 controls . this might be interpreted as evidence that steady - state granulopoiesis and response to dexamethasone are unrelated processes ( i.e. , their perforin deficiency entails two distinct defects in granulopoiesis , rather than one defect with distinct manifestations ) . we do not support this interpretation , however , because the duration of the experiment might be insufficient for an effect of lymphocyte transfer alone on steady - state granulocyte numbers to become noticeable . it is also possible that demonstration of a durable effect on steady - state granulocyte numbers would require not only an extended observation period but also an increased or repeated input of wild - type lymphocytes and an interaction of exogenous lymphocytes with endogenous adrenal glucocorticoids as well .

exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone - marrow . a hematological response dependent on endogenous glucocorticoids underlies bone - marrow eosinophilia induced by trauma or allergic sensitization / challenge . we detected a defect in granulopoiesis in nonsensitized , perforin - deficient mice . in steady - state conditions , perforin- ( pfp- ) deficient mice showed significantly decreased bone - marrow and blood eosinophil and neutrophil counts , and colony formation in response to gm - csf , relative to wild - type controls of comparable age and/or weight . by contrast , peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency . dexamethasone enhanced colony formation by gm - csf - stimulated progenitors from wild - type controls , but not pfp mice . dexamethasone injection increased bone - marrow eosinophil and neutrophil counts in wild - type controls , but not pfp mice . because perforin is expressed in effector lymphocytes , we examined whether this defect would be corrected by transferring wild - type lymphocytes into perforin - deficient recipients . short - term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild - type donors . transfer of the same amount of splenic lymphocytes from perforin - deficient donors was ineffective . this demonstrates that the perforin - dependent , granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations .

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62.4 million indians were reported to have type 2 diabetes mellitus ( t2 dm ) putting india on the forefront of diabetic epidemic across globe . fear of hypoglycaemia and gain in body weight are barriers for initiation of insulin therapy . modern insulin analogues are a convenient new approach or tool to glycaemic control , associated with low number of hypoglycaemia and favourable weight change . a1chieve , a multinational , 24-week , non - interventional study , assessed the safety and effectiveness of insulin analogues in people with t2 dm ( n = 66,726 ) in routine clinical care . please refer to editorial titled : the a1chieve study : mapping the ibn battuta trail . the patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in the table 1 . the majority of patients ( 73.9% ) started on or switched to biphasic insulin aspart . other groups were insulin detemir ( n = 313 ) , insulin aspart ( n = 144 ) , basal insulin plus insulin aspart ( n = 53 ) and other insulin combinations ( n = 41 ) . overall demographic data after 24 weeks of treatment , overall hypoglycaemic events reduced from 1.5 events / patient - year to zero events in insulin user group while hypoglycaemia remained nil in insulin nave group similar to that of baseline . no hypoglycaemic episode in insulin naive group even at 24 weeks suggests low event rate than insulin users at baseline . blood pressure decreased whereas overall lipid profile and quality of life improved at week 24 in the cohort [ tables 2 and 3 ] . all parameters of glycaemic control improved from baseline to study end in the total cohort [ table 4 ] . overall efficacy data of the total cohort , 1561 patients started on biphasic insulin aspart ogld , of which 1471 ( 94.2% ) were insulin nave and 90 ( 5.8% ) were insulin users . after 24 weeks of starting or switching to biphasic insulin aspart , hypoglycaemic events reduced from 1.2 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia was nil in insulin naive group similar to baseline . quality of life improved after 24 weeks of treatment [ tables 5 and 6 ] . biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [ table 7 ] . biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort , 53 patients started on basal + insulin aspart ogld , of which 27 ( 50.9% ) were insulin nave and 26 ( 49.1% ) were insulin users . after 24 weeks of starting or switching to basal + insulin aspart , hypoglycaemic events reduced from 1.0 events / patient - year to 0.0 events / patient - year in insulin user group , while hypoglycaemia was nil in insulin naive group similar to baseline . quality of life improved at the end of the study [ tables 8 and 9 ] . basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [ table 10 ] . basal+insulin aspartoral glucose - lowering drug efficacy data of the total cohort , 313 patients started on insulin detemir ogld was 313 , of which 302 ( 96.5% ) were insulin nave and 11 ( 3.5% ) were insulin users . after 24 weeks of starting or switching to insulin detemir , hypoglycaemic events was nil in both insulin nave and user groups similar to baseline . body weight decreased and quality of life improved at 24 weeks [ tables 11 and 12 ] . insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [ table 13 ] . insulin detemiroral glucose - lowering drug efficacy data of the total cohort , 144 patients started on insulin aspart ogld , of which 131 ( 91.0% ) were insulin nave and 13 ( 9.0% ) were insulin users . after 24 weeks of starting or switching to insulin aspart , hypoglycaemic events reduced from 2.0 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia remained nil in insulin nave group similar to baseline . quality of life improved at the end of the study [ tables 14 and 15 ] . insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [ table 16 ] . of the total cohort , 1561 patients started on biphasic insulin aspart ogld , of which 1471 ( 94.2% ) were insulin nave and 90 ( 5.8% ) were insulin users . after 24 weeks of starting or switching to biphasic insulin aspart , hypoglycaemic events reduced from 1.2 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia was nil in insulin naive group similar to baseline . quality of life improved after 24 weeks of treatment [ tables 5 and 6 ] . biphasic insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for both insulin nave and insulin user groups [ table 7 ] . of the total cohort , 53 patients started on basal + insulin aspart ogld , of which 27 ( 50.9% ) were insulin nave and 26 ( 49.1% ) were insulin users . after 24 weeks of starting or switching to basal + insulin aspart , hypoglycaemic events reduced from 1.0 events / patient - year to 0.0 events / patient - year in insulin user group , while hypoglycaemia was nil in insulin naive group similar to baseline . quality of life improved at the end of the study [ tables 8 and 9 ] . basal+insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for both insulin nave and insulin user groups [ table 10 ] . of the total cohort , 313 patients started on insulin detemir ogld was 313 , of which 302 ( 96.5% ) were insulin nave and 11 ( 3.5% ) were insulin users . after 24 weeks of starting or switching to insulin detemir , hypoglycaemic events was nil in both insulin nave and user groups similar to baseline . body weight decreased and quality of life improved at 24 weeks [ tables 11 and 12 ] . insulin detemiroral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin detemir oglds for both insulin - nave and insulin user groups [ table 13 ] . of the total cohort , 144 patients started on insulin aspart ogld , of which 131 ( 91.0% ) were insulin nave and 13 ( 9.0% ) were insulin users . after 24 weeks of starting or switching to insulin aspart , hypoglycaemic events reduced from 2.0 events / patient - year to 0.0 events / patient - year in insulin user group , whereas hypoglycaemia remained nil in insulin nave group similar to baseline . quality of life improved at the end of the study [ tables 14 and 15 ] . insulin aspartoral glucose - lowering drug safety data all parameters of glycaemic control improved from baseline to study end in those who started on or were switched to insulin aspart oglds for both insulin nave and insulin user groups [ table 16 ] . our study reports improved glycaemic control and quality of life following 24 weeks of treatment with any of the insulin analogues ( biphasic insulin aspart ; basal + insulin aspart ; insulin detemir ; insulin aspart ) with or without ogld . sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients . overall , body weight increased in insulin nave group while there was no change in body weight for insulin user group . though the findings are limited by number of patients , still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in mumbai , india .

background : the a1chieve , a multicentric ( 28 countries ) , 24-week , non - interventional study evaluated the safety and effectiveness of insulin detemir , biphasic insulin aspart and insulin aspart in people with t2 dm ( n = 66,726 ) in routine clinical care across four continents.materials and methods : data was collected at baseline , at 12 weeks and at 24 weeks . this short communication presents the results for patients enrolled from mumbai , india.results:a total of 2112 patients were enrolled in the study . four different insulin analogue regimens were used in the study . patients had started on or were switched to biphasic insulin aspart ( n = 1561 ) , insulin detemir ( n = 313 ) , insulin aspart ( n = 144 ) , basal insulin plus insulin aspart ( n = 53 ) and other insulin combinations ( n = 41 ) . at baseline glycaemic control was poor for both insulin nave ( mean hba1c : 8.7% ) and insulin user ( mean hba1c : 9.2% ) groups . after 24 weeks of treatment , both the groups showed improvement in hba1c ( insulin nave : 1.4% , insulin users : 1.8% ) . sadrs including major hypoglycaemic events or episodes did not occur in any of the study patients.conclusion:starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia .

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over the past decade , many cell types have been evaluated in an effort to find the best source for cardiac regeneration . of these , the most extensively studied have been embryonic stem cells , mesenchymal stem cells , skeletal myoblasts , and bone marrow - derived progenitor cells , while more recent possibilities have included induced pluripotent stem cells and resident cardiac stem cells . embryonic stem cells ( escs ) can be obtained from the inner cell mass of a pre - implantation blastocyst and expanded in vitro almost indefinitely without loss of pluripotency [ 13 ] . when allowed to differentiate as embryoid bodies , escs give rise to most somatic lineages , including cardiovascular lineages [ 48 ] . culture of embryoid bodies with specific growth factors or small molecules can drive differentiation toward cardiovascular phenotypes , thereby enriching the pool of esc - derived cardiac cells available for transplant [ 813 ] . both pluripotent escs and committed esc - derived cardiac cells have been tested in rodent models with encouraging results in terms of engraftment , survival , and improvement in ventricular function [ 1416 ] . however , no clinical trials have been attempted , for concerns of teratoma formation [ 16 , 17 ] , graft - versus - host disease , and bioethics . while improved in vitro differentiation of escs may eliminate the first of these concerns , recent discoveries indicating that adult somatic cells can be reprogrammed to yield esc - like cells may eliminate the second and third of these issues . induced pluripotent stem cells ( ipscs ) can be generated from murine and human adult somatic cells by overexpression of transcription factors critical for maintenance of esc pluripotency . while initial ipsc protocols required retro- or lentiviral transfer of four factors , namely octamer - binding transcription factor 3/4 ( oct 3/4 ) , sry - related hmg - box transcription factor ( sox2 ) , krppel - like factor 4 , and cellular myelocytomatosis oncogene [ 19 , 20 ] or oct 3/4 , sox2 , nanog , and lin28 , subsequent reports have reduced the risk of tumorigenesis introduced during the de - differentiation process by utilizing non - integrating technologies [ 2225 ] or by transferring fewer transcription factors [ 2628 ] while still achieving pluripotency . once established , ipscs differentiate much like escs , yielding functional cardiovascular lineages from embryoid bodies in vitro [ 2832 ] and contributing to normal cardiovascular development from chimeric blastocysts in vivo [ 28 , 31 , 33 ] . in an initial proof - of - principle study , four - factor ipscs successfully restored myocardial structure and function after coronary artery ligation in mice . thus , ipscs may provide an unlimited supply of autologous donor cells , once technological advancements eliminate the risk of teratoma or other tumor formation . mesenchymal stem cells ( mscs ) can be separated from bone marrow and adipose tissues based on their adherence to a culture dish . mscs most readily differentiate into osteoblasts , chondrocytes , and adipocytes [ 35 , 36 ] but can be induced to differentiate into cardiomyocytes under special conditions [ 3739 ] . they can be rapidly expanded in culture , thereby allowing for autologous transplantation , and they may be less immunogenic than other cell populations , thereby allowing for allogenic transplantation [ 40 , 41 ] . but , their propensity to differentiate into non - cardiac tissues ( e.g. , heterotopic ossification ) currently limits their use [ 42 , 43 ] . nonetheless , at least one clinical trial has shown improved ventricular function at 3 and 6 month follow - up after intracoronary infusion of autologous mscs postinfarction . skeletal myoblasts , also known as satellite cells , can be harvested from muscle biopsies , expanded in culture , and autologously reimplanted , albeit on a timescale more appropriate for chronic heart failure therapy than for early postinfarction management . as these cells are further differentiated than escs , they are less prone to teratoma formation ; they are also more resistant to ongoing ischemia and more apt to function in a contractile capacity postengraftment . however , true cardiomyocyte differentiation has not been observed in vivo . as a result , although skeletal myoblasts may be incorporated into an infarct site , their contractions are dyssynchronous due to failure of electromechanical coupling with the surrounding myocardium [ 45 , 46 ] . thus , no significant benefit has been seen in large - scale clinical trials , while a trend toward more frequent arrhythmias has been observed . bone marrow - derived mononuclear cells , including hematopoietic and endothelial progenitor cells , can be mobilized with cytokine therapy or harvested by standard collection techniques . in culture , they can differentiate into cardiomyocytes , endothelial cells , and smooth muscle cells [ 4851 ] , and after transplant , they can supply a broad range of paracrine factors with proangiogenic , positive remodeling , and antiapoptotic properties . a meta - analysis of 18 randomized and non - randomized trials involving 999 patients with acute myocardial infarction or chronic ischemic cardiomyopathy found that intracoronary infusion of adult bone marrow - derived stem cells improved left ventricular ejection fraction by 3.7% , decreased infarct scar size by 5.5% , and lowered left ventricular end - systolic volume by 4.8 ml ( p < 0.01 for each index ) . among these pooled studies , the largest randomized trial , repair - ami , was particularly notable for not only demonstrating improvement in ventricular function but also for reducing the combined endpoint of death , myocardial infarction , or revascularization at 12 months when compared with placebo . nonetheless , controversy remains regarding the fates of bone marrow - derived cells used for cardiac regeneration . identification of the most safe and efficacious means of stem cell delivery remains an important challenge , both in terms of route and timing of administration . current methods of stem cell delivery include non - invasive mobilization from resident niches ( especially bone marrow ) with cytokine therapy , non - selective intravenous or intracavitary injection , selective intracoronary infusion , and intramyocardial injection from either surgical or percutaneous access . although some intravenously injected bone marrow - derived stem cells do home to the infarct border zone , only a small fraction ( < 5% ) of intravenously injected cells is retained in this intended destination . rather , the majority of intravenously injected cells are trapped by the lungs , liver , kidneys , and spleen [ 56 , 57 ] . more proximal but still non - selective delivery via intracavitary or intra - aortic ( with the distal aorta occluded ) injection marginally improves cardiac uptake [ 56 , 57 ] , suggesting that closer may be better . indeed , intracoronary delivery appears superior to intravenous delivery in man [ 58 , 59 ] , although overall cell retention remains low as rapid washout follows balloon deflation . clinical trials assessing the biodistribution of infused cells have recently been reviewed . in animal studies , intramyocardial delivery yields better retention than intracoronary delivery [ 57 , 62 , 63 ] . but so far , no clinical trial has directly compared these two modalities , although such results should be forthcoming . in theory , intramyocardial delivery may provide more precisely targeted therapy without need for patent coronary arteries , while intracoronary delivery may benefit from operator familiarity and lower cost . since intramyocardial injections carry risk of myocardial perforation and electrical instability shortly after infarction , these two strategies in fact may be complementary , with intracoronary cell delivery provided early and intramyocardial cell delivery provided late . despite the potential importance of correctly timing stem cell administration , few studies have directly addressed this issue . on one hand , stem cell survival may be compromised shortly after infarction by transient microvascular obstruction , recurrent ischemia , or reperfusion injury . on the other hand , stem cell homing to the infarct border zone may be enhanced shortly after injury by transient upregulation of cytokines such as stromal cell - derived factor 1 ( sdf-1 ) , vascular endothelial growth factor ( vegf ) , hepatocyte growth factor ( hgf ) , and monocyte chemotactic protein-3 ( mcp-3 ) [ 6568 ] . in rodents , this latter phenomenon appears to have the upper hand , as the benefits of msc therapy are seen only when administered within 4 days postmyocardial infarction , paralleling myocardial sdf-1 expression . in man , so far , stem cell administration within 1 day of reperfusion therapy has not shown clinical benefit . therefore , the nih cardiovascular cell therapy research network 's time trial will compare an early time point of 3 days postmyocardial infarction to an intermediate time point of 7 days postmyocardial infarction for intracoronary stem cell infusion [ ; http://ccct.sph.uth.tmc.edu/cctrn/public/publichome.aspx ] . this trial will run in parallel with a related study called late - time , which will focus on a late time point of 23 weeks postmyocardial infarction for intracoronary cell infusion . beyond that time point , a recent substudy of the mystar trial found no significant differences in the long - term improvements in infarct size and ventricular function observed after bone marrow mononuclear cell delivery at 36 weeks versus 34 months . treatments designed to improve stem cell homing to diseased myocardium have the potential to markedly enhance both the safety and efficacy of cardiovascular cell therapy . by increasing cardiac retention , stem cell capture by non - cardiac tissues may be reduced , thereby limiting potential side effects . this may allow stem cell dosages to be increased , even as outcomes on a per cell basis are improved . many current strategies to improve cardiovascular homing aim to enhance adhesion protein and cytokine interactions between donor cells and host tissues . ex vivo conditioning of donor cells , for example , by incubation of endothelial progenitor cells with a 2-integrin activating antibody , can improve retention and transcapillary migration at sites of injury , where intracellular adhesion molecule-1 ( icam-1 ) and fibrinogen are upregulated . alternatively , in vivo conditioning of the host vasculature , for example , by infusion of adenosine , can promote cardiac retention of endothelial progenitor cells by upregulating coronary artery expression of p - selectin . increased display of this cell adhesion protein captures endothelial progenitor cells that constitutively express p - selectin glycoprotein ligand-1 [ 73 , 74 ] . adenosine can be safely infused into the human coronary circulation ; thus , it could be an adjuvant to many existing cell therapy protocols . these include master regulators of ischemic responsiveness such as integrin - linked kinase ( ilk ) and hypoxia inducible factor-1 ( hif-1 ) , as well as secreted homing factors such as sdf-1 , mcp-3 , and vegf . in response to hypoxia increased ilk activity then leads to increased expression of sdf-1 and icam-1 through hif-1- and nuclear factor b - mediated signaling . while this beneficial property of ilk has been demonstrated only in a hindlimb ischemia model thus far , its cardiac application is likely not far off . already , it is known that sdf-1 recruits endothelial progenitor cells expressing cxcr4 to injured myocardium , and icam-1 binding to its receptor , 2 integrin , on endothelial progenitor cells is required for engraftment after myocardial infarction . therefore , given the functional improvements seen with ilk gene therapy after coronary artery ligation in rats , utilization of ilk in stem cell therapy would seem a logical step . indeed , encouraging results have been obtained when local myocardial therapy has been combined with stem cell transplantation . downstream of ilk , hif-1 regulates both sdf-1 and its receptor cxcr4 , as well as icam-1 [ 76 , 79 , 80 ] . combining intramyocardial injection of adenovirus - encoded hif-1 with transplantation of skeletal myoblasts results in improved cell engraftment and cell survival after coronary artery ligation in rats . this results in marked improvement in ejection fraction ( by 27% ) at 1 month posttherapy over baseline at 1 week postinfarct . in comparison , monotherapy with hif-1 or myoblasts alone serves only to prevent further decline of the ejection fraction over this interval . further down this ischemic response pathway , both sdf-1 and its receptor cxcr4 have been used successfully as adjuncts to cardiac cell therapy . in mice , endogenous sdf-1 is upregulated for less than 1 week after myocardial infarction [ 6567 ] . re - establishment of sdf-1 expression at later time points can enhance stem cell recruitment and retention when the ischemic border zone might be less hostile to cell engraftment and survival . indeed , intramyocardial delivery of scaffold - bound sdf-1 after coronary artery ligation in rats and mice results in enhanced c - kit+ cell homing and improved ventricular function [ 82 , 83 ] . while it may seem counterintuitive for a stem cell to deliver its own homing factor , considerable benefit has been obtained when genetically modified stem cells recruit additional native and transplanted cells to injured myocardium . for example , improved cell engraftment and survival , as well as improved ventricular function , has been observed after transplantation of sdf-1expressing skeletal myoblasts ( as opposed to unmodified skeletal myoblasts ) at both early and late time points after myocardial infarction in rats [ 84 , 85 ] . similarly , transplantation of sdf-1-expressing cardiac fibroblasts promotes homing of endogenous cd117 + stem cells to injured myocardium . this results in improved ventricular function even without cardiomyocyte regeneration . rather , improved engraftment and differentiation of transplanted sdf-1-expressing mscs ( as opposed to unmodified mscs ) into myofibroblasts , but not true cardiomyocytes , results in improved ventricular function by providing trophic support for cardiomyocyte preservation but not regeneration after infarction . similar results are obtained after transplantation of cxcr4-overexpressing mscs , indicating that improved stem cell homing , engraftment , and paracrine activity can be obtained via cell - based gene therapy with both stem cell homing factors and their receptors . other growth factor chemokine - receptor pairs , which may be utilized to improve outcomes of cardiac cell therapy , include vegfflk-1 , hgfc - met , mcp-3ccr1/2 , and growth - related oncogene-1cxcr1/2 [ 66 , 68 , 8891 ] . of these , the most extensively studied factor is vegf . vegf is transiently upregulated after myocardial infarction and serves to mobilize endothelial progenitor cells while promoting neovascularization [ 92 , 93 ] . vegf is both induced by sdf-1 and an inducer of sdf-1 's receptor cxcr4 [ 9496 ] . early clinical trials demonstrated that intramyocardial delivery of naked plasmid - encoded vegf gene therapy is safe but results in only marginal clinical benefit [ 97 , 98 ] . subsequent human and large animal studies of combination intramyocardial vegf gene delivery plus stem cell therapy have yielded mixed results [ 99101 ] . meanwhile , vegf - transfected stem cells have consistently outperformed untransfected control cells after coronary artery ligation in rodents [ 102107 ] . and in those studies that have compared cell - based gene therapy to direct gene delivery , vegf - transfected skeletal myoblasts and vegf - transfected mscs have improved ventricular function to a greater degree than either direct adenoviral injection or liposomal plasmid delivery , respectively [ 106 , 107 ] . taken together , these results suggest that cell - based gene therapy may be more effective than either unmodified stem cell therapy or acellular gene therapy alone . the degree despite the structural and functional benefits of stem cell therapy in animal and human studies of acute myocardial infarction and chronic heart failure , limited transplanted cell survival has been observed , especially in the long - term . less than 1% of transplanted cells survive in patient hearts , and even in the idealized scenario of syngeneic neonatal cardiomyocyte intramyocardial injection in healthy rats , only 15% of transplanted cells survive for 12 weeks . nonetheless , recent discoveries demonstrative of the heart 's capacity for regeneration and repair suggest that these challenges are not insurmountable . measurement of c in the dna of individuals born prior to cold war era nuclear testing estimates that nearly half of all cardiomyocytes are exchanged during a normal life span . since c is incorporated equally by the division of resident cardiomyocytes and circulating progenitor cells , carbon dating can not distinguish the cellular origins of such impressive turnover . rather , the phenomenon of cardiac chimerism seen after sex - mismatched heart or bone marrow transplant confirms that extra - cardiac progenitor cells are capable of long - term engraftment and survival in the healthy human heart . thus , the challenge for cardiac cell therapy is to strengthen donor cells for the harsh microenvironment of the infarcted heart . in addition to activating survival pathways in cardiomyocytes , ischemic preconditioning recruits endothelial progenitor cells to serve as mobile reservoirs of cardioprotective cytokines . proponents of cardiac cell therapy now seek to harness this property to enhance donor cell survival . hypoxic preconditioning of mesenchymal stem cells and cardiac progenitor cells prior to transplant improves not only their recruitment , survival , and differentiation at the infarct border zone but also overall ventricular structure and function [ 114 , 115 ] . similar benefits might be obtained by hypoxic preconditioning of endothelial progenitor cells , based on results from an ischemic hindlimb model . this is because , in each of these cell types , hypoxic preconditioning upregulates a diverse array of survival , angiogenic , and migratory proteins , including hif-1 , akt-1 , bcl-2 , ang-1 , vegf and its receptor flk-1 , sdf-1 's receptor cxcr4 , and hgf 's receptor c - met [ 114 , 115 , 117 ] . together , these factors allow donor cells not only to endure but also to heal the injured myocardium . extending this concept , hypoxic preconditioning can be mimicked by genetic modification of donor cells to overexpress hypoxia - responsive survival factors . akt-1 is activated by hypoxia and other stimuli , including cytokines . as a general mediator of survival signaling , blockade of akt-1 activation with the phosphoinositol 3-kinase ( pi3k ) inhibitor wortmannin attenuates the cardioprotective effects of endothelial progenitor cell therapy after ischemic injury in pigs . on the other hand , genetic modification of mscs to overexpress akt-1 results in marked improvement in intracardiac msc retention and survival in rat and pig infarct models [ 120 , 121 ] . in both species , transplantation of akt-1-overexpressing mscs follow - up studies have demonstrated that akt-1-overexpressing mscs release paracrine factors that promote myocardial survival and recovery after infarction [ 122 , 123 ] . by means of functional genomics , secreted frizzled related protein 2 ( sfrp2 ) was identified as the key akt-1msc - expressed paracrine factor mediating myocardial survival and repair after ischemic injury . when sfrp2 is suppressed in akt-1mscs , alternatively , genetic modification of mscs to specifically overexpress sfrp2 promotes their retention and survival within the infarct border zone , as well as facilitates restoration of cardiac structure and function after infarction . these benefits may be attributable to sfrp2-mediated inhibition of wnt binding to the frizzled receptor , which results in accumulation of -catenin and upregulation of antiapoptotic and proangiogenic signaling . other paracrine factors upregulated in akt-1mscs include thymosin 4 ( t4 ) and insulin - like growth factor-1 ( igf-1 ) . t4 is a secreted , g - actin sequestering , 43 amino - acid peptide that stimulates the migration and survival of cardiomyocytes and endothelial cells , as well as the migration and differentiation of epicardial coronary vascular progenitors . after coronary artery ligation in mice , intramyocardial injection of t4 protein exemplary of t4 's importance in cell therapy , shrna - mediated knockdown of t4 in endothelial progenitor cells blocks the cardioprotective actions of these cells after ischemia - reperfusion in mice . in addition to stimulating myocardial growth and contractility , igf-1 activates the pi3k / akt-1 pathway to promote cardiomyocyte survival and proliferation in response to injury [ 128 , 129 ] . igf-1 protein has been combined with cell therapy to improve donor cell survival and differentiation in host myocardium [ 130 , 131 ] . extending these benefits , igf-1 cell - based gene therapy has improved donor cell engraftment and proliferation and enhanced host cell recruitment to ischemic myocardium [ 132 , 133 ] . this has been accompanied by decreased infarct size , increased angiogenesis , and improved ventricular function . molecular studies have attributed igf-1 's therapeutic benefit , in part , to activation of pi3k / akt-1 and bcl-2 and to release of sdf-1 and vegf [ 132 , 133 ] . downstream of igf-1 and pi3k / akt-1 , but not sfrp2 , bcl-2 has been identified as a key antiapoptotic protein with potential utility in cardiac cell therapy . cell lines derived from escs which overexpress bcl-2 self - renew continuously , even in the absence of serum and feeder cells . indeed , mscs , smooth muscle cells , and cardiomyoblasts genetically modified to overexpress bcl-2 have all demonstrated reduced cell loss and improved ventricular function after transplant into ischemic myocardium [ 136138 ] . but while such results may be encouraging , cell - based gene therapy utilizing cell survival factors is not without its challenges , as these proteins may predispose to tumorigenesis . much work still lies ahead to regulate the expression of these factors before their clinical application . however , recent experience has shown that cell therapy is capable of providing significant structural and functional benefit with only minimal cardiomyocyte regeneration . as we have discussed , donor cells can recruit host cells to the site of injury , especially if they are modified to overexpress stem and progenitor cell homing factors . donor cells can secrete paracrine factors to promote host tissue preservation and repair , especially if they are modified to overexpress survival or growth factors [ 52 , 122 , 123 , 133 ] , and donor muscle cells can provide contractile reserve , especially if they are modified to overexpress connexins essential for electromechanical coupling . given these capabilities , one might legitimately ask if the last piece of the cardiac cell therapy puzzle cardiomyocyte regeneration even needs to be solved . as we have discussed , recent measurements of c incorporation into the heart indicate that nature provides for cardiac regeneration throughout a normal human lifetime . genetic fate - mapping studies have shown that nearly 20% of cardiomyocytes bordering a murine myocardial infarction are derived from endogenous stem or precursor cell differentiation . this may be accomplished by utilization of more committed cardiac progenitor cells isolated from the adult heart or derived in culture from pluripotent stem cells . the adult heart is home to a side population ( sp ) of stem cells identified by exclusion of the vital dyes hoechst 33342 and rhodamine 123 [ 141 , 142 ] . isolated cardiac sp cells that express stem cell antigen-1 ( sca-1 + ) , but are negative for the endothelial marker cd31 , are capable of differentiation into functional cardiomyocytes after co - culture with adult cardiomyocytes or treatment with a cocktail of bone morphogenetic protein 2 , fibroblast growth factors 4 and 8 , the wnt antagonist dickkoff-1 , and 5-azacytidine . transplantation of both pre - differentiated and undifferentiated sca-1+/cd31 cells after myocardial infarction in mice results in efficient homing , engraftment , and differentiation into cardiomyocytes and endothelial cells , as well as improved ventricular function [ 144146 ] . a second resident progenitor population comprises c - kit ( also known as cd117 ) positive cells , which are located in small clusters within the adult heart . isolated c - kit+ cells do not differentiate into cardiovascular cell types in culture but showed impressive regenerative potential in some studies after transplantation into the injured rat heart , where they gave rise to cardiomyocytes , endothelial cells , and smooth muscle cells , while also improving ventricular function [ 147150 ] . nonetheless , a rare sca-1+/c - kit+ cardiac cell type that differentiates into cardiomyocytes upon oxytocin treatment has been identified in mice . from a technical standpoint , cardiac progenitor cells can be isolated from mouse hearts and human endomyocardial biopsies by enzymatic digestion to release round cells that form so - called cardiospheres in suspension [ 152 , 153 ] . cardiosphere - derived cells express endothelial and stem cell markers , show contractile activity in culture , and can differentiate into cardiomyocytes , endothelial cells , and smooth muscle cells . transplantation of these cells improves ventricular function after myocardial infarction in mice and swine , due to both direct cardiomyocyte regeneration and indirect paracrine action [ 153155 ] . to test whether these presumably committed cardiac progenitor cells have benefit in man , enrollment in the first clinical trial of cardiosphere - derived autologous stem cells to reverse ventricular dysfunction an alternative to laborious isolation of committed cardiac progenitor cells from the adult heart is differentiation of committed cardiac progenitor cells from embryonic stem cells , induced pluripotent stem cells , or other multipotent cellular resources such as adipose tissue - derived mscs . after study of many different growth factor cocktails , a growing consensus now agrees that modulation of bone morphogenetic protein ( bmp ) signaling may be the key to reliable ex vivo cardiomyocyte differentiation . however , consensus is lacking with regard to identification of the critical bmp agonists and antagonists , as well as their appropriate dosing . treatment of murine escs or mscs with bmp2 in the presence of a second growth factor such as transforming growth factor-1 , fibroblast growth factor 2 or 4 , or leukemia inhibitory factor promotes cardiomyocyte differentiation and improves cardiac structure and function following transplantation into infarcted myocardium [ 8 , 9 , 11 , 157 ] . however , it is also known that the endogenous bmp antagonist noggin and the synthetic bmp inhibitor dorsomorphin also increase cardiomyocyte differentiation [ 1012 ] , in apparent contrast to studies that blocked the inductive properties of bmp2 and bmp4 with noggin [ 8 , 13 ] . a unifying hypothesis for these observations suggests that bmp signaling is necessary for at least two steps in cardiomyocyte development : mesodermal induction and cardiomyocyte differentiation [ 158 , 159 ] , between which transient block of bmp signaling is essential . thus , the optimal timing and dosage of bmp agonist and antagonist treatments may be critical . finally , recent evidence that adult cardiomyocytes may be induced to proliferate could provide a complementary strategy for cardiac regeneration . ongoing research at the bench and the bedside will surely continue to perfect these pieces of the cardiac cell therapy puzzle : comparing sources of donor cells , testing methods of cell delivery , augmenting myocardial homing , bolstering cell survival , and promoting cardiomyocyte differentiation , and while much more work needs to be done , we have confidence that stem cell therapies in conjunction with current treatment modalities will ultimately reduce the mortality and improve the quality of life of patients with cardiovascular disease .

acute ischemic injury and chronic cardiomyopathies can cause irreversible loss of cardiac tissue leading to heart failure . cellular therapy offers a new paradigm for treatment of heart disease . stem cell therapies in animal models show that transplantation of various cell preparations improves ventricular function after injury . the first clinical trials in patients produced some encouraging results , despite limited evidence for the long - term survival of transplanted cells . ongoing research at the bench and the bedside aims to compare sources of donor cells , test methods of cell delivery , improve myocardial homing , bolster cell survival , and promote cardiomyocyte differentiation . this article reviews progress toward these goals .

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the mineral polyhalite ( chemical formula [ k2ca2mg(so4)4.2h2o ] ) commonly occurs in sedimentary evaporite successions and in rare cases as a sublimate in volcanic successions ( e.g. vesuvius , campania , italy ) ( anthony et al . , 1996 ) . polyhalite was first described from the salt mine bad ischl - perneck in austria ( province upper austria ) by the german chemist stromeyer ( 1818 ) and has since been known from many salt deposits ( e.g. warren , 2006 ) . in evaporites , polyhalite is commonly associated with halite , anhydrite , glauberite , carnallite , kieserite , langbeinite , vanthoffite , chlorides and sulphides . the crystal structure was first determined in samples from altaussee in austria ( schlatti et al . , 1970 ) and later confirmed ( bindi , 2005 ) . polyhalite can be synthesised under laboratory conditions by a reaction of gypsum with appropriate solutions in the ternary system k2so4mgso4h2o at temperatures above 70 c ( freyer and voigt , 2003 ) . at lower temperatures ( 2008 ) found under laboratory conditions the dehydration temperature of polyhalite to start at 255 c to a maximum of 343 c . substitution of divalent cations by other elements like zn , cu , cu , ni significantly changes dehydration reactions to higher or lower temperatures ( wollmann et al . , 2008 ) . further chemical data on the conditions of formation and stability of polyhalite were presented inter alia by krupp ( 2005 ) and wollmann et al . polyhalite most commonly forms early - diagenetically ( e.g. holser , 1966 ; busson and perthuisot , 1977 ; pierre , 1985 ; khn and mller - schmitz , 1987 ) by brine - back reactions of k mg so4 brine with earlier formed gypsum , anhydrite or glauberite ( e.g. braitsch , 1962 ; schauberger , 1986 ; warren , 2006 ) after a prolonged contact ( over months or years ) of the brine with the precursor rock ( hardie , 1984 ) . for example , all alpine polyhalites are of secondary origin ( leitner , 2011 ; leitner et al . a formation of polyhalite in marginal marine shallow water sedimentary environments is only suspected ( hryniv et al . , 2007 ) . polyhalite is stable between room temperature ( 025 c ) and 255343 c ( wollmann et al . , 2008 ) or 285 c ( fischer et al . , 1996 ) and , therefore , within nearly the whole temperature range , the haselgebirge sulphates have experienced ( 200300 c , table 1 , see below ) . polyhalite contains potassium in stochiometric amounts rendering the mineral interesting for absolute age dating essential for deciphering the geological history of a region . since polyhalite was considered to be rare in most salt deposits , published ages are scarce and dating was carried out with other methods . in austria and southern germany , ( in the following named as haselgebirge mlange ) , a tectonically mixed evaporite succession at the stratigraphic base of the northern calcareous alps ( in the following abbreviated as nca ) ( fig . 1 ) . there , polyhalite is a minor , but widespread and important constituent and is accessible in several salt mines including the type locality of bad ischl , hallstatt , berchtesgaden drrnberg and hall in tirol ( schauberger , 1986 ) . at the localities berchtesgaden and altaussee , polyhalite mainly occurs intergrown with anhydrite or as vein fillings and , in press a , b ) , for example intergrown with fine - grained anhydrite , in veins , in nodules , and recrystallised in foliated polyhalite rocks along shear zones . grgey ( 1910 ) distinguished three different types of polyhalite occurrences : ( 1 ) fine - fibrous , acicular or lamellar , yellowish - red to reddish polyhalite layers , which form 120 cm thick layers within the haselgebirge , often with an extension of several square metres ; ( 2 ) compact fine - to - medium - grained yellowish - brown to dark red coloured polyhalite layers associated with anhydrite and ( 3 ) fibrous - acicular or granular polyhalite areas , which form seams around anhydrite crystals ( like in hall in tirol ) . schindl - neumayer ( 1984 ) examined the grain structures of anhydrites , polyhalites and halites of the alpine salt deposits . she distinguished five different polyhalite types : ( 1 ) fine - felty polyhalite aggregates , ( 2 ) fine - grained polyhalite groundmasses , ( 3 ) polyhalite twinnings in a polyhalite groundmass and in anhydrite porphyroblasts , ( 4 ) polyhalites as felty conversion seams round anhydrite and ( 5 ) polyhalite and clay / mudrock . the most recent work on polyhalite fabrics from altaussee and berchtesgaden ( in press b ) distinguished three main polyhalite microfabric types : ( 1 ) fibrous polyhalite in veins , ( 2 ) porphyroblasts of polyhalite in anhydrite with subdivisions ( 2a ) ( round polyhalite blasts ) and ( 2b ) ( fibrous polyhalite blasts ) and ( 3 ) dense polyhalite comprising corroded large anhydrite grains with subdivisions ( 3a ) ( fine - grained , mylonitic polyhalite rock ) , ( 3b ) ( non - foliated polyhalite rock with local zones ) and ( 3c ) ( non - foliated polyhalite rock ) . ( in press a ) described a diagenetic reaction after halite hopper crystals , which forms anhydrite and polyhalite and added another polyhalite microfabric type : polyhalite in pseudomorphic anhydrite cubes after halite hopper crystals and polyhalite from within the retaining shape of deformed halite hopper shaped cubes . ( in press b ) demonstrated , on a limited number of examples , that polyhalite occurs in polyhalite rocks and as vein filling with various fabric types , which also gave distinct ar / ar ages . one of the most important results of these papers is that polyhalites of the alpine haselgebirge formed at ca . 235210 ma ( leitner et al . , in press b ) , ca . 2025 ma after the sedimentation of the haselgebirge evaporites ( leitner , 2011 ) and with a maximum estimated overburden of approximately 800 m ( e.g. rantitsch and russegger , 2005 ) . with this study , we further explore the variety of microfabric types of mono- and polymineralic polyhalite rocks and vein and cavity fills from the hallstatt mine as we found that the variety is significantly larger than elsewhere in the nca . the hallstatt salt mine is within the upper permian lower triassic haselgebirge fm .. the classic division within the nca defines the bajuvaric , tirolic and juvavic nappe complexes ( tollmann , 1985 , 1987 and references therein ; mandl , 2000 ) ( fig . 1 ) . the permian to lower triassic haselgebirge occurs mainly in juvavic units of the central and eastern nca ( schauberger , 1986 ; leitner and neubauer , 2011 and references therein ) and subordinately in tirolic units . the age of formations in the nca ranges from late carboniferous ( ? ) or early permian to eocene . during the middle and late triassic , the upper juvavic nappes comprise mostly reefs and deposits next to reefs , the tirolic and bajuvaric nappes comprise lagoonal facies types and subordinate reefs . the lower juvavic nappe unit represents solely an outer shelf or a deep - sea facies type ( mandl , 2000 ) . sedimentation of the lower juvavic unit occurred in basins with basinal limestones ( ptschen limestone ) and on intrabasinal ridges , with reduced sedimentary thickness ( the pelagic hallstatt limestone ) . the ridges were suggested to relate to salt diapirism in triassic times ( mandl , 1982 , 2000 ; plchinger , 1984 ) . the westernmost part of the expanding triassic tethys ocean is called hallstatt - meliata ocean , which comprises rare deep - sea ( ophiolitic ? ) sequences in the eastern parts of the nca ( faupl and wagreich , 2000 ; neubauer et al . , 2000 and references therein ) . most authors interpret the hallstatt limestone as an outer shelf ( tollmann , 1985 , 1987 ; mandl , 2000 ) . others propose a position of the hallstatt - meliata relics between the upper juvavic and tirolic units ( schweigl and neubauer , 1997 ) . the permian / middle triassic to lower upper triassic rift stage and passive margin formation was associated with widespread synsedimentary and diagenetic pb the hallstatt - meliata ocean was being closed during the late jurassic ( dallmeyer et al . , 2008 and references therein ) . coevally , the sea floor dropped and reached maximum water depths with the formation of radiolarites . gravitational sliding is reported from different places ( e.g. mandl , 1982 ; plchinger , 1990 ) , and this concept was developed until recent years ( missoni and gawlick , 2011 and references therein ) . these authors argue for a late jurassic age of shortening . during early cretaceous , nappe thrusting prograded from south to north , respectively from ese to wnw ( ratschbacher , 1986 ; linzer et al . , 1995 ; mandl , 2000 ; neubauer et al . , 2000 ) . the mechanism and the time of emplacement of the juvavic units are still a matter of controversy . the classic hypothesis assumes that both juvavic nappes took their position during the eo - alpine deformational event ( late early to early late cretaceous ) by means of thrust tectonics ( kober , 1955 ; pichler , 1963 ; schweigl and neubauer , 1997 ; mandl , 2000 ) . a further model explains the emplacement of all juvavic units by gravity sliding since late jurassic times , as haselgebirge clasts of various sizes have been found in the upper jurassic oberalm and lower cretaceous rossfeld fms . this concept was extended to large mountain - like blocks also explained by emplacement by simple gravity sliding ( gawlick and lein , 2000 ; missoni and gawlick , 2011 and references therein ) . frank and schlager ( 2006 ) propose the emplacement as a consequence of late jurassic strike - slip movements related to the opening of the penninic ocean . schorn and neubauer ( 2011 ) proposed an early cretaceous emplacement of the haselgebirge - bearing nappe over tirolic units . in the eocene , the second paroxysm of the alpine orogeny occurred , when continental basement slices ( middle penninic ) and parts of the north penninic ocean ( rhenodanubian flysch ) adriatic microcontinent ( faupl and wagreich , 2000 ; linzer et al . , 2002 and references therein ) . the present nca were partly detached from their austroalpine basement ( e.g. greywacke zone ) and thrusted over the rhenodanubian flysch and helvetic domain resulting in a wide thin - skinned tectonic nappe complex ( linzer et al . , 1995 ; mandl , 2000 ; neubauer et al . , 2000 ) . the familiar rocksalt deposits , which are all located in the interior of the nca mostly within the lower juvavic unit , were considered to be only slightly affected by these cenozoic deformation stages , since the detachment of the nca domain occurred beneath the lowermost unit , the bajuvaric nappe ( fig . 1 ) . deformation of upper cretaceous to eocene gosau basins deposited on uppermost nappes ( tirolic and juvavic nappes ) suggests significant deformation in late eocene to early miocene ( linzer et al . , 1995 , 1997 ; the salt body of hallstatt ( figs . 2 and 3 ) , which is part of the hallstatt nappe north of the echern valley ( i.a . habermller , 2005 ) , was described recently by leitner and neubauer ( 2011 ) and we follow their description . w direction and is around 600 m wide ( maps and sections : schauberger , 1955 ; schffer , 1982 ; scheidleder et al . , 2001 ; habermller , 2005 and references therein ) . the highest point is 1350 m above sea level , the lowest erbstollen level is at around 500 m above sea level ( section of schauberger in habermller , 2005 ) . the salt body suitable for mining diminishes with depth , mostly because of incorporated country rocks . the base has not yet been reached at 100 m below the level of lake hallstatt ( pers . is around 55 wt ( weight ) % ( schauberger , 1931 , 1949 , 1955 ) . the mining started in celtic times , as indicated by a bronze - age staircase found in the mine , which was dated accurately by dendrochronology to 1344 bc ( grabner et al . , 2007 ) . the sedimentary succession of the lower juvavic unit is nearly complete and comprises , beside the basal upper permian lower triassic haselgebirge fm . contains blocks of meta - basalts ( melaphyre ) ( e.g. schauberger , 1960b ; zirkl , 1957 and references therein ) within the buntes salztongebirge ( = colored salt - bearing claystone ) ( pak and schauberger , 1981 ; schauberger , 1986 ) which are also assumed to be of upper permian age ( e.g. klaus and pak , 1974 ) , but no reliable dating was performed . based on palynological evidence and a sulphur isotopic signature , the sediments of the haselgebirge were deposited during late permian and early triassic times ( klaus , 1955 , 1965 ; klaus and pak , 1974 ; pak , 1974 , 1978 , 1981 ; pak and schauberger , 1981 ; gtzinger and pak , 1983 ; sptl , 1987 , 1988a , 1989 ; sptl and pak , 1996 ) . sptl ( 1988a ) carried out detailed studies on relict sedimentary structures in the drrnberg and hallstatt mines , and interpreted the evaporites to have formed in a sabkha - like environment , situated in an aborted rift of the tethys ocean ( sptl , 1989 ) . pak and schauberger ( 1981 ) postulated , based on s - isotopy , that the rock types rotsalzgebirge ( = red salt rock ) and grntongebirge ( = green salt - bearing clay ) ( following the nomenclature of schauberger , 1986 ) of an older phase ( schauberger , 1986 ) were deposited during late permian ( probably until earliest triassic times ) , while the stinkdolomitisch / anhydritische grausalzgebirge ( bituminous dolomitic / anhydritic grey salt rock ) and probably also the bunte salztongebirge ( = colored salt - bearing claystone ) of a younger phase ( schauberger , 1986 ) were sedimented during the early triassic and earliest anisian times . for hallstatt , pak and schauberger ( 1981 ) and pak ( 1981 ) assumed a continuous saliniferous sedimentation from the upper permian to the lower triassic / anisian boundary . ( krystyn , 2008 ) are often associated with jurassic rocks ( sandlingalm fm . ) . , are covered by an undeformed lid of upper jurassic rocks ( gawlick and schlagintweit , 2006 ; suzuki and gawlick , 2009 ) . a sedimentary contact between the sandlingalm fm . and the overlying plassenkalk fm . is missing ( suzuki and gawlick , 2009 ) . on a map view of hallstatt , the lower juvavic unit , the rocksalt and the jurassic cover are found within the area of the upper juvavic nappe ( fig . 2 ) . based on vitrinite reflectance studies , maximum temperatures for the central sectors of the nca in the surroundings of the hallstatt mine are estimated between 160 and 180 c , and with 200300 c in the surroundings ( see table 1 , gtzinger and grum , 1992 ; gawlick et al . , 1994 ; wiesheu and grundmann , 1994 ; wiesheu , 1997 ; sptl and hasenhttl , 1998 and references therein ) . proposed by schauberger ( 1986 ) , polyhalite is mainly part of the rotsalzgebirge ( red salt rock ) , which is composed of a mixture of reddish - grey salt , anhydrite , polyhalite , glauberite , na mg sulphates ( like bldite / simonyit ) , red and black claystones and grey - brown sandstones . there , polyhalite ( and bldite [ na2mg(so4)2.4h2o ] ) are very common but in low proportions ( schauberger , 1986 ) . but polyhalite is also reported from the rock types buntes salztongebirge ( colored salt - bearing clay ) , grausalzgebirge ( grey salt - bearing claystone ) and rot - grnes bergangsgebirge ( reddish - green salt rock ) ( nomenclature after schauberger , 1986 ) . most of the described 27 samples were taken from the lower and middle levels of the hallstatt salt mine : kaiser franz josef , nusko , kaiserin elisabeth , kaiserin maria theresia and kaiserin christina . for an overview of the sample locations see supplementary material , table sm 1 and fig . 2 , fig . 3 , fig . 4 . electron microprobe analysis empa on polyhalite , anhydrite , bassanite and bldite was carried out on a jeol electron microprobe ( jxa-8600 ) at the department geography and geology , university of salzburg , using a wavelength dispersive system . because sulphates are unstable under the electron beam , we used an acceleration voltage of 15 kv and a low sample current of 20 na . natural and synthetic mineral standards were used to calibrate the microprobe and raw data was reduced using standard zaf correction . the detection limits ( 3 ) for the elements na , k , mg , mn , ca , fe and s are 0.025 wt ( weight ) % and 0.045 wt% for sr and ba . after being covered with carbon the thin section blocks of the three samples ht-8b , ht-10 and ht-16 were examined by scanning electron microscopy ( sem ) and qualitative energy dispersive x - ray analysis ( edx ) with the leica stereoscan 430 equipped with rntec - edwin 98 winshell / wintools at the department geography and geology , university of salzburg . the x - ray diffraction analysis was carried out with the automatic diffractometer siemens d500 using a semi - automatic divergence slit and a graphite - based secondary monochromator using cu k-radiation ( 40 kv , 35 ma ) . the measurements were performed in the step - scan mode in an angular range of 375 2 , step length 0.03 with a measurement time of 12 s per step . the program eva 3.0 ( bruker axs ) and the pdf-2-database ( international centre for diffraction data ) were used for evaluation of the x - ray diffraction results . in the hallstatt mine , polyhalite rocks occur in tectonic lenses within the protocataclasite to protomylonite matrix . thin section analysis of hallstatt samples reveals many different fabric types ( table 2 , supplementary material , table sm 1 ) . very often , one rock sample exposes more than one polyhalite microfabric type . ( in press b ) , although their polyhalite rock type 2a ( porphyroblasts of polyhalite in anhydrite , round blasts ) only occurs subordinately . in the following , we distinguish between mono- and polymineralic polyhalite rocks , and furthermore , vein and cavity - filling polyhalite ( table 2 , supplementary material , table sm 1 ) . the thin sections of monomineralic polyhalite mylonites ( fig . 5 , fig . 6a and b ) show the following main features and are often characterised by large grain size variations ( from 0.05 to 0.5 mm long ) : ( 1 ) fine - grained ( 0.05 0.1 mm on average ) , well - recrystallised elongated ( long axis parallel to the foliation ) polyhalite grains with a shape preferred orientation ( spo ) ; ( 2 ) fibrous polyhalite pressure fringes ( 0.05 0.5 mm on average ) around large , rigid subeuhedral opaque mineral grains ( 0.5 0.5 mm up to maximally 20 20 mm ) within a fine - grained mylonitic fabric ( average grain sizes of 0.02 0.1 mm ) ( fig . 6a ) and ( 3 ) fine - grained ( 0.03 0.1 mm on average ) grain boundary migration ( gbm ) with amoeboid grain boundaries ( fig . crenulation cleavage usually shows a two - phase fabric indicating two different deformation stages and these structures can be found in fine - grained polyhalite rocks ( fig . 6c and d and 7a ) of mono- und polymineralic polyhalite rocks show a secondary overprint by a second deformation stage . such features include : ( 1 ) a refolded foliation in a fine - to - medium - grained ( ca . 0.1 0.3 mm long ) polyhalite rock with a crenulation cleavage ; ( 2 ) microfolds folding the foliation s1 of polyhalite grains ( fig . 6c ) and ( 3 ) a crenulation cleavage and partly folded clay - rich layers , coarse - grained ( 0.1 0.2 mm long on average ) polygonal anhydrite fabric and fine - grained ( 0.02 0.07 mm long on average ) polyhalite ( fig . 5b ) from hallstatt show various effects of recrystallisation , respectively annealing ( fig . 7a c ) : fine - grained ( 0.02 0.1 mm long on average ) , fibrous polyhalite grains partly show crenulation cleavage (= microfabric type a-2 ) and overgrow coarse - grained ( 0.5 1.54 mm long on average ) polyhalite crystals typical for microfabric type a-3 ( fig . coarse - grained , recrystallised polyhalite aggregates ( 0.7 0.9 mm up to 1 2.5 mm long ; single fibres on average 0.2 0.6 mm long ) are in contact with coarse - grained anhydrite ( 0.1 0.2 mm on average ) ( fig . 7b ) and well - recrystallised polyhalite grains still preserve a shape preferred orientation ( spo ) ( fig . 5c ) a number of effects related to pressure solution can be observed ( fig . 7d f ) : folded dark clay - rich layers and very fine - grained polyhalite grains ( 0.02 0.05 mm on average ) , showing pressure solution and grain boundary migration ( = gbm ) . pressure solution foliation of very fine - grained ( 0.01 0.05 mm on average ) polyhalite grains surrounding coarse - grained ( 0.1 0.1/0.2 mm on average ) polyhalite crystals ( fig . 7d ) . pressure solution of extremely fine - grained ( 0.001 0.1 mm representing an ultramylonite ) polyhalites and more coarse - grained polyhalite grains ( 0.05 0.1/0.2 mm long on average ) of sample ht-16 ; the layer in the center represents a mylonite ( microfabric type a-1 ) with a polyhalite fish ( 0.05 0.3 mm ) ( fig . in other cases , pressure solution of extremely fine - grained ( 0.01 0.1 mm on average ) polyhalites around a coarse - grained polyhalite twin ( 0.2 0.4 mm long ) is observed ( fig . cataclastic fabrics of angular fine and coarse ( 0.0050.08 mm ) polyhalite grains with serrated grain boundaries and many grain size classes ( fig . 8a ) of microfabric type a-5 are only observed in sample ht-6 ( fig . generally , the presence of other mineral phases inhibits the mineral growths , resulting in large grain size variations ( if no phases are removed ) . some samples from hallstatt show fine - grained polyhalite ( 0.003/0.004 mm thick and up to 0.2 mm long ) with shape preferred orientation ( spo ) overgrowing older , large anhydrite porphyroblasts ( fig . the anhydrite porphyroblasts ( 0.4 0.6 mm ) show a sieve structure with polyhalite inclusions ( with no shaped preferred orientation ) and crystallographic preferred orientation ( cpo ) within the anhydrite grain . this indicates that the fabric formed prior to the growth of anhydrite porphyroblasts did not show an spo or cpo ( pre - tectonic porphyroblast ) ( fig . folded clay - rich layers and an extremely fine - grained ( 0.001 0.01 mm ) polyhalite - rich matrix are observed in some polyhalite rocks . fabric type b-1 shows a typically mylonitic fabric of polyhalite layers interbedded with clay , carbonate or anhydrite layers . 8c e ) of polymineralic mylonites show a number of distinct features , which include inter alia coarse - grained ( 0.1 0.4 mm on average ) anhydrite layers with grain boundary migration ( gbm ) , which are embedded in fine - grained ( 0.003 mm thick and up to 0.2 mm long ) mylonitic polyhalite layers of microfabric type a-1 ( whole sample : microfabric type b-1 ) ( fig . fine- ( 0.1 0.15/0.3 mm on average ) and coarse - grained ( 0.2 0.5 mm on average ) polyhalite layers of another sample show a foliation and amoeboid grain boundaries as well as a pressure solution , probably aligned to microfabric type b-1 ( polymineralic mylonite ) ( fig . clay - rich layers are embedded in a fine - grained ( 0.01 0.05 mm on average ) mylonitic polyhalite fabric with shape preferred orientation ( spo ) , together constituting microfabric type b-1 ( fig . reaction fabrics are commonly characterised by the control of the orientation of one mineral by another . they might have been formed by exsolution , ( re-)crystallisation , parallel growth or replacement ( lauder , 1961 ) . the yellow - coloured mineral aggregates are composed of bldite [ na2mg(so4)2.4h2o ] , the red ones of polyhalite and large , red anhydrite ( fig . fine - grained ( 0.05 0.1/0.2 mm on average ) , fibrous polyhalites ( fig . 8f ) probably grew in 0.5 1.2 mm large areas as a reaction product between coarse - grained bldite and a large ( 2 2.5 mm ) anhydrite ( single crystal ) , indicating static p bldite constitutes large areas ( 17 20 mm ) , with single grains measuring between 1 1 mm and up to 4 8 mm . mainly polyhalite or anhydrite , subordinate clay and carbonate minerals , which are growing into cavities , represent cavity infilling . commonly , large polyhalite fibres ( fibrous aggregates : 0.1/0.5 2.5/3 mm ; single fibres are only 0.020.1 mm thick ) grew into cavities , which were filled afterwards by halite ( see also figs . in other cases , a cavity filled with halite and coarse - grained ( 0.1 0.2/0.4 mm on average ) , polygonal anhydrite grains occurs in a matrix of fine - grained ( 0.03/0.04 0.2 mm ) , folded polyhalite grains ( fig . furthermore , polyhalite and anhydrite ( 0.05 0.1/0.2 mm on average ) grains grew together into halite - filled cavities . 9d ) and is consistent with polyhalite type 1 ( fibrous polyhalite in veins ) described by leitner et al . the vein - filling polyhalite samples of altaussee and berchtesgaden described by leitner ( 2011 ) and leitner et al . sample hst-21 does not show a well - developed median suture line , which means that the large ( 0.2 1 mm on average , up to 0.2 1.5 mm ) , in part sheaf - shaped polyhalite fibres ( following the description of leitner , 2011 ) , which are orientated about perpendicular to the vein walls of clay and mudstone , have grown in only one direction ( fig . three fine - grained samples ( ht-8b , ht-10 and ht-16 ) were examined by sem and qualitative edx measurements in order to check the nature of fine - grained minerals . polyhalite ultramylonite between coarse - grained magnesite breccia fragments , with halite cavity - filling and fine - grained dark carbonate or clay schlieren / layers ( fig . sample ht-8b represents a recrystallised polyhalite rock with many questionable clay schlieren and layers and salt cavity filling . rock sample ht-10 contains polyhalite mylonites , rare anhydrite , questionable fine - grained polyhalite clay layers / schlieren and halite cavity filling . similar to samples ht-8b and ht-16 the dark , fine - grained schlieren of sample ht-10 ( fig . 10b ) contain a polyhalite clay mineral mixture composed of s , mg , k , ca , si , al and fe . the elements si and al argue particularly for the presence of a clay mineral ( fig . measurements the light - grey breccia fragments of sample ht-16 are composed of the elements mg , c , o and traces of fe , constituting the mineral magnesite ( fig . magnesite grains are isometric with an average size of 0.005 mm ( fig . furthermore , the sem - measurements clearly show that the fine - grained polyhalite fibres and coarse - grained polyhalites and anhydrites ( at least of sample ht-16 ) partly overgrow halite ( microfabric type c-1 ) indicating an older relative age compared to halite ( fig . electron microprobe ( emp ) measurements were performed on some polyhalite types ( samples ht-2 , ht-3 , ht-12 , ht-16 , ht-27 ( 2 ) and ht-30 ) , associated anhydrite and on bldite in order to characterize compositional variations during various stages of mineral growth ( see table 3 ) . polyhalite grains of the microfabric types a-1 , a-4 , b and c-1 show only minor variation in the polyhalite composition ( see also supplementary material , table sm 2 ) . only the sro abundances exhibit some minor variation which is usually between 0.05 and 0.28 wt ( weight ) % . in samples ht-12 and ht-16 , the sro content is slightly elevated ranging from 0.28 to 0.74 wt% . the na2o content ranges between 0.025 and 0.48 wt% , and is increased up to 1.80 respectively to 4.1 wt% in sample ht-12 ( although a measurement error is conceivable as well ) . a reaction fabric of bldite , a polyhalite seam and associated anhydrite is observed in sample ht-12 , representing a metamorphic reaction fabric ( fig . the presence of bldite was found by both emp analysis ( table 3 ) and x - ray diffraction ( supplementary material , fig . bldite of the haselgebirge fm . may occur primarily as nodules or more common intergrown with the mineral lweite ( schauberger , 1986 ) , which has a nearly identical chemical composition and is stable at higher temperatures . in sample ht-3 , fibrous bassanite ( caso4.0.5h2o ) partial exsolution from an unidentified small mineral of round shape within polyhalite is observed in sample ht-16 ( fig . microprobe measurements demonstrate a nearly polyhalite - like chemistry ( supplementary material , fig . in this chapter , we first highlight the significance of various polyhalite fabrics and try to establish the timing of tectonic events by comparison with dated polyhalite fabrics from other alpine salt deposits ( leitner et al . , in press a , b ) as unfortunately no ar / ar ages are available from hallstatt . then , we compare these fabrics with such from calcite and quartz , and , finally , we discuss the regional implications of the new observations . finally , we debate the formation conditions of some of the observed particular mineral compositions , which in part allow us to deduce formation temperatures and the secondary origin of polyhalite . the examined polyhalite samples of hallstatt show a wide variety of microfabrics and many of them , the mylonitic ones , resemble closely to such observed from rock - forming constituents of continental crust , like quartz , feldspar and calcite . similarly , the microfabrics may have resulted from a wide range of external conditions like temperature , strain rate and differential stresses ( passchier and trouw , 2005 ) . the aim is now also to deduce conditions of formations of these microfabrics within the temperature stability field of polyhalite . we combine here all observations and show models for the suggested mode of origin in fig . the vein - filling , fibrous polyhalite samples of microfabric type c-2 ( hst-21 ) and probably some of microfabric type c-1 ( e.g. ht-2 , ht-3 ) are expected to show the oldest ages . these rocks can be compared with the fibrous polyhalites in veins ( polyhalite rock type 1 ) of leitner et al . ar / ar age dating of similar types mainly yielded ages between 208 and 235 ma . thus , 2025 million years after the deposition of the haselgebirge fm . during the main extensional stages of the nca and a maximum overburden of 800 m ( rantitsch and russegger , 2005 ) migrating brines of likely hydrothermal origin were reacting with the haselgebirge sulphates to form polyhalite . following the interpretation of leitner ( in press b ) and leitner ( 2011 ) , these circulating brines and heating were probably generated by the opening of the meliata ocean ( rifting ) , which lasted until 225 ma ago . during recrystallisation , temperatures of 6090 c might have been reached as authigenic quartz originating from a transformation of mudrock minerals indicates ( sptl et al . , 1998a , b ) . some of the fibrous polyhalites of altaussee and berchtesgaden yield ar / ar age steps of 204209 ma , which can probably be explained by a separate polyhalite growing event during norian times , when subsidence took place in the sedimentation area ( and the 1.5 km thick dachstein limestone was deposited over the haselgebirge fm . the veins might have formed due to extension during middle triassic times . during evaporation of seawater the polyhalite fibres probably formed by back reactions of the k mg cl so4 brine ( the k and mg might originate from pre - existing clay minerals or clay stones ) with earlier formed gypsum , anhydrite or glauberite ( inter alia warren , 2006 ) on the vein walls ( fig . the polyhalite mylonites ( e.g. samples ht-1 , ht-11 , ht-30 ) of microfabric types a-1 and b-1 were most likely formed along shear zones . these foliated and lineated rocks ( fig . 5a and b ) can be characterised by their comparatively small grain sizes ( varying from very fine - grained ultramylonitic to medium - grained fabrics ) and distinct penetrative or closely spaced and planar foliation with a shape preferred orientation ( spo ) . porphyroclasts , derived from a relatively rigid material , are quite common for mylonites , which usually indicate one deformation stage . so4 brines infiltrated ( upwards and downwards ) the evaporite rock along opened faults and joints and polyhalite - forming brine - back reactions took place . the mylonites were formed at high strain rates due to intracrystalline deformation , may have developed from a more coarse - grained precursor metamorphic rock and probably suffered several stages of deformation . the low crystallographic preferred orientation ( cpo ) of polyhalite may indicate some recrystallisation postdating mylonitisation . furthermore , the mylonites are partly overprinted by recrystallised polyhalite grains ( microfabric type a-3 ) , which probably indicate a static growth . these polyhalite mylonites can probably be compared to polyhalite rock type 3a ( fine - grained , mylonitic polyhalite rock ) and 3b ( non - foliated polyhalite rock with local shear zones ) described by leitner et al . ar / ar age dating shows two stages of growth of fine - grained polyhalite 152158 ma , the later between 105 and 118 ma ( leitner et al . ages between 152 and 158 ma may be related to the closure of the meliata ocean and the initiation of alpine nappe stacking and accompanying ductile deformation ( leitner et al . furthermore , these ages correlate well with the 145154 ma old authigenic feldspar reported from moosegg quarry by sptl et al . ( 1996 , 1998a , b ) . a second age of 105118 ma of fine - grained polyhalites in shear zones is related to cretaceous - aged deformation ( low - grade metamorphic ductile overprint during eo - alpine nappe stacking ) and corresponds well to ar / ar white mica ages of 114120 ma reported from the southern margin of the nca ( frank and schlager , 2006 ) . the overburden must have been relatively high , which contradicts the small overburden deduced by fluid inclusion studies . the argon loss of the fine grains of mylonitic polyhalites at about 44 ma is explained by leitner et al . ( in press b ) by a low temperature thermal overprint in the eocene , which was postulated inter alia by hejl and grundmann ( 1989 ) and pueyo et al . ( 2007 ) due to a regional thrusting processes in the nca ( for example , linzer et al . , 1995 ) . ( in press b ) published a wide range of ar / ar age data ( 15844 ma ) for this rock type the mylonitisation was most likely induced by well reported early cretaceous thrusting events ( mandl , 2000 ; schorn and neubauer , 2011 and references therein ) ( fig . 12 ) because of nearly complete resetting of the ar isotopic system at 113 ma ( leitner et al . the polyhalite samples showing crenulation cleavage and folds ( microfabric type a-2 ) , experienced a microfolding of the existing foliation and , therefore , at least two different deformation stages . the recrystallised polyhalites of microfabric type a-3 were formed due to healing processes of the fabric after the main deformation process . crenulation cleavage is commonly induced by microfolding of an existing foliation ( e.g. a slaty or phyllitic cleavage ) , typically due to shortening of foliation about parallel to its layering . many samples , especially polyhalite mylonites ( of microfabric type a-1 ) , also show pressure solution of microfabric type a-4 , which indicates high strain rates , the presence of intergranular fluids and a second deformation stage under lower p t - conditions . so4 brines infiltrated polyhalite rocks ( upwards and downwards ) along joints and faults , which were opened during several tectonic deformation stages ( some of them were probably the same events which were also responsible for the mylonitisation and cataclastic fabrics ) and induced the polyhalite recrystallisation ( microfabric types a-2 , a-3 and a-4 ) at elevated temperatures ( fig . the microfabric type a-5 ( sample ht-6 ) has to be relatively young ( compared with the other microfabric types ) , otherwise its cataclastic fabric would not have been preserved . in the course of the mylonitisation during early cretaceous thrusting , or in younger geological times , a cataclastic deformation took place , which is still preserved in the boudinaged polyhalite anhydrite layer and the cataclastic polyhalite microfabric ( type a-5 ) of sample ht-6 . it is characterised by equidimensional angular grains of variably size ( 0.0050.08 mm ) and areas with secondary grain size reduction . this fabric was formed due to relatively high strain rates , high fluid pressures at non - metamorphic to very low - grade metamorphic conditions . so4 brines were circulating in the cataclastically opened voids , inducing / generating the polyhalite precipitation ( fig . the polyhalite grains , which marginally grow into halite - filled cavities ( microfabric type c-1 , e.g. samples ht-2 , ht-3 , ht-16 , fig . 9a ) , are definitely younger than the microfabric types c-2 , a-1 and b-1 and probably also younger than a-2 , a-3 and a-4 . otherwise , the salt filling could not have been preserved until now . the k mg cl so4 brines may have infiltrated the rock ( upwards and downwards ) after the main alpine deformation stages along already opened cracks or joints ( fig . the metamorphic reaction fabric ( microfabric type b-2 ) of sample ht-12 is constituted by bldite , a fine - grained polyhalite seam and large anhydrite crystals . the decomposition reaction of bldite might have taken place at 61.4 c ( if a saturation in halite is assumed or 71 c without nacl - saturation ) or slightly above ( for a detailed discussion see chapter 7.3 ) . it is not clear whether this polyhalite type is younger or older than microfabric type c-1 or a-3 . however , as this decomposition reaction took place at relatively low temperatures , a large overburden can be excluded . therefore , this microfabric type is certainly younger than the microfabric types c-2 , a-1 and b-1 and probably also younger than a-2 , a-3 and a-4 types . as no ar however , microprobe measurements and photos show isomorphic polyhalite fibres and corroded anhydrite and bldite crystals ( fig . 11c and d ) , which strongly argue for a very young ( probably cenozoic ) polyhalite age ( in comparison with the other minerals ) and a progressive metamorphism at low temperatures and elevated pressures . so4 brines might have infiltrated the host rock along already existing cracks or faults ( of a former tectonic event ) . the polyhalite fibres were precipitated by brine - back - reactions with earlier formed gypsum or anhydrite ( fig . at least the microfabric types a-1 ( and b-1 , probably a-5 ) , b-2 , c-1 and c-2 formed during different geological events , which were accompanied by fluid migration . each of these brines , which infiltrated the haselgebirge evaporites showed a different chemical composition , which , as we expected at the beginning of our study , would result in the precipitation or formation of polyhalite rocks with an appropriate and varying chemical composition . but microprobe measurements only showed a very small chemical variation ( supplementary material , fig . sm 2 ) of polyhalite samples from microfabric types a-1 , a-3 , a-4 , b , b-2 , c-1 and c-2 ( table 3 , supplementary material , table sm 2 ) . obviously , brines of different chemical compositions within the haselgebirge precipited polyhalites of nearly the same mineral chemistry . the examined polyhalite samples of hallstatt show a wide variety of microfabrics and many of them , the mylonitic ones , resemble closely to such observed from rock - forming constituents of continental crust , like quartz ( stipp et al . , 2002 ) , similarly , the microfabrics may have resulted from a wide range of external conditions like temperature , strain rate and differential stresses ( passchier and trouw , 2005 ) and the temperatures at hallstatt remained low ( < 250 c ) . we also note that recrystallisation and annealing plays an important role veiling preferred crystal lattice patterns , which could not observed in or mylonitic samples . the influence of migrating brines to various microfabrics ( see discussion in following sections ) is more obvious in polyhalite rocks than in usual continental rocks with their predominance of quartzite , feldspar and calcite . in oceanic salt deposits , bldite is mainly formed secondarily from kieserite due to solution metamorphism and often observed in kainite hats or associated with halite , lweite and mg sulphates ( braitsch , 1962 ) and may also precipitate primarily from normal marine water ( braitsch , 1962 ; zayani et al . , 1999 ) , na2so4-rich salt lakes or nitric acid deserts . in the haselgebirge fm . of the eastern alps , polyhalite intergrown with bldite ( samples ht-12 and ht-15 , ht-27 ) was , inter alia , described by grgey ( 1909 ) from hall in tirol . the lower crystallisation temperature of bldite is given as 6.26.4 c ( under laboratory conditions ) in the presence of halite ( autenrieth and braune , 1960a ; charykova et al . , 1992 ) ( 4.5 c van't hoff , 1912 ) or 20.0 c in the ternary system na2so4mgso4h2o ( charykova et al . , 1992 ) . the upper formation temperature ranges between 61.4 c ( with nacl - saturation ) ( autenrieth and braune , 1960b ) and 71 c ( without nacl - saturation ) ( van't hoff , 1912 ) . this decomposition reaction of bldite and probably anhydrite , which was observed in sample ht-12 ( microfabric type b-2 ) might have taken place , therefore , at 61.4 c the upper formation temperature of bldite , if a halite saturation is assumed ( or 71 c without nacl - saturation ) or slightly above . the polyhalite fibres of this metamorphic reaction fabric might have formed after the partly decomposition of the low temperature salt bldite . as bldite has formed at relatively low temperatures , this decomposition reaction might also indicate a weak static reheating of the rock , during tectonic processes in young geological ( probably cenozoic ) times and the age of ca . 44 ma found in the altaussee deposit may represent this event ( leitner et al . furthermore , this microfabric type might indicate , that the polyhalite rocks of the alpine haselgebirge fm . have been infiltrated by extraordinary na - rich brines , which induced the bldite formation at low temperatures , during late stages of the alpine deformation probably during cenozoic times . bldite sample ht-12 were extraordinarily rich in na ( table 3 , supplementary material , fig . autenrieth ( 1958 ) , who examined the six component system k na mg ca so4cl h2o , reported the formation of a new , metastable sediment na - polyhalite ( formula : 25caso4.2k2so4.3na2so4.15h2o ; autenrieth , 1958 , 1959 ) during polyhalite precipitation . after a while , these prismatic to rod - shaped , na - polyhalites are converted to fine - grained , milky to dumbbell - shaped polyhalite . in most cases na - polyhalite may be intermediary formed at temperatures above 50 c from gypsum or brines with high caso4-concentrations within nearly the entire stability field of polyhalite ( autenrieth , 1958 ) . polyhalite may only convert directly ( and quite fastly ) to na - polyhalite at temperatures above 50 c in the presence of mg - free or very mg - poor brines , which are saturated with respect to kcl and nacl ( autenrieth , 1958 ) or above 90 c in the presence of cacl2 and low mgcl2-concentrations ( autenrieth , 1959 ) . the tendency of na - polyhalite formation might increase with rising temperatures and is relatively high at 90 c ( autenrieth , 1959 ) . according to gudowius and hodenberg ( 1979 ) na - polyhalite shows structural similarities with the minerals bassanite ( caso4.0.5h2o ) and -caso4 , which was later verified by reisdorf and abriel ( 1987 ) . for further information on the stability of na - polyhalite , see supplementary material sm-4 . therefore , we conclude tentatively , that na - polyhalite was most likely formed at temperatures above 5090 c , most likely from gypsum or at very low mg - concentrations from polyhalite . metamorphic reaction fabric b-2 and might indicate another , probably older stage of polyhalite formation . as it forms no oceanic salt minerals it is very suitable as index element ( khn , 1968 ) . therefore , khn ( 1972 ) examined the rubidium contents of sulphate salts like sylvite , carnallite , langbeinite , leonite and polyhalite of several salt deposits . primary polyhalites usually show low rubidium contents of < 0.0001 wt ( weight ) % ( khn , 1972 ) , while secondary polyhalites are rubidium - rich and this observation may potentially allow distinction between primary and secondary polyhalite . the polyhalites of hallstatt constitute 0.0012 wt% rb ( khn , 1972 ; schauberger , 1986 ) , implying the reworking of rb - richer salts , and strongly arguing for a secondary origin of the polyhalites within the haselgebirge fm .. our examined hallstatt samples did not show high enough rubidium concentrations for microprobe measurements . due to the lack of sedimentary structures during thin section analysis leitner et al . ( in press b ) also proposed a clearly secondary origin of the polyhalites of the alpine haselgebirge . according to our microprobe measurements the sro contents of the polyhalite of hallstatt are slightly elevated and range from 0.28 to 0.74 wt ( weight ) % . the variation likely originates from exchange with anhydrite , which has a higher sro content ( schauberger , 1986 ) . the sr content of all examined anhydrite samples of the alpine salt deposits mainly ranges between 0.1 and 0.2% ( ruscha , 1976 , unpublished data quoted from schauberger , 1986 ) or 0.305% for hall in tirol ( sptl , 1988b ) or even lower , strongly arguing for a diagenetic and secondary formation of the alpine anhydrites ( due to conversion of gypsum , which was formed in the caso4-phase ) ( schauberger , 1986 ) . strontium ions of the seawater may isomorphously replace ca and sr ( due to a linear function ) in the crystal lattice of aragonite , calcite , anhydrite and gypsum ( usdowski , 1973 ) . according to usdowski ( 1973 ) anhydrite , which was formed by conversion of gypsum , shows average sr - contents of 0.2 wt% , while primary anhydrite constitutes sr - concentrations as high as 2.9 wt% . we conclude , therefore , that the relatively low sr content of polyhalite is of secondary origin . the magnesites of the haselgebirge fm . from the eastern alps are of secondary origin and may have formed by exchange reactions between mg - bearing solutions and primary low mg - carbonates during several stages of diagenesis or metamorphism ( niedermayr et al . , 1989 ) . within the haselgebirge fm . , fine - grained magnesite is one of the major minerals of the carbonate fraction ( niedermayr et al . , 1989 ) . magnesite ( mgco3 ) is inter alia reported from the evaporitic claystone of hallstatt salt mine ( schauberger , 1986 ) . sptl ( 1988a ) described traces of magnesite ( by x - ray diffraction ( xrd ) measurements ) in dolomitic limestones of a lower triassic bituminous dolomitic / anhydritic grey salt rock profile in the level kaiser franz josef . within the alpine haselgebirge fm . , magnesite may have formed by epigenetic saliniferous processes ( which means post - sedimentary - diagenetic recrystallisation ) ( schroll , 1961 ; schauberger , 1986 ) . sptl ( 1988a ) suggested that the examined succession of fossil - free , bituminous , laminated siltstones , anhydrites and carbonates of the level kaiser franz josef ( hallstatt ) might have been deposited in a subtidal , hypersaline and anaerobe basin milieu ( sptl , 1988a ) . ( during ca - sulphates and salt precipitation ) by the infiltration and percolation of mg - bearing brines in the basin center , which induced the dolomite and even magnesite formation of carbonate sediments . salt diapirism and several alpine metamorphic stages may have led to mobilisation and recrystallisation of predominantly coarse - grained magnesite ( of the nca ; see also ebner et al . , 2000 ) . we conclude therefore , that the fine - grained magnesite associated with polyhalite is of secondary origin . in this paper , we present for the first time a polyhalite microfabric classification particularly to the hallstatt salt mine , which shows a much wider variety of fabrics as reported in recent works from other salt mines in the eastern alps ( leitner et al . , in press a , b ; leitner , 2011).(1)polyhalite microfabrics from hallstatt include : ( 1 ) vein - filling , fibrous polyhalite of likely middle to late triassic age , ( 2 ) polyhalite mylonites of early cretaceous age , ( 3 ) metamorphic reaction fabrics of a post - early cretaceous age and ( 4 ) cavity - filling polyhalite of uncertain age.(2)the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock.(3)the mylonites are partly overprinted by recrystallised , statically grown polyhalite grains.(4)metamorphic reaction fabrics ( microfabric type b-2 ) of fine - grained polyhalite seams , which have grown due to the decomposition of bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports , bldite may occur primarily as nodules or , more commonly , intergrown with lweite , which has a nearly identical chemical composition and is stable at higher temperatures . reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . this fabric type was only found in one sample in relation with bldite , and has a formation temperature of 6.261.4 c in the presence of halite.(5)for the first time , we demonstrate that microfabrics like mylonitic shear fabrics , porphyroclast systems , annealing etc . , which are described for other minerals like quartz , etc . , are also preserved in relatively rare sulphates like polyhalite , which is stable between low temperatures and ca . 255 c.(6)in general , coarse - grained polyhalite grains or twins in a fine - grained polyhalite matrix ( microfabric type a-4 ) were only observed in samples from hallstatt . the cataclastic polyhalite microfabric a-5 was also only found in one hallstatt sample ( ht-6 ) and the metamorphic reaction fabric b-2 was also reported from one polyhalite polyhalite microfabrics from hallstatt include : ( 1 ) vein - filling , fibrous polyhalite of likely middle to late triassic age , ( 2 ) polyhalite mylonites of early cretaceous age , ( 3 ) metamorphic reaction fabrics of a post - early cretaceous age and ( 4 ) cavity - filling polyhalite of uncertain age . the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock . metamorphic reaction fabrics ( microfabric type b-2 ) of fine - grained polyhalite seams , which have grown due to the decomposition of bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports , bldite may occur primarily as nodules or , more commonly , intergrown with lweite , which has a nearly identical chemical composition and is stable at higher temperatures . reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . this fabric type was only found in one sample in relation with bldite , and has a formation temperature of 6.261.4 c in the presence of halite . for the first time , we demonstrate that microfabrics like mylonitic shear fabrics , porphyroclast systems , annealing etc . , are also preserved in relatively rare sulphates like polyhalite , which is stable between low temperatures and ca . 255 c . in general , coarse - grained polyhalite grains or twins in a fine - grained polyhalite matrix ( microfabric type a-4 ) the cataclastic polyhalite microfabric a-5 was also only found in one hallstatt sample ( ht-6 ) and the metamorphic reaction fabric b-2 was also reported from one polyhalite

in the hallstatt salt mine ( austria ) , polyhalite rocks occur in 0.51 m thick and several metre long tectonic lenses within the protocataclasite to protomylonite matrix of the alpine haselgebirge fm .. thin section analysis of hallstatt polyhalites reveals various fabric types similar to metamorphic rocks of crust - forming minerals , e.g. quartz and feldspar . polyhalite microfabrics from hallstatt include : ( 1 ) polyhalite mylonites , ( 2 ) metamorphic reaction fabrics , ( 3 ) vein - filling , fibrous polyhalite and ( 4 ) cavity - filling polyhalite . the polyhalite mylonites contain a wide range of shear fabrics commonly known in mylonitic quartzo feldspathic shear zones within the ductile crust and developed from a more coarse - grained precursor rock . the mylonites are partly overprinted by recrystallised , statically grown polyhalite grains . metamorphic reaction fabrics of polyhalite fibres between bldite ( or astrakhanite ) [ na2mg(so4)2.4h2o ] and anhydrite have also been found . according to previous reports , bldite may occur primarily as nodules or intergrown with lweite . reaction fabrics may have formed by exsolution , ( re-)crystallisation , parallel growth or replacement . this fabric type was only found in one sample in relation with the decomposition of bldite at ca . 61 c in the presence of halite or slightly above , testifying , therefore , a late stage prograde fabric significantly younger than the main polyhalite formation .

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oxidative stress can be described as an imbalance between the production of free radicals and antioxidant defence . over the last decades , it has become amply evident that oxidative stress , usually in the form of reactive oxygen species ( ros ) , is a critical pathogenic factor in the development of several systemic diseases [ 1 , 2 ] . therefore , inhibition of ros formation , scavenging of ros , or interfering with ros pathogenic signaling pathways might be the potential ways to protect against oxidative stress - induced ailments . propofol ( 2,6-diisopropylphenol ) ( figure 1 ) , a highly lipid - soluble anaesthetic , is widely used for induction and maintenance of general anaesthesia . propofol ameliorates oxidative injury in several organs , including the heart , lungs , brain , liver , and testis . propofol is chemically similar to the endogenous antioxidant -tocopherol ( vitamin e ) and , theoretically , it should have similar properties . it is not surprising therefore that many studies have demonstrated antioxidant effects of propofol in vitro [ 1012 ] and in vivo . propofol has also been shown to elicit antiapoptotic effect in human umbilical vein endothelial cells by acting as an antioxidant . to the best of our knowledge , the effects of propofol at high doses on the lipid profile , cardiovascular marker enzymes , and enzymatic and nonenzymatic antioxidant indices in rodents have not been determined . propofol also called disoprivan by astrazeneca ( switzerland ) used for this study was obtained from one of the coauthors who is an anaesthetist ( dr . olugbenga akinwonmi of the department of anaesthesia , university college hospital , ibadan , nigeria ) . glutathione , hydrogen peroxide , 5 , 5-dithios - bis-2-nitrobenzoic acid ( dntb ) , and epinephrine were purchased from sigma chemical co. , saint louis , mo , usa . other chemicals were of analytical grade and purest quality available . inbred male wistar rats weighing between 200 and 220 g were purchased from the central animal house located in the department of physiology , university of ibadan , nigeria . the animals were kept in well - ventilated cages at room temperature ( 2830c ) , and maintained on laboratory chow ( ladokun feeds , ibadan , nigeria ) and water ad libitum . rats handling and treatments protocol conforms to the guidelines of the faculty of basic medical sciences , university of ibadan animals ' ethical committee as well as the national institute of health ( nih publication 85 - 23 , 1985 ) for laboratory animals ' care and use . twenty - one male albino rats ( wistar strain ) were randomly distributed into three groups of seven animals each . the first group served as the control and was given corn oil ( vehicle for the drug ) . the second group received propofol at a dose of 2 mg / kg body weight ( equivalent of human therapeutic dose ) , and the third group received propofol at a dose of 4 mg / kg body weight ( twice therapeutic dose ) . propofol was prepared with corn oil and was administered intraperitoneally three times in a week for four consecutive weeks . twenty - four hours after the last dose of the drug , the animals in each group were sacrificed by cervical dislocation , and blood was collected from the inferior vena cava of the heart into plain centrifuge tube . blood was allowed to stand for 1 h and was then centrifuged at 3000 g for 15 min in a bench centrifuge to obtain serum . the serum samples were used for the analysis of biochemical indices and lipid profile of the animals . liver and kidney from the animals were quickly removed and washed in ice - cold 1.15% kcl solution , dried , and weighed . the tissues were homogenized in 4 volumes of 50 mm phosphate buffer , ph 7.4 , and centrifuged at 10,000 g for 15 min to obtain postmitochondrial fraction ( pmf ) of the liver and kidney , which were used for assay of the antioxidant parameters . protein levels in the samples were assayed by the method of lowry et al . using bovine serum albumin as standard . the activities of serum alanine and aspartate aminotransferases ( alt and ast ) were assayed by the combined methods of mohun and cook and , reitman and frankel . pmf lipid peroxidation levels were assayed by the reaction between 2-thiobarbituric acid and malondialdehyde , an end product of lipid peroxides , as described by walls et al . . pmf - reduced glutathione ( gsh ) level was assayed by measuring the rate of formation of chromophoric product in a reaction between 5,5-dinitrobis-2-nitrobenzoic acid and free sulphydryl groups at 412 nm as described by moron et al . . pmf superoxide dismutase ( sod ) activity was measured by the nitro blue tetrazolium reduction method of mccord and fridovich . pmf catalase ( cat ) activity was assayed spectrophotometrically by measuring the rate of decomposition of hydrogen peroxide at 240 nm as described by aebi while glutathione - s - transferase ( gst ) activity was determined by the method of habig et al . . the activities of serum creatinine phosphokinase ( ck - mb ) were estimated using immune - inhibition kinetic assay according to the method of okinaka et al . . lactate dehydrogenase ( ldh ) activities in the serum were determined according to the method of zimmerman and weinstein . the total bilirubin levels were assayed by the method of rutkowski and debaare while serum creatinine and urea were estimated by the methods of jaffe and talke and schubert , respectively . serumtriglyceride ( tg ) and total cholesterol ( tc ) levels were assayed using commercial diagnostic kits ( randox ) . for the determination of hdl level , vldl and after centrifugation , the supernatant containing the hdl fraction was assayed for cholesterol using randox diagnostic kit . ldl - cholesterol ( ldl - c ) was calculated using the formula of friedewald et al . . data were analyzed using one - way anova followed by the post - hoc duncan 's multiple - range test for analysis of biochemical data using spss version 10.0 ( spss inc . reactive oxygen species ( ros ) attack cellular components containing polyunsaturated fatty acid residues of phospholipids , which are sensitive to oxidation . the peroxyl radicals formed by the attack can be rearranged via a cyclization reaction to endoperoxides which are precursors of mda , which subsequently lead to lipid peroxidation . lipid peroxidation ( lpo ) has been implicated in the pathogenesis of several injuries by many toxicants and may be responsible for cell membrane alterations which can result into enzymes leakage in animals . in this study , administration of propofol did not significantly ( p > 0.05 ) affect the levels of serum and renal and hepatic mda in the rats ( table 3 ) , indicating that the metabolism of propofol may not be linked to the generation of free radicals within the animal tissues . this observation is not strange since propofol has a very active phenolic ring within its structure which resembles -tocopherol and may release electron to terminate free radical reactions during its metabolism in the rats . however , table 2 shows that the levels of serum total cholesterol , triglycerides , and ldl - cholesterol were significantly ( p < 0.05 ) increased following administration of propofol . precisely , propofol at 2 and 4 mg / kg increased the levels of serum total cholesterol by 74% and 55% , triglycerides by 97% and 115% , and ldl - cholesterol by 45% and 73% , respectively . in addition , propofol at both doses significantly ( p < 0.05 ) decreased the levels of serum hdl - cholesterol in the rats by 41% and 54% . lipids are a heterogeneous group containing active metabolic substances that play an important role in the pathogenesis of drugs - induced liver disease . the most common lipid abnormalities during drug toxicity are hypercholesterolemia and hypertriglyceridemia , which was confirmed in this study . also , in propofol - treated rats , ldl - c was remarkably increased in the serum , while hdl - c was found to be reduced . the increased cholesterol level during propofol administration may be linked to increase in alpha - hydroxyl methyl glutaryl coa reductase activity , which is the rate limiting step in cholesterol biosynthesis . likewise , the increased triglycerides levels may be due to the increased availability of free fatty acid , glycerophosphates , decreased triglycerides lipase activity , or decreased fatty oxidation . in the present study propofol at 2 and 4 mg / kg increased the levels of hepatic gsh by 42% and 37% , respectively , when compared to the control ( table 3 ) . in addition , propofol at 2 and 4 mg / kg dose dependently and significantly ( p < 0.05 ) increased the activities of hepatic glutathione peroxidase by 1.9 and 2.1 folds and glutathione - s - transferase by 2.3 and 2.4 folds , respectively ( figure 3 ) . however , there were no significant ( p > 0.05 ) differences in the activities of renal and hepatic sod and cat of propofol - treated rats relative to controls ( figure 2 ) . reduced glutathione acts as a free radical scavenger and regenerator of alpha - tocopherol , and it plays a significant role in sustaining protein sulfhydryl groups in proteins and other key molecules . in addition , gsh may act as an essential cofactor for antioxidant enzymes , especially gpx and gst . the increase in the levels of hepatic gsh observed in this study may result in increased detoxifying and antioxidant capability of the liver . the gst and gpx are groups of multifunctional proteins that play a central role in the detoxification of electrophilic chemicals and the hepatic removal of potentially harmful hydrophobic compounds from blood . the increased activities of hepatic gst and gpx observed may partly be due to increase in their substrate or induction of de novo synthesis of the enzymes . importantly , propofol increased the levels of gsh and activities of gst and gpx in the liver and thus enhancing the detoxifying potential of the animals . the cardiotoxicity of xenobiotics can be evaluated using the serum activities of marker enzymes especially ldh and ck - mb , which are distributed throughout the body and have isoenzymes that are recognized as markers for muscle and heart lesion . results from this study showed that propofol at 2 and 4 mg / kg significantly ( p < 0.05 ) increased the activities of serum ldh by 1.7 and 1.8 folds and ck - mb by 2.0 and 2.1 folds , respectively ( figure 4 ) . in their studies , hayden and tyagi linked the increase in serum ck - mb and ldh of diabetic rats to cardiac muscular damage caused by the disease . similarly , wiernsperger stated that the activities of serum ck - mb and ldh are a measure of the state of necrosis in cardiac tissues . in view of the observed increase in ck - mb and ldh activities in propofol - treated rats administration of propofol at 2 and 4 mg / kg significantly ( p < 0.05 ) increased the level of total bilirubin by 52% and 53% , respectively , while serum urea , alanine and aspartate aminotransferases ( alt and ast ) , and creatinine levels were insignificantly ( p > 0.05 ) affected ( table 1 ) . it is established that ast can be found in the liver , cardiac muscle , skeletal muscle , kidney , brain , pancreas , lungs , leukocytes , and erythrocytes , whereas alt is predominantly present in the liver . also , serum creatinine and urea levels are the sensitive and reliable biochemical indices for evaluation of renal function in animal models . the insignificant effects of propofol on the levels of ast , alt , and creatinine indicate that the drug has no adverse effect on the hepatic and renal tissues of the rats . our data showed that propofol promotes the antioxidant status of the rats by increasing the levels of reduced glutathione and gsh - dependent enzymes . however , propofol elicited a detrimental effect on the lipid profile resulting in hypercholesterolemia which subsequently leads to abnormally high activities of serum creatinine phosphokinase and lactate dehydrogenase in the rats . the study , therefore , suggests that the use of propofol in patients with cardiovascular or lipid disorders should be carefully reviewed .

in recent years , the activity of anaesthetic propofol on biological processes has been attracting attention . the effect of propofol on biochemical indices in animals is unknown . in this study , we examined the effects of propofol on lipid profile , antioxidant indices , and cardiovascular marker ( cvm ) enzymes in rats . the study consists of three groups of seven rats each . group one received corn oil ( control ) while groups two and three received propofol ( doses of 2 and 4 mg / kg body weight , resp . ) . results showed that administration of propofol caused a significant ( p < 0.05 ) and dose - dependent increase in the levels of total bilirubin . propofol at 2 and 4 mg / kg increased the levels of serum total cholesterol by 74% and 55% , triglycerides by 97% and 115% , and ldl - c ( low - density lipoprotein - cholesterol ) by 45% and 73% , respectively , while hdl - c ( high - density lipoprotein - cholesterol ) decreased by 41% and 54% , respectively . propofol significantly ( p < 0.05 ) increased the levels of the hepatic reduced glutathione ( gsh ) and activities of gsh - dependent enzymes . propofol at 2 and 4 mg / kg increased the activities of cvm enzymes : lactate dehydrogenase by 1.7 and 1.8 folds and creatinine phosphokinase by 2.0 and 2.1 folds , respectively . taken together , propofol increased the levels of gsh and gsh - dependent enzymes but adversely affected the lipid profile of the rats .

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idiopathic hypogonadotropic hypogonadism ( ihh ) refers to gonadal dysgenesis due to deficiency of gonadotropin - releasing hormone ( gnrh ) release from the hypothalamus . the prevalence of ihh is about 1/10,000 in men and 1/50,000 in women with a m / f ratio of 4 - 5 : 1 . since ihh is rare , the number of ihh patients is small in any one geographical area . it is often difficult for a single clinical center to perform comprehensive assessment of the disease due to the limited number of cases . internationally , large - scale clinical studies on this rare disease are usually conducted in the form of multicenter trials . however , these kinds of studies are time consuming and require large amount of resources as well as close coordination among study groups . with the advances in information technology in the recent years , people have utilized networks for different research purposes . by december 2011 , the number of internet users in china had exceeded five hundred million . with more wide - spread availability of networks and the dramatic increase in the number of internet users , they form disease - specific social groups to share their knowledge and experience spontaneously , which makes it possible for medical researchers to conduct medical research through networks [ 3 , 5 ] . network investigation is a new modality of means of medical research that can cover large populations with low investigation costs , especially for investigation of a rare disease in a particular network social group . it can collect large numbers of clinical cases within a short period for the research purpose . in the present study , we collected the clinical data of ihh patients in china by interviewing ihh patients through network questionnaire and their medical records . we compared the obtained data with those reported in previous studies in the literature for the purpose of improving our understanding , diagnosis , and treatment of ihh . the kallmann liangjian social group is the largest ihh social networking group in china ( qq as the real - time network communication tool , id7685615 ) with 103 members . the items of the questionnaire investigation were designed by endocrinologists on the basis of the data obtained from literatures of ihh in china and abroad . the information obtained through the questionnaire survey includes the following : demographic characteristics , family history , age , clinical symptoms and laboratory tests at onset of disease , particular therapeutic protocols ( testosterone ( t ) , human chorionic gonadotropin ( hcg ) , human menopausal gonadotropin ( hmg ) , luteinizing - hormone - releasing hormone ( lhrh ) , or random combination of them ) , developmental stage of the testis and secondary sex characteristics , sexual function , level of sexual hormones , and quality - of - life ( qol ) rating . using the tanner classification pictures , patients made a self - assessment of the development of the external genital organs , pubic hair , and breasts . self - reported data was referred to the same standard , while the laboratory examination results and disease diagnosis were actually obtained from medical records . online questionnaire survey submission included electronic consent as part of the website log - on process . this study was approved by the institutional review board of huashan hospital in shanghai , china . a total of 75 questionnaires answering all of the 64 questions were obtained online between october 2011 and march 2012 . all of the participants were asked to provide copies of their medical records ; then , the records were examined by one endocrinologist , and only ihh patients were allowed to join the qq group . all activities and exchanges involved in the investigation were voluntary and free of charge . participants can submit the questionnaire survey only after all of the 64 questions were answered ; thus , there was no missing data for the investigation . all participants had to hand in their medical records for ihh and provide their contact means such as the e - mail address and telephone number , so that the investigators could check whether or not the information that the participants provided was authentic . finally , the computer system generated tables automatically , and the investigated data were stored in the data bank directly . differences between categorical variable groups were compared by chi - square test or bilateral probability of fisher 's exact test . scores for overall qol and correlations of clinical parameters were analyzed by spearman 's correlation analysis . all of the 74 patients participating in the ihh investigation were males with the mean age being 27.3 3.4 years ( ranging from 20 to 35 years ) . the mean age at disease onset was 15.2 6.6 years , and the duration of the disease ranged from 1 to 25 years with a mean of 12.1 7.2 years . the height of the 74 patients ranged from 157 to 188 cm with a mean of 174.4 6.9 cm . ( mean : 24.27 3.73 kg / m ) , belonging to overweight ( bmi of 24 kg / m is used as cutoff point for the chinese adults to define overweight ) . of the 74 patients , 28 ( 37.8% ) patients had a history of smoking ( 2 cigarettes per day on average ) . all patients had junior middle school or higher education level , 25 ( 33.8% ) patients had senior middle school education , and 37 ( 50% ) patients had college education . all the 74 patients were diagnosed with ihh , and 84% of them were diagnosed in general hospitals , 13.5% in district hospitals , and 2.7% in community hospitals . 42 ( 56.8% ) patients were diagnosed and treated in endocrinology unit , 25 ( 33.5% ) patients in urinary surgery unit , and 6 ( 8.1% ) patients in pediatrics unit . of the 74 patients , 38 ( 51.4% ) patients had hyposmia or anosmia , and 69 ( 93.2% ) patients presented with obvious pubertal delay . the serum testosterone levels at disease onset were decreased with varying degrees in all of the 74 patients , as the medical reference ranges in different laboratories are different . serum follicle - stimulating hormone ( fsh ) levels were decreased in 71 ( 95.9% ) patients , and serum luteinizing hormone ( lh ) levels were decreased in 69 ( 93.2% ) patients . of the 74 patients , 68 patients received hormone replacement therapy , and the therapeutic protocols are shown in table 1 . 20 ( 27.0% ) patients had cryptorchidism , and 11 patients received cryptorchidism surgery . except for the family history , there was no significant difference in gender composition , age , bmi , testosterone level at disease onset , pubertal development , small penis , testicular volume , breast enlargement , cryptorchidism , and number of patients receiving cryptorchidism surgery between the populations in our study and the study reported by the reproductive endocrine unit of massachusetts general hospital ( table 2 ) , while , in one multicenter study from england , more patients had family history of ihh , and the testicular volumes of ihh patients are smaller than those in our study . in the 74 patients , the mean length of the penis before erection was 3.98 1.73 cm , and the mean testicular volume was 4.11 2.12 ml . based on the tanner classification criteria , there are 38 patients at genital development stage i and 36 patients at stage ii , and nobody was at stage iii , iv , or v. there are 46 patients at pubic hair development stage i and 28 patients at stage ii , and nobody was at stage iii , iv , or v. all of the 74 patients had no or small laryngeal prominence and no or sparse facial hair including 12 ( 16.2% ) patients with small laryngeal prominence and 9 ( 12.2% ) with sparse beard . of the 74 patients , only 27 ( 36.5% ) patients had the male erection function , and 8 ( 10.8% ) patients had physiological nocturnal spermatorrhea . among the 68 patients who received hormone replacement therapy , for example , they had more masculine body , more facial hair and pubic hair , larger laryngeal prominence , more developed external genital organs , larger penis and scrotum , stronger erections , and nocturnal spermatorrhea . there was significant difference in penis length , testicular volume , development of external genital organs , pubic hair , beard , laryngeal prominence , penis erection , and spermatorrhea before and after treatment ( p < 0.001 , table 3 ) . the normalization of testosterone level rose from 0% before treatment to 66.2% after treatment , excluding 3 patients who are now receiving exogenous testosterone therapy . of the 74 patients , 38 ( 51.4% ) patients now have sexual activities , including 14 ( 18.9% ) patients whose spouses became pregnant . thirty - seven patients who received hcg or hcg + hmg treatment were included into a single gonadotropin group ( g group ) , and 25 patients who received hcg + t or hcg + hmg + t treatment were included into a gonadotropin + androgen replacement group ( g + a group ) . those who were still receiving testosterone replacement therapy were excluded from the g + a group , and the remaining 22 patients who once received testosterone replacement therapy discontinued its use for 18 years ( mean : 4.5 2.6 years ) . there was no significant difference in age at the time of investigation , age at disease onset , testosterone level before treatment , and decrease rate of fsh and lh between g group and g + a group . it was found that g + a group was significantly superior to g group in terms of penis length , pubic hair , laryngeal prominence development , and normalization of testosterone level ( p < 0.05 , table 3 ) . compared with g group , g + a group also exhibited more improvement in testicular volume , genital organs , beard , and spermatorrhea , though the difference was not statistically significant . qol of the ihh patients was assessed with respect to physical strength , appetite , sleep , libido , and sense of happiness . the mean score of physical strength was 5.1 1.7 , that of appetite was 7.3 1.8 , that of sleep was 6.8 2.3 , that of libido was 7.2 2.0 , and that of sense of happiness was 5.7 2.6 . the overall qol scored was 1045 , and the mean value was 32.1 7.5 . the ihh patients reported that they still felt poor in the sense of happiness and physical strength after treatment . there was a significant difference in the physical strength score between g group and g + a group ( 5.40 0.36 versus 4.65 0.30 , p = 0.025 ) , while there was no significant difference in appetite , sleep , libido , and sense of happiness . the overall qol score of the ihh patients was positively correlated with the level of testicular volume ( r = 0.324 ; p = 0.005 ) and the developmental state of the external genital organs ( r = 0.281 ; p = 0.015 ) after treatment . the most unique characteristic of the present study is the utilization of a web - based disease - specific social networking group for our investigation . to the best of our knowledge , the continuous expansion of internet users provides a good foundation for the development of network investigation , a new approach for medical research in china and elsewhere . there had been multiple studies comparing network studies with conventional studies , and they found that there was no significant difference in the study population characteristics and statistical results [ 11 , 12 ] . table 2 shows that the results obtained in the present network investigation are similar to those reported in the literature with respect to sex , age , bmi , and clinical characteristics of the ihh patients , which further confirms that group data obtained from network investigation are representative . however , ihh patients in the study reported no family history , which may be related to the age and sporadic characteristics of the patients . the patients ' parents did not have ihh , or else they would not have offspring as there was no domestic treatment method for ihh several decades ago . this finding is consistent with those of the previous large - sample studies on ihh in china . ( 1 ) it can include more participants , especially when network investigation is directed at a particular disease - specific social group . in addition , it is not restricted by time , space , or location ( i.e. , a single medical center ) , so that more patients with different severities and courses of the disease distributed in different regions of china or around the world can be investigated , and , therefore , the data obtained are more representative . ( 2 ) information can be obtained more quickly and conveniently , because network investigation saves amount of time in patient recruitment . in this study , we only spent five months to collect the data of the 74 ihh patients compared with 20 years for the massachusetts general hospital . in addition , network questionnaire investigation has a quick response rate , and the data obtained can be imported into the data bank directly , thus saving time , material , and manpower and reducing the cost of research effectively as compared with the conventional research methods . ( network investigation can be completed in a relatively independent situation , devoid of external interferences , so that the patients are able to express freely their sexual function and other private information [ 3 , 15 ] . network investigation can be carried out by a relatively small and independent research team , and any clinician can conduct the research as long as he / she has a reasonable study design . although network investigation is a new method for medical research and carries many advantages , it has some disadvantages . ( 1 ) as only network users can participate in the study , there may be some bias in patients selection . the participants need to be able to get on networks and have a certain education level . ( 2 ) network investigation has to use the layman 's language for communication , and , therefore , it is not suitable for studies that strongly depend on medical terminology . prolonged sex hormone deficiency may otherwise produce adverse effects on the development , metabolism , and psychology . all treated patients reported their improvements in the penis , testis , external genital organs , pubic hair , beard , laryngeal prominence development , erection , spermatorrhea , and testosterone normalization . these findings are basically consistent with those of the previous studies of us massachusetts general hospital and uk manchester royal infirmary [ 8 , 19 ] . more than 50% of the patients in this study have regained their sexual function after treatment , and part of the patients ' spouses have got pregnant . the possible reason is that most of the ihh patients in our study were overweight ; with physical development of the body and accumulation of the adipose tissue , the breasts became larger , which made self - assessment of the mammary development according to the tanner classification criteria difficult . for this reason , ultrasonography should be used to evaluate mammary gland development more accurately . in addition , as the physical strength of ihh patients is generally weak and the sense of happiness is usually low , it is necessary to provide psychosocial support to ihh patients . unfortunately , there is a lack of such studies in both china and other countries . in the present study , we interestingly found that the therapeutic effect in g + a group was superior to that in g group with respect to the penis length , pubic hair growth , testosterone normalization rate , and self - assessment scores of physical strength . the testosterone normalization rate in the g + a group ( excluding the three patients who were still receiving testosterone replacement therapy in the recent three months ) was significantly higher than that in g group ( 90.9% versus 51.7% ) , indicating that improvement of the clinical symptoms in g + a group was not due to the immediate exogenous testosterone treatment but due to the therapeutic effect of the testosterone treatment that they previously received . whether exogenous testosterone replacement therapy is beneficial to the response of ihh patients to gonadotropin therapy remains inconclusive , but the report from harvard reproductive endocrine sciences center seems to support this . the conditions of ihh can be reversed in about 10% of the patients , and all of these reversed cases received androgen replacement therapy , although the course of treatment may vary from individual to individual . therefore , they postulated that androgen may be able to improve the plasticity of the brain nerve network in generating gnrh , thus promoting reversal of the ihh condition . androgen has been found in regulating the generation of neurons in human olfactory epithelium , olfactory bulb , and hippocampal dentate gyrus and maintaining a normal number of neuronal synapses . in our investigation , 62/68 patients received exogenous hcg and/or hmg therapy , and the feedback inhibition of exogenous testosterone does not exist , while only 6 patients receiving lhrh may be mildly influenced . another simple reason is that the hcg dose may not have been adequately titrated up , so androgen was being used as a kind of top - up therapy to increase spermatogenesis efficiency and fertility in the males . similar to a recent study , recombinant fsh pretreatment followed by gnrh is successful in inducing testicular growth and fertility in men with congenital hypogonadotropic hypogonadism with prepubertal testes . the mechanism(s ) for the more favorable clinical outcome of g + a treatment over the simple g treatment can not be elucidated in the present study . however , the phenomenon of ihh reversal as observed by harvard reproductive endocrine sciences center suggests that testosterone treatment may improve the therapeutic effect of gonadotropin through an unknown pathway or mechanism in a particular stage . future randomized controlled clinical trials and mechanistic studies are needed before any definitive conclusions can be made . in conclusion , disease - specific network investigation can be used as an alternative method of medical research , although its effectiveness needs to be further verified . in the present study , we made use of the network information technology to conduct an investigation on ihh and found that hormone replacement therapy could improve the conditions of ihh patients effectively . in addition , the commonly used gonadotropin + androgen therapy appears to be superior to androgen replacement alone , although the actual clinical significance needs to be confirmed in prospective randomized control studies .

idiopathic hypogonadotropic hypogonadism ( ihh ) is a rare condition in which puberty does not take place naturally . we aimed to develop and follow an internet - based cohort and to improve our understanding of the disease . we established an internet - based questionnaire survey . a total of 74 male ihh patients were recruited from the chinese largest ihh network social group . the clinical symptoms before treatment mainly included small testis , underdeveloped secondary sexual characteristics , and sexual dysfunction . after treatment , the penis length , testicular volume , external genital organ development , pubic hair , beard , laryngeal prominence , erection , and spermatorrhea were improved significantly ( p < 0.001 ) . 18.9% of the patients completed fertility ; however , more than half of the patients still complained of poor happiness and low physical strength . in addition , improvements in penis and pubic hair development , testosterone normalization and the physical strength in ihh patients who received gonadotropin and androgen replacement therapy were better than in those who received single gonadotropin therapy ( p < 0.05 for all ) . in conclusion , disease - specific network investigation can be used as an alternative method of medical research for rare diseases . the results of our cross - sectional study showed the effectiveness of hormone replacement therapy for ihh and implied that gonadotropin and androgen replacement therapy may be superior to gonadotropin treatment alone .

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nonsurgical endodontic retreatment is indicated as the primary choice to eliminate or substantially reduce the microbial content from endodontically failed teeth . nevertheless , well - compacted root fillings offer resistance to the instruments used for retreatment , leaving root filling residues . this restricts access to the apical foramen and impairs root canal disinfection and complete reshaping . these include hand files , engine - driven rotary retreatment files ( rfs ) , ultrasonic tips / files , and heat - carrying instruments . the use of rotary files successfully facilitates an operative removal of the bulk of gutta - percha - based fillings from the root canal system . however , many studies have reported that complete removal is not gained . endodontic retreatment might result in pushing the irritants in the form of root canal filling materials , necrotic pulp tissues , bacteria , or root canal irrigants into the periapical region . these extruded irritants are responsible for postoperative inflammation , flare - ups , and even impaired healing . according to the literature , all instrumentation techniques exhibit some degree of apical extrusion of debris , the amount varying with different instrumentation techniques . recent studies have reported the inability of the universal protaper rfs ( d1-d3 ) to entirely remove the root canal fillings and proposed the use of supplementary files to aid removal of the root filling residues . hence , the current study was formulated to evaluate the extrusion of debris apically after the use of protaper universal rfs and different finishing files for removing the residual root canal fillings . the finishing files used in the current study were protaper next ( ptn ) , waveone ( wo ) , and self - adjusting file ( saf ) . the null hypothesis tested was that the three different supplementary files used would exhibit no significant differences in the amount of debris extruded . standard oval access was gained with access cavity burs ( endo z access kit , dentsply tulsa , oklahoma , usa ) . the canal patency was accomplished and working length ( wl ) was determined as 1 mm short of the length of a size 15 k - file that was visible beyond the apical foramen . the teeth were cleaned and shaped using protaper universal rotary files ( sx - f3 ; dentsply maillefer , ballaigues , switzerland ) . irrigation was performed using a 27-gauge side - vented needle placed 2 mm short of the apex between each file with sodium hypochlorite ( naocl ; 5.25% ) . the shaping procedure was continued till size f3 reached wl , followed by a final irrigation with 1 ml naocl . ethylenediaminetetraacetic acid ( edta ) gel ( rc help , prime dental products , mumbai , india ) served as a lubricant for the file with every reinsertion . the canals were then irrigated with 3 ml of 17% aqueous edta solution , which was followed by flushing the canal with distilled water and drying the canal with paper points . the root canal obturation was done by standardized f3 master cones ( dentsply maillefer ) and ah - plus sealer ( dentsply - detrey , konstanz , germany ) by warm lateral compaction , adding accessory cones . the excess gutta - percha was sheared by hot hand plugger and the access cavities were sealed using composite resin . the teeth were then kept for 30 days at 37c and 100% humidity to allow complete setting of the sealer . following this , these 80 samples were randomly divided into four experimental groups for retreatment . the present study used the model described by myers and montgomery for evaluating apical extrusion of debris . an analytical balance ( single pan k-15 , k roy instruments pvt . ltd . , varanasi , india ) with an accuracy of 10 g was used to help determine the initial weights of the tubes . three consecutive weights were obtained for each tube ( pre- and postinstrumentation ) , and the mean was computed . the root fillings in this group were removed using protaper universal rfs : d1 , d2 , and d3 at 2-ncm torque and 500-rpm speed using x - smart plus endomotor ( dentsply maillefer ) . the files were used in a brushing motion resting against the walls in a crown - down direction until it reached the wl . d1 was used only for the cervical third , d2 only for the middle third , and d3 through the entire wl . the rfs were used for removing the bulk of the root canal fillings as described in group 1 . then , ptn files ( x2 , x3 , and x4 ) were used to remove the root filling residues that remained . these files were used in an x - smart plus endomotor ( dentsply maillefer ) at 300 rpm and 3-ncm torque . whenever the file met resistance the file was withdrawn , the canal was irrigated , the canal was dried , and the procedure was continued . the above procedure was performed till no root filling material debris was seen on the files after the x4 reached the wl . the initial retreatment protocol was performed in a similar way to that in group 1 . following the removal of bulk , a wo large file this file was used in a preprogrammed endomotor x - smart plus ( dentsply maillfer ) . after every three pecks the file was withdrawn , the canal was irrigated , and the file was reintroduced into the canal . the above procedure was performed till no root filling debris was seen on the file after the file reached the wl , as in group 2 . after the removal of the bulk of root canal filling by rfs , the residual filling was removed using saf ( 2.0 mm ) in this group . this file instrumentation was accompanied by continuous irrigation ( bidistilled water ) provided by a peristaltic in - built pump in the endostation ( redent nova , raanana , israel ) for 1 min , removing the coarse root fillings . following this , the pump was turned off , the canal was filled with endosolv r ( septodont , paris , france ) , and saf was operated for 1 min . again the canal was irrigated by bidistilled water , and dried and filled with drops of solvent ; saf was operated for additional 1 min . next , the pump was turned on and the file was operated for the last 1 min . the file was used in the canal till it reached the wl . after each instrument ( rotary ) or after three pecks ( reciprocating ) , the irrigation needle was placed as deep as possible into the canal but not deeper as the predetermined wl minus 1 mm . for all the groups , endosolv r was used as a gutta - percha solvent . the debris extruded by groups 2 , 3 , and 4 the teeth were removed from the tube and the debris adhering to the root surface was collected by washing off the apical area of the tooth with 1 ml of distilled water into the centrifuge tube . the centrifuge tube was stored in an incubator at 70c for 5 days to allow the moisture to evaporate , before weighing the dry debris , using an electronic balance [ figure 1 ] . three consecutive weight measurements were then taken for each collection assembly , with the mean value recorded . collected debris after retreatment procedure the one - way analysis of variance ( anova ) and post hoc test were used to determine the significant group ( spss 20 , spss inc , chicago , il , usa ) with the alpha - type error set at 0.05 . the weights obtained after the use of three experimental supplementary files ( ptn , wo , and saf ) were compared to the control group to find out the percentage ( % ) of the debris extruded . the root fillings in this group were removed using protaper universal rfs : d1 , d2 , and d3 at 2-ncm torque and 500-rpm speed using x - smart plus endomotor ( dentsply maillefer ) . the files were used in a brushing motion resting against the walls in a crown - down direction until it reached the wl . d1 was used only for the cervical third , d2 only for the middle third , and d3 through the entire wl . the rfs were used for removing the bulk of the root canal fillings as described in group 1 . then , ptn files ( x2 , x3 , and x4 ) were used to remove the root filling residues that remained . these files were used in an x - smart plus endomotor ( dentsply maillefer ) at 300 rpm and 3-ncm torque . whenever the file met resistance the file was withdrawn , the canal was irrigated , the canal was dried , and the procedure was continued . the above procedure was performed till no root filling material debris was seen on the files after the x4 reached the wl . the initial retreatment protocol was performed in a similar way to that in group 1 . following the removal of bulk , this file was used in a preprogrammed endomotor x - smart plus ( dentsply maillfer ) . after every three pecks the file was withdrawn , the canal was irrigated , and the file was reintroduced into the canal . the above procedure was performed till no root filling debris was seen on the file after the file reached the wl , as in group 2 . after the removal of the bulk of root canal filling by rfs , the residual filling was removed using saf ( 2.0 mm ) in this group . this file instrumentation was accompanied by continuous irrigation ( bidistilled water ) provided by a peristaltic in - built pump in the endostation ( redent nova , raanana , israel ) for 1 min , removing the coarse root fillings . following this , the pump was turned off , the canal was filled with endosolv r ( septodont , paris , france ) , and saf was operated for 1 min . again the canal was irrigated by bidistilled water , and dried and filled with drops of solvent ; saf was operated for additional 1 min . next , the pump was turned on and the file was operated for the last 1 min . after each instrument ( rotary ) or after three pecks ( reciprocating ) , 2 ml of bidistilled water was used as irrigant . the irrigation needle was placed as deep as possible into the canal but not deeper as the predetermined wl minus 1 mm . for all the groups , endosolv r was used as a gutta - percha solvent . the debris extruded by groups 2 , 3 , and 4 following the completion of the retreatment procedure , the teeth were removed from the tube and the debris adhering to the root surface was collected by washing off the apical area of the tooth with 1 ml of distilled water into the centrifuge tube . the centrifuge tube was stored in an incubator at 70c for 5 days to allow the moisture to evaporate , before weighing the dry debris , using an electronic balance [ figure 1 ] . three consecutive weight measurements were then taken for each collection assembly , with the mean value recorded . collected debris after retreatment procedure the one - way analysis of variance ( anova ) and post hoc test were used to determine the significant group ( spss 20 , spss inc , chicago , il , usa ) with the alpha - type error set at 0.05 . the weights obtained after the use of three experimental supplementary files ( ptn , wo , and saf ) were compared to the control group to find out the percentage ( % ) of the debris extruded . the median and standard deviation of debris extrusion by the groups is shown in table 1 . the weights obtained after the supplementary use of ptn , wo , and saf were subtracted from the control group , giving as the result the actual amount of debris extruded by the supplementary files used for retreatment [ table 2 ] . according to the statistical results of the twofold retreatment protocols , the wo file used after rfs extruded significantly more debris , followed by ptn and saf ( p < 0.01 ) . saf was associated with the least extrusion of debris among the three experimental groups ( p < 0.01 ) [ graph 1 ] . when compared to the control group , wo was associated with 27% increase in the extrusion of debris , ptn with 19% increase , and saf with 12% increase [ graph 2 ] . in addition , the debris extruded by twofold retreatment protocol using three investigational files after the rfs median and standard deviation of debris extruded by the twofold retreatment procedure subtracted from the control group . all the groups were statistically significant ( p < 0.01 ) meanwise distribution of the weights of apically extruded debris by the three experimental files ( ptn , wo , and saf ) after the use of universal protaper rfs . statistically significant difference seen in all the three groups with waveone ( * ) exhibiting the highest and saf ( # ) exhibiting the lowest extrusion of debris when used in retreatment percentage of increased debris extruded by the experimental files after subtracting from the weights obtained from the control group . wo was associated with 27% , ptn with 19% , and saf with 12% increase in debris when used as supplementary files after using the pt rfs the results of the current study exhibited that debris extruded apically regardless of the retreatment protocol . the current study aimed to conclude the debris extrusion using supplementary files after using protaper universal rfs . this twofold retreatment protocol was formulated , as previous studies stated that the rfs alone fail to render the canals free of root fillings . using wo after rfs resulted in more extrusion of the debris apically when compared to ptn and saf ( p < 0.01 ) . after the use of rfs , it is important to use supplementary files , in order refine the apical preparation because of the diameter of a d3 file being equal to size 20 , impairing any reasonable cleaning of the apical portion . using supplementary files for finishing provides a cleaner root canal , facilitating removal of the residual root fillings that cause periapical extrusion of debris . hence , the current study compared debris extrusion by supplementary files to the control group . the results obtained for the control group are in accordance with earlier studies ; however , to the best of the authors knowledge , there is no report on apical extrusion of debris using supplementary files ptn , wo , or saf . additionally , in the present study bidistilled water was used as an irrigation solution to prevent possible crystallization of naocl . the saf group was associated with the lowest percentage of debris extruded apically in the current study ( 12% ) . it is a single - file system , devoid of a central metal core and any cutting edge or flutes , and instead has an abrasive surface . this hollow file adapts to the canal irregularities and the abrasive surface helps in creating fine dust while shaping , which is easily removed by associated , continuous irrigation . the continuous flow of irrigant does not build up any pressure in the canal as the metal meshwork allows free escape of the irrigant . in the narrowest apical part , the saf leaves more than 38% of the canal cross section free for backflow of fluid and dentin debris . de melo ribeiro et al . stated that in the apical third , the saf created cleaner canal walls when compared to the rotary system . the high amount of debris formed in the apical third may also cause its extrusion periapically . higher extrusion of debris by wo has been reported in the literature by various studies on primary endodontic in vitro studies , which can be attributed to the results of the present retreatment study as well . the wo files are characterized by a modified triangular cross section , which results in decreased cutting efficacy and smaller chip space , resulting in auguring the formed debris after instrumentation , periapically . the wo files also exhibit a larger taper of 0.08 at the apical 3 mm , which can be attributed to excessive debris formation apically , and extrusion periapically . the ptn file system has very rare reports on the apical extrusion of debris with its instrumentation . this design provides more cross - sectional space for enhanced cutting , loading , and successfully allowing the debris to travel out of a canal ( coronally ) . in addition , the swaggering motion of the file while in rotation may help the file to touch a greater root canal area . this may aid in better removal of the root canal filling residue and hence was used in one of the experimental groups in the current study . the samples in the group instrumented by ptn resulted in less extrusion of the debris when compared to the wo group . the current study was an attempt to find the amount of debris extruded by the use of supplementary files after using protaper universal rfs . under the experimental conditions of the present study , it can be possible to conclude that all retreatment systems caused apical debris extrusion . saf instrumentation after the rfs results in less extrusion than ptn and wo . there are no conflicts of interest .

aim : this study evaluated whether using supplementary files for removing root canal filling residues after protaper universal retreatment files ( rfs ) increased the debris extrusion apically.materials and methods : eighty mandibular premolars with single root and canal were instrumented with protaper universal rotary system ( sx - f3 ) and obturated . the samples were divided randomly into four groups ( n = 20 ) . group 1 served as a control ; only protaper universal rfs d1d3 were used , and the extruded debris was weighed . groups 2 , 3 , and 4 were the experimental groups , receiving a twofold retreatment protocol : removal of the bulk , followed by the use of supplementary files . the bulk was removed by rfs , followed by the use of protaper next ( ptn ) , waveone ( wo ) , and self - adjusting file ( saf ) for removal of the remaining root filling residues . debris extruded apically were weighed and compared to the control group . statistical analysis was performed using one - way analysis of variance ( anova ) and post hoc tukey 's test.results:all the three experimental groups presented significant difference ( p < .01 ) . the post hoc tukey 's test confirmed that group 4 ( saf ) exhibited significantly less ( p < .01 ) debris extrusion between the three groups tested.conclusion:saf results in less extrusion of debris when used as supplementary file to remove root - filling residues , compared to wo and ptn .

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traditionally , maxillary teeth have been anesthetized by administering an injection in the mucobuccal supraperiosteal fold in the proximity to the apices of teeth to be anesthetized . it is a convenient , safe , and effective way to achieve pulpal anesthesia in maxillary teeth and associated tissues . the disadvantage is that it requires multiple injections to cover more than one tooth and causing concomitant anesthesia of the lip and facial muscles . however , the maxillary teeth can also be anesthetized with troncular techniques ( eg , infraorbital , maxillary ) or intraosseous and intraligamentary injection . the literature did not describe a technique that is produced with a single injection , multiple teeth anesthesia in maxilla , and without causing collateral anesthesia of the face . therefore , friedman and hochman ( 1998 ) proposed an anesthetic technique in maxilla , the anterior and middle superior alveolar ( amsa ) nerve block . the authors state effective pulpal anesthesia from the central incisor to second premolar through palatal approach with a single injection and an expected anesthesia duration of 45 minutes to an hour [ figure 1 ] , using a computer - controlled injection system : the wand plus ( milestone scientific , deerfield ) . furthermore , the authors affirm that the anesthesia is achieved without causing lip numbness and no interference with the muscles of the facial expression . anesthetic area of amsa injection : buccal gingiva , teeth ( central incisor , lateral incisor , canine , first premolar , second premolar and mesiobuccal root of first molar ) , palatine gingiva and mucosa to midline amsa nerve block derives its name from the supposedly anesthesia of the anterior and middle superior alveolar nerves due to diffusion of the anesthetic solution via numerous nutrient channels on the palatal process of the maxillary bone [ figure 2 ] . both nerves are collateral branches of the infraorbital nerve in the homonymous canal and part of the maxillary nerve.[1710 ] the anterior superior alveolar ( asa ) nerve originates approximately 5 - 8 mm posterior to the infraorbital foramen . it is responsible for the pulpal innervation of the central incisor , lateral incisor , and canine.[1710 ] meanwhile , the middle superior alveolar ( msa ) nerve originates approximately 10 mm posterior to the infraorbital foramen . it is responsible for pulpal innervation of the premolars and the mesiobuccal root of the first molar.[1710 ] however , the msa nerve is not always present in several human dissection studies , which shows their presence in a range of 30 - 72%.[710 ] when the msa nerve is absent , a plexus is formed between the posterior superior alveolar nerve and the asa nerve to supply its innervation.[710 ] palatal nutrient channels ( arrows ) the amsa nerve block injection site is located palatally at a point that bisects the premolars and is approximately halfway between the midpalatine raphe and the crest of the free gingival margin [ figure 3 ] . this injection site is the confluence area of the asa and msa nerves , or the plexus when the last nerve is absent . theoretically , this technique would be beneficial , because a bilateral amsa nerve block technique supposedly anesthetizes 10 maxillary teeth extending from the second premolar of one side to the opposite side without causing anesthesia of the facial muscles , making it a great advantage in restorative dentistry . injection site ( arrow ) , halfway along an imaginary line ( a ) connecting the mid - palatal suture ( b ) to the free gingival margin ( c ) fukayama et al . and lee et al . evaluated the efficacy of this technique using the computer - assisted injection system ( wand ) , but the percentages of pulpal anesthesia obtained were questionable . other studies have reported its application in periodontal surgery but using the conventional syringe instead of the computer - assisted injection system . the benefits are excellent hemostatic control of palatal soft tissue , avoiding collateral facial anesthesia and fewer cumulative injections . due to few studies about this technique in literature , the aim of our study is to determine the success rates of pulpal anesthesia with the amsa nerve block technique using the conventional syringe ( carpule type ) . in this controlled clinical study , 30 caucasians patients ( 16 men and 14 women ) , with an average age of 22 years - old ( range , 21 - 25 years - old ) , belonging to the school of dentistry of los andes university ( santiago , chile ) , were selected . during the selection of patients , the following exclusion criteria were established : presence of systemic pathologies that contraindicate local anesthetics with vasoconstrictors , pregnancy , use of medications that alter pain perception , active orthodontic treatment , extractions , fixed prostheses , extensive fillings , and/or endodontic treatment in the experimental teeth ( maxillary central incisor , lateral incisor , canine , and first and second premolars ) . this study was approved by the ethics committee of the school of dentistry ( los andes university ) , and patients voluntarily decided to participate in the study by signing the written informed consent . the 30 patients received an amsa nerve block on one side of the maxilla randomly selected using the conventional syringe ( carpule type , hu - friedly ) , 1 ml of lidocaine 2% with epinephrine 1:100,000 ( octocaine100 , novocol pharmaceutical ) was injected to all patients . the teeth that were tested are , the maxillary central incisor , lateral incisor , canine , and first and second premolars of the side previously chosen . the mandibular canine was used as the non - anesthetized control tooth to ensure that the pulp tester was operating adequately during the study . at the beginning of each appointment and before any injections were given , the experimental teeth and control canine were tested with the pulp tester ( sybron endo , vitality scanner ) to record baseline vitality . after isolation with cotton rolls , the topical anesthetic ( benzocaine 20% ; alpha dental ) was applied to the tip of the pulp tester , which was placed midway between the gingival margin and the incisal or occlusal border of the tooth to be tested . the current rate was set on the pulp tester at 25 seconds to increase from no output ( 0 ) to the maximum output ( 80 ) . to establish the injection site on the palate , the amsa injection site was centered halfway between the midpalatine raphe and the gingival margin of the first and second premolars [ figure 3 ] . a cotton - tip applicator was used to apply the topical anesthetic ( benzocaine 20% ; alpha dental ) at the injection site for a minute . the injection was given with a 30-gauge needle , 22 mm ( terumo dental needle ) . the needle with the bevel against the bone was oriented at an angle of 45. then slowly the needle penetrated the palatal mucosa to establish contact with the palatal bone , to deposit 1 ml of anesthetic solution ( lidocaine 2% with epinephrine 1:100,000 ; octocaine100 , pharmaceutical novocol ) in three minutes [ figure 4 ] . execution of the technique the anesthetic success was monitored with the electric pulp tester ( sybron endo , vitality scanner ) , and we followed the protocol established by lee et al . in their study . a minute after the amsa injection , pulp test readings were obtained for the first and second premolars . at two minutes the testing continued in four - minute cycles for a period of an hour . no response from the subject to the maximum output ( no response at the 80 reading ) of the pulp tester was used as the criterion for pulpal anesthesia . anesthetic success only considers when two consecutive 80 readings occur at some point over an hour . the induction time ( time it takes to succeed anesthetic ) and the duration of anesthetic success was also registered . additionally , we verify that there was anesthesia in the palatal soft tissue , upper lip , and the surface of the face . thirty adult patients participated in this study , 16 men and 14 women with an average age of 22 years- old ( range , 21 - 25 years- old ) . all the patients received an amsa nerve block on one side of the maxilla randomly selected using the conventional syringe ( carpule type , hu - friedly ) , 1 ml of lidocaine 2% with epinephrine 1:100,000 ( octocaine100 , novocol pharmaceutical ) was injected to all patients . the anesthetic success of the amsa nerve block technique using conventional syringe is presented in table 1 . the anesthetic induction times ranged from 6 to 12 minutes and the duration of pulpal anesthesia ranged between 23 - 40 min [ table 1 ] . percentages of anesthetic success ( two consecutive no response at the 80 reading of the pulpal tester ) , mean anesthetic induction time ( min ) and mean anesthetic duration time ( min ) for every experimental tooth with amsa nerve block all the subjects obtained palatal soft tissue anesthesia , extending from the central incisor to the mesial of the first molar , never crossing the midline . no patient obtained pulpal anesthesia from the second premolar to the central incisor and facial anesthesia . while , in eight patients ( 26.7% ) had no anesthetic success in any of the experimental teeth . no severe pain was recorded during the administration of the local anesthetic in the palate . any complications or adverse reactions during and/or after completion of the anesthetic technique the main theoretical advantage of this amsa nerve block is that it reduces the number of injections and the quantity of anesthetic solution administered in comparison with the conventional supraperiosteal infiltrative anesthesia applied in multiple injections for each tooth . in addition , it would be ideal to use in cosmetic dentistry as it does not cause numbness of the lip and face . however , our results show pulpal anesthetic success rates ( two readings in 80 of the pulp tester ) ranging from 16.7 to 66.6% . failure to respond to 80 ( maximum output of the pulp tester ) was the criterion for pulpal anesthesia based on clinical studies by dreven et al . and certosimo et al . , because their studies showed that the readings below 80 resulted in pain during operatory procedures . in our study , the anesthetic blockade of the five experimental teeth was not obtained in any of our patients as expounded by friedman and hochman . the duration of pulpal anesthesia was gradually declining during the 60 minutes ; we can not confirm the clinical impression of the authors that there is duration of pulpal anesthesia for 60 minutes . on the other hand , we can say that there is no anesthesia of the lips and facial muscles of expression . studies of this technique using a computer - controlled system , which was made by fukayama et al . , obtained anesthetic success in the range of 42 - 86% and 35 - 58% by lee et al . in our study , the success rates were significantly low with the use of conventional syringe ( 17 - 66% ) . the low success rates of conventional syringe system could be due to the superiority of the computer - controlled system to inject the anesthetic solution with a controlled and continuous flow . with the conventional syringe system , the flow pressure depends on the operator although all injections were performed by the same investigator . the anesthetic flow can not be controlled as precisely as with the computer - controlled system . speculated that the injection with the computer - controlled system creates an improved pressure gradient environment for diffusion of the anesthetic solution through the numerous nutrient channels of the palatal vault . in the case of the low anesthesia percentages , the central and lateral incisor could be attributed to an increased presence of the msa nerve in the patients of the study . thus , in such cases , the msa nerve is anesthetized , and not the asa nerve , due to its distance from the puncture site . anatomical studies on dissection of cadavers have found the presence of the msa nerve in the range of 30 - 72% , and when it is absent , its innervation is supplied from a plexus formed by the asa and the posterior superior alveolar nerves.[1810 ] the exact role of the absence of the msa nerve in the amsa nerve block success is not known . malamed defines a nerve block when the solution is deposited in the vicinity of the main nerve trunk , so that the anesthetic should be placed close to the asa or msa nerves , resulting in high rates of anesthetic success , but we saw that this did not happen because the solution is deposited on the palatine process of the maxilla for its diffusion , searching the terminal branches of the alveolar nerves . so based on our clinical observations , it is more of an infiltrative technique than a nerve block . on the other hand , the reduction of multiple injections reduces the total amount of delivered vasoconstrictor and may be useful for cardiovascular - compromised patients that required maxillary anesthesia . for maxillary mucogingival procedures , the amsa nerve block with palatal delivery of anesthetic with vasoconstrictor provides excellent hemostasis and reduces the need for multiple re - injections to obtain hemostatic control during periodontal procedures or graft harvest . one of the main disadvantages of the amsa nerve block is that palatal injections are generally considered the most painful injections . wahl et al . showed that palatal injections caused significantly more pain than other intraoral injections , probably due to the result of pressure . however , in our study , severe pain during the technique was not reported , possibly due to the prior application of topical anesthetic and the slow and controlled injection of the anesthetic solution . other problem of this technique is that it is restricted only for the five anterior maxillary teeth and procedures such as periodontal surgeries require anesthesia of the full maxillary arch that may complement with another technique . a second technique that would produce anesthesia from the central incisor to second premolar with a single injection is the infraorbital nerve block ( ionb ) . having the advantage that the injection site is not in the palate , it is therefore less painful , but with the inconvenience of causing facial numbness . karkut et al . in their study found that ionbs were ineffective in providing profound pulpal anesthesia of the maxillary central incisor ( 15% success rate ) and lateral incisor ( 22% success rate ) . the pulpal anesthesia success rate was 92% for the canine and 80% for first and second premolars . moreover , berbeich et al . obtained similar results in 40 patients with ionb where pulpal anesthesia was ineffective in providing profound anesthesia of maxillary central and lateral incisors . successful pulpal anesthesia of the canine and first and second premolars ranged from 75 - 92% . these previously discussed studies occupy anesthetic evaluation protocols similar to those in our study , but getting better rates of success with the ionb . although corbett et al . conducted a study to compare the efficacy of the amsa nerve block with that of the ionb in achieving pulpal anesthesia in the anterior maxilla , anesthetic success was significantly greater with the amsa nerve block than with the ionb in central and lateral incisors ; however , anesthesia was achieved in only 42.9% of central incisors with the amsa nerve block . the authors observed a significantly greater number of anesthetic success episodes in the premolar and canine teeth after ionb . the incidence of subjective lip numbness was 100% after ionb and 14.3% after amsa nerve block . there was no significant difference in the rates of injection discomfort between the two techniques . the authors conclude that the ionb has better success rates of pulpal anesthetic , but amsa has the advantage that the same injection has the same potential to provide pulpal anesthesia from central incisor to second premolar . in addition , the amsa nerve block anesthetize palatine tissue without causing facial numbness . the shape of the palate was not recorded but we believe that there was a homogeneous distribution between the deep and shallow palate in this study . because this anesthetic technique was applied to young population , the results may not be applied to children or the elderly . because of the unpredictable anesthetic success of the experimental teeth and variable anesthesia duration , the technique is disadvantageous for clinical application as the first choice , counting with other techniques that have greater efficacy in the maxilla . however , it may be clinically useful in restorative dentistry ( by not anesthetizing the facial muscles and not affecting the smile line ) and in periodontal surgery due to the excellent hemostatic control in palatal soft tissues .

background : dental procedures in the maxilla typically require multiple injections and may inadvertently anesthetize facial structures and affect the smile line . to minimize these inconveniences and reduce the number of total injections , a relatively new injection technique has been proposed for maxillary procedures , the anterior and middle superior alveolar ( amsa ) nerve block , which achieves pulpal anesthesia from the central incisor to second premolar through palatal approach with a single injection . the purpose of this article is to provide background information on the anterior and middle superior alveolar nerve block and demonstrate its success rates of pulpal anesthesia using the conventional syringe.materials and methods : thirty caucasian patients ( 16 men and 14 women ) with an average age of 22 years - old , belonging to the school of dentistry of los andes university , were selected . all the patients received an amsa nerve block on one side of the maxilla using the conventional syringe , 1 ml of lidocaine 2% with epinephrine 1:100.000 was injected to all the patients.results:the amsa nerve block obtained a 66% anesthetic success in the second premolar , 40% in the first premolar , 60% in the canine , 23.3% in the lateral incisor , and 16.7% in the central incisor.conclusions:because of the unpredictable anesthetic success of the experimental teeth and variable anesthesia duration , the technique is disadvantageous for clinical application as the first choice , counting with other techniques that have greater efficacy in the maxilla . although , anesthetizing the teeth without numbing the facial muscles may be useful in restorative dentistry .

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malaria continues to be a serious public health problem in south - east asia including india . about 36% of the world 's population ( 2020 million ) the south - east asian region contributes 2.5 million cases to the global burden of malaria , of which india 's contribution is 76% . according to the world malaria report 2014 , 22% ( 275.5 m ) of india 's population live in high transmission ( > 1 case/1000 population ) areas , 67% ( 838.9 m ) live in low transmission ( 01 cases/1000 population ) areas , and 11% ( 137.7 m ) live in malaria - free ( 0 cases ) areas . in 2013 , 0.88 million cases have been recorded , with 128 million tests being conducted on the suspected cases , with plasmodium falciparum causing 53% and plasmodium vivax causing 47% of the infections . the incidence of malaria in india accounted for 58% of cases in the south - east asia region of who . at present , official figures for malaria in india , available at national vector borne disease control programme , indicate 0.71.6 million confirmed cases and 4001,000 deaths annually . however , a study conducted by teams from the office of the registrar general of india , centre for global health research at st . michael 's hospital and university of toronto , canada , published in the lancet on november 20 , 2010 has reported that malaria causes 205,000 malaria deaths per year in india before age 70 years ( 55,000 in early childhood , 30,000 at ages 514 years , and 120,000 at ages 1569 years ) with a 1.8% cumulative probability of death from malaria before age 70 years . the report says that 90% of the deaths were recorded in rural areas , of which 86% occurred at home without any medical attention . beliefs and practices of malaria are often related to culture which can influence the effectiveness of control strategies . thus , local knowledge and practices related to the disease are key to implementation of culturally appropriate , sustainable , and effective interventions . community perception , beliefs , and attitude about malaria control , symptom identification , treatment , and prevention influence efforts to address malaria and are often overlooked in control efforts ; and vary from country to country and among individual households . failure to consider community 's knowledge , attitude , and practice ( kap ) about malaria may contribute to the inability of the program to achieve sustainable control . hence , kap can be an important step in developing strategies aimed at controlling malaria . extensive pubmed search reveals very little in terms of evidence regarding the current kaps of the population in general and rural population , in particular , in this part of the country . keeping this in view , further , the state of jammu and kashmir falls in the low endemic zone with annual parasite incidence ( api ) of 0.11 only . however , the major concentration of malaria cases in jammu and kashmir is restricted to jammu division of the state only with kashmir division recording an api of < 0.1 . in this , the jammu division of the state resembles the rest of north india more than the kashmir division in the same state . this differential distribution of malaria cases within a state with a low incidence of malaria is of great public health importance . the importance will lie in our ability to identify the potential contributors to this differential distribution . it also opens up the potential for developing preventive strategies across different parts of india . a community - based descriptive cross - sectional study was carried out in rs pura health block of jammu district in jammu and kashmir state in north india . the main outcome of interest was assessment of kap regarding malaria among inhabitants rs pura health block . rs pura block is located in the south - west of jammu city adjacent to the indo - pak border with a total area of 273 km and average density of 658/km . there are 176 villages and 1 town ( 11 wards ) in the block with an estimated population of 179,636 . the methodology used for the purpose of this study has been developed by institute for research in medical statistics as an alternative to probability proportionate to size ( pps ) methodology . the pps methodology although self - weighing in nature has a somewhat centrist bias which distorts this property . to obviate this bias , alternative methodology ( irms ) was developed and tested with beneficial results across different parts of india by scientists from indian council of medical research . a sample size of 300 distributed as described below is in agreement with this methodology . hence , no separate sampling strategy was adopted to identify the required number of households to be included in the study . the same methodology has been used in the current study . for the purpose of this study , only the rural area of the block the stratification was done in accordance with population size of villages located in rs pura block . the villages were identified as the primary sampling units while households located within the villages were identified as the secondary sampling units . the stratification was conducted as detailed below : stratum 1 small villages - villages with population of < 500stratum 2 moderately small villages - villages with population of 500999stratum 3 medium size villages - villages with population of 10001999stratum 4 large size - villages with population 2000 . stratum 1 small villages - villages with population of < 500 stratum 2 moderately small villages - villages with population of 500999 stratum 3 medium size villages - villages with population of 10001999 stratum 4 large size - villages with population 2000 . , 15 households with at least one member above age of 18 years at the time of the study were selected . the village selected was mapped , and a house - to - house survey was conducted until 15 households were completed . only one adult per household was interviewed . study villages from rs pura block from these 300 households , 300 adult respondents were identified for administration of the study questionnaire . the questionnaire was administered either to the head of the household or in his absence to a responsible adult above 18 years of age . the questionnaire included questions on : demographic details , on knowledge and understanding of malaria transmission , on recognition of sign and symptoms , on perception of cause , treatment seeking patterns , preventive measures , and protective patterns . stratum 1 small villages - villages with population of < 500stratum 2 moderately small villages - villages with population of 500999stratum 3 medium size villages - villages with population of 10001999stratum 4 large size - villages with population 2000 . stratum 1 small villages - villages with population of < 500 stratum 2 moderately small villages - villages with population of 500999 stratum 3 medium size villages - villages with population of 10001999 stratum 4 large size - villages with population 2000 . , 15 households with at least one member above age of 18 years at the time of the study were selected . the village selected was mapped , and a house - to - house survey was conducted until 15 households were completed . study villages from rs pura block from these 300 households , 300 adult respondents were identified for administration of the study questionnaire . the questionnaire was administered either to the head of the household or in his absence to a responsible adult above 18 years of age . the questionnaire included questions on : demographic details , on knowledge and understanding of malaria transmission , on recognition of sign and symptoms , on perception of cause , treatment seeking patterns , preventive measures , and protective patterns . however , data on 4 households was found to be incomplete at the time of analysis and , therefore , were excluded . out of 296 study participants interviewed 194 ( 65.5% ) were males , while 102 ( 34.5% ) females [ table 1 ] . the demographics revealed that the maximum participants ( 33.7% ) belonged to the 4150 years age group . education wise , one - third ( 29.7% ) of the participants were illiterate , and an almost equal proportion ( 29.1% ) had studied up to middle standard ( 8 grade ) . maximum ( 31% ) number of study participants was agriculturists followed by homemakers ( 29.7% ) . more ( 56% ) number of study participants belonged to nuclear families . the demographic details of the study participants all of the study participants had heard of malaria [ table 2 ] . importantly , 97% of the study participants correctly associated malaria with mosquito bite [ table 2 ] . the majority ( 76.35% ) of the study participants had knowledge of malaria symptoms , and a majority of them had knowledge that malaria could be both prevented ( 90.5% ) as well as cured ( 93.2% ) . further , a majority ( 77.7% ) of the study participants knew that malaria could be fatal if not treated . an overwhelming ( 95% ) number of the study participants had knowledge regarding various modes of disease prevention . knowledge about malaria among respondents the response to questions on knowledge with multiple options drew multiple responses . however , the most common response ( 68.2% ) to questions on the source of information regarding malaria was television ( tv)/newspaper . adequate knowledge was also found among them regarding resting habits of mosquitoes with only 2.8% of responses registering the responses to other questions are detailed in table 2 . a majority ( 92.5% ) of the study participants considered malaria to be a serious health problem , thus reflecting their attitude to the disease [ table 3 ] . more than two - third of them would consult a doctor in case of child 's illness though the majority of them had no knowledge regarding malaria treatment . the overall attitude of the study population toward the use of bed nets was positive and similar was the case with vector control program . attitude of respondents towards malaria regarding practices , a majority ( 71.6% ) of the study participants preferred going to doctors at government hospitals for malaria treatment , and 56% of study participants were willing to seek medical help in < 24 h in case of a child having a febrile episode [ table 4 ] . with regard to personal protective measures , regarding preventive practices , more than half of the respondents would avoid collections of stagnant water while a quarter of them would prefer indoor spraying . this is of greater value in diseases such as malaria , wherein awareness about the cause and spread is a major stakeholder for prevention . it assumes significance in a state like jammu and kashmir , wherein the distribution of malaria cases within this state is not geographically uniform . therefore , the strength of this study lies in its ability to capture the probable contribution of disease awareness in lower local disease burden . the findings of this study could shape the future discourse of research on local approaches to prevention of endemic diseases in india . this is in agreement with findings of some studies but is in contrast to other studies . this finding assumes importance in view of the fact that the study participants belong to a low incidence state and , therefore , have a lower exposure to cases of malaria . this may be because the main source of information in the current study population was mass media ( tv / newspaper ) followed by friends / neighbors . this finding is in contrast to the findings reported by singh et al . where an individual 's experience with malaria was the main source . creation of awareness through mass media is an excellent model of intervention in the prevention of malaria . reported health facility ( 29% ) as the main source of information among the respondents . knowledge regarding causation of disease , its cure and prevention were very high in this study , though knowledge that it can be fatal was a bit on the lower side . this again may be because of less number of personal interactions with patients of malaria . the results from our study regarding the transmission of malaria are in contrast to a nigerian study where only a small proportion of respondents correctly answered questions about malaria transmission and its cause . the study results have demonstrated that respondents had good knowledge about malaria signs and symptoms , and the results are consistent with some other studies . this high level of awareness can best be explained by increased access to mass media and health education by government agencies . 97.2% respondents made correct association between malaria and mosquito bite which is in agreement with those reported by hlongwana et al . knowledge regarding breeding and resting places of mosquitoes were high among the study population in line with the results reported by some other studies like the one by chirebuv et al . similarly , knowledge regarding prevention of mosquito breeding and prevention of malaria was very high , and the results concur with those reported by other authors . it must be mentioned that excellent knowledge does not guarantee better preventive measures as observed by the authors . this could be explained by the low literacy levels and poor socioeconomic status of the study population and is reflective of the trend followed by health seekers in other health initiatives . therefore , probably the key lies in creating awareness across all socioeconomic domains and identifying the possible bottlenecks . the respondents attitude toward malaria was reflected when more than 90% labeled it as a serious health problem . the majority ( 70.9% ) consulted a doctor in case the child was found to be febrile , and only 18.2% reported using a home remedy . the results are in contrast to those reported by eversole et al . where treatment involved traditional medicines more frequently . the reasons for better access to registered medical practitioner in our study could be attributed to the better availability of health care in rs pura block as the block is a part of medical college field practice area . that may be the probable reasons for only 2.7% reporting to a traditional healer . another positive finding was that the motivation to seek care for a febrile child was the condition of the child in an overwhelming 91.8% of the study population . however , only 15.5% knew chloroquine to be the best treatment for malaria , and the most obvious reason could be low literacy in this rural population . in contrast , khan et al . reported 90% respondents could name drugs used in malaria treatment . despite this attitude toward vector control program was very positive . the practice of going to government hospital / doctor for malaria treatment was found at a healthy rate of 72% among the respondents and 56% would seeking help in < 24 h of the febrile episode . the results are in agreement with those reported by hlongwana et al . though a higher rate ( 88% ) sought treatment within 24 h of onset of malaria symptoms . the majority of the respondents in the current study had adequate knowledge about protective as well as preventive measures against malaria which concur with those reported by hlongwana et al . in the study conducted in south africa . probably , a large study sample with distribution in rural , urban , and tribal areas of jammu and kashmir will provide us with a better understanding on the role knowledge and awareness on differential distribution malaria . probably , a large study sample with distribution in rural , urban , and tribal areas of jammu and kashmir will provide us with a better understanding on the role knowledge and awareness on differential distribution malaria . results revealed that kap among respondents were reasonably good and key sociocultural , and related indicators need to be identified as a part of malaria elimination strategy .

introduction : an extensive search on pubmed reveals very little in terms of evidence regarding the current knowledge , attitude , and practices ( kap ) of the population in general and rural population , in particular , in this part of the country . therefore , a study was conducted with the aim to assess the communities knowledge of malaria transmission , recognition of signs and symptoms , treatment seeking.materials and methods : a stratified two - stage design was used to conduct a house - to - house survey using a semi - structured questionnaire in rs pura block of jammu district of jammu and kashmir state in north india.results:a total of 300 households were included in the study . however , data on 4 households was found to be incomplete at the time of analysis and , therefore , were excluded . out of 296 study participants interviewed 65.5% were males , while 34.5% females . all of the study participants ( 100% ) had heard of malaria , and the main source of their information was television / newspaper . 92.5% of the study population considered malaria to be a serious health problem , thus reflecting their attitude to the disease . regarding practices , 71.6% of the study participants preferred going to doctors at government hospitals for malaria treatment , and 56% were willing to seek medical help in < 24 h in case of a child has a febrile episode.conclusions:results revealed that kap among respondents were reasonably good and key sociocultural , and related indicators need to be identified as a part of malaria elimination strategy .

pubmed

recent advances in molecular biology and microscopy technologies have enhanced our understanding about the higher - order folding of the genome in an unprecedented manner . it is now widely accepted that the dynamic folding of the chromatin is fundamental in regulating gene expression and dna replication . the genome is folded in a hierarchical manner into chromosome territories , genomic compartments , and topologically associating domains ( tads ) , in which specific long - range looping interactions occur . each of these structures can be dynamically regulated during development , and perturbations in these folding units are associated with multiple diseases and cancer . one interesting feature that was revealed upon fine mapping of the folding of the genome is the tad structures , which range from 100 kb up to 1 mb in size . tads are units of chromosomes that exhibit higher frequency of physical contacts between genes and their cognate regulatory regions . the tads have been shown to be stable across different species , cell types , and cellular conditions . the replication timing , the presence of different histone modifications and the expression of the genes inside a tad are highly correlated . however , the underlying features that generate invariant tad boundaries remain unknown . in human cells , tad boundaries have been associated with the enrichment of ctcf , cohesin , dnase i hypersensitivity , certain histone marks and the timing of replication domains . furthermore , in drosophila , the combinatorial binding of different types of insulators ( also referred to as architectural proteins ) , such as beaf32 and tfiiic , is associated with the strength of the tad boundary . the boundaries serve as barriers to long- and short - range interactions between dna sequences , thus the most likely interaction partner for a genomic region is another region within the same tad . a strong tad boundary limits interactions between the two adjacent tad domains , whereas a weak tad boundary allows a higher frequency of inter - tad interactions . although a relationship between tad boundaries and the binding of insulators has been demonstrated , the effects of enzymes that modify or remodel chromatin are largely unknown . the major atpase subunit of the swi / snf chromatin remodeling complex , brg1 ( also known as smarca4 ) , is required for proper nucleosome occupancy and positioning at target regions as well as for establishing numerous long - range chromatin interactions . , we highlight our recent finding that brg1 contributes to tad boundary strength and discuss the implications of brg1 loss on nucleosome occupancy and higher order chromatin organization . to delineate the role of brg1 in genome architecture , we performed rna - seq and hi - c in control and brg1 knockdown mammary epithelial mcf-10a cells . moreover , to probe brg1 localization , we performed chip - seq in wild type parental mcf-10a cells . we reported that brg1 regulates the expression of genes related to extracellular matrix and lipid synthesis . in addition , brg1 knockdown resulted in the differential expression of many long non - coding rnas , which are thought to be involved in genome organization . chip - seq analysis of brg1 demonstrated binding primarily ( 60% ) at genic regions . intergenic brg1 peaks included super - enhancers , which are defined as clusters of regulatory regions with unusual enrichment of factor binding sites and/or histone modifications that are in close proximity , though this classification has recently been challenged . comparison of the control and brg1 knockdown hi - c data sets revealed that the majority of the tad boundaries were largely overlapping between control and brg1 knockdown cells , consistent with prior results . however , more than 10% of the tad boundaries were unique to either the control or the brg1 knockdown cells , providing evidence that brg1 may contribute to the integrity of tad boundaries . in support of this concept this result may be expected since tad boundaries have been shown to be enriched for both housekeeping and developmentally regulated genes . surprisingly , the strength of the tad boundaries , as assessed using the insulation method , was significantly reduced in brg1 knockdown human mammary epithelial mcf-10a cells . in other words , upon brg1 knockdown , there was a higher frequency of inter - tad genomic interactions . we further validated this result by intersecting tad boundaries with brg1 chip - seq data and categorizing the boundaries as either brg1 bound or not bound . the strength of tad boundaries that were bound by brg1 was stronger than the boundaries that lacked brg1 localization . an explanation for the effect of brg1 knockdown is that brg1 loss may disrupt chromatin accessibility and preclude the binding of transcription factors and/or chromatin modifiers at tad boundaries . to examine this possibility , we analyzed a publicly available mnase - seq data set from wildtype and brg1 knockdown mouse embryonic fibroblast ( mef ) cells . by intersecting the nucleosome occupancy data with the encode mef ctcf chip - seq data set , we showed that there was decreased nucleosome occupancy at ctcf - bound regions in brg1 knockdown cells when compared to control cells . thus , our results suggested that brg1 plays a role at tad boundaries by regulating nucleosome occupancy and possibly ctcf localization . dixon et al . recently hypothesized that the orderly positioning and occupancy of nucleosomes at tad boundaries renders the local chromatin at the boundary less flexible , and thus prevents long - range interactions surpassing the boundary . the orderly positioning and occupancy of nucleosomes at tad boundaries may be achieved by enriched ctcf binding , which positions 20 nucleosomes around its binding sites . our observation of the relationship between brg1 knockdown and the reduction of nucleosome occupancy around the ctcf sites supports such a mechanism . it was recently shown that the directionality of ctcf binding is very strongly associated the formation of tads , though the relationship between brg1 binding and ctcf binding site orientation remains to be determined . we propose that loss or knockdown of brg1 results in the reduction of nucleosome occupancy at ctcf sites either through loss of its atp - dependent chromatin remodeling activity or by negatively impacting ctcf binding ( fig . 1 ) . such an effect of brg1 may also occur at regions where other chromatin organizers ( e.g. cohesin ) are bound . figure 1.a schematic figure depicting the possible connection between brg1 , ctcf and topoisomerases . in the presence of brg1 ( top panel ) , ctcf and topoisomerases can efficiently bind to tad boundaries and promote proper nucleosome occupancy and uncoiling of the dna , resulting in a strong tad boundary . therefore , we propose that upon brg1 knockdown , ctcf and topoisomerases may interact with tad boundary sequences , but the lack of atp - dependent remodeling activity may alter nucleosome occupancy and affect boundary strength ( middle panel ) . alternatively , the binding of ctcf and topoisomerases may be perturbed , resulting in altered nucleosome occupancy and reduced boundary strength ( bottom panel ) . a schematic figure depicting the possible connection between brg1 , ctcf and topoisomerases . in the presence of brg1 ( top panel ) , ctcf and topoisomerases can efficiently bind to tad boundaries and promote proper nucleosome occupancy and uncoiling of the dna , resulting in a strong tad boundary . therefore , we propose that upon brg1 knockdown , ctcf and topoisomerases may interact with tad boundary sequences , but the lack of atp - dependent remodeling activity may alter nucleosome occupancy and affect boundary strength ( middle panel ) . alternatively , the binding of ctcf and topoisomerases may be perturbed , resulting in altered nucleosome occupancy and reduced boundary strength ( bottom panel ) . the model proposed by dixon et al . , is reinforced by accumulating evidence that highlights the role of the topoisomerase complex as a regulator of tad boundaries . recent results demonstrate that topoisomerase ii beta ( top2b ) binding overlaps with almost half of ctcf / cohesin binding sites , and that top2b may facilitate supercoiling at ctcf sites in a transcription - dependent manner . direct links between topoisomerases and brg1 are provided by a proteomic profiling using mass spectrometry that identified a significant interaction between brg1 and top2b . consistent with this finding , brg1 also binds to topoisomerase ii alpha ( top2a ) . furthermore , brg1 is required for the recruitment of topoisomerase i ( top1 ) to chromatin , and in the case of both top2a and top1 , the atpase activity of brg1 has been shown to be essential for the recruitment of the topoisomerase proteins . these findings suggest an interplay and functional cooperativity between ctcf , brg1 and topoisomerases in the organization of tads via the active regulation of tad boundary regions . additional evidence for engagement of topoisomerases in genome organization is provided by studies from bacteria and yeast . a hi - c study in prokaryotes examined the biology of chromosomal interaction domains ( cids ) , which are prokaryotic chromatin folding structures that are analogous to the eukaryotic tads . treatment of the prokaryotic cells with novobiocin , a drug that inhibits dna gyrase ( a homolog of topoisomerase ii ) and thus supercoiling , perturbed the sharpness and the positions of cid boundaries . in yeast , fine resolution nucleosome mapping determined that self - associating domains similar to but smaller than tads exist and that their boundaries were bound by the rsc chromatin remodeling enzyme . the rsc atpase is structurally related to brg1 , and the genetic analyses performed confirmed a role for rsc in yeast genome organization . moreover , hrp1 , an atp - dependent chromatin remodeling protein from the chd subfamily , was shown to collaborate with top1 to maintain open chromatin at active gene regions in yeast . taken together , these findings suggest a strong link between chromatin remodeling enzymes , including the mammalian swi / snf complex , architectural proteins , and topoisomerases in genome organization . we propose that the association between brg1 knockdown and reduction in tad boundary strength may be due to perturbations in the recruitment of ctcf and topoisomerases , and may therefore affect integrity of the chromatin structure and the stiffness of the chromatin at tad boundaries ( fig . 1 ) . in further support of the chromatin hypothesis , a recent study showed that a small deletion of a tad boundary was not sufficient to disrupt the tad domain , as it remained stable . this result suggests that the boundary is not defined by the exact boundary sequence or length , but instead depends either on the supercoiling or the overall composition of the factors present at the boundary . our recent data indicating that brg1 , and hence the mammalian swi / snf chromatin remodeling enzyme , binds to tad boundaries and promotes boundary strength adds a novel biochemical activity , atp - dependent chromatin remodeling , to the complex structure that regulates tad formation and function . other chromatin remodeling complexes may play similar roles , as these enzymes can function in a redundant manner . continued examination of the factors found at tad boundaries will yield important insights into the biophysical properties of tads and their boundaries , as well as into chromatin folding and overall genome organization that supports biological control .

abstractthe eukaryotic genome is partitioned into topologically associating domains ( tads ) . despite recent advances characterizing tads and tad boundaries , the organization of these structures is an important dimension of genome architecture and function that is not well understood . recently , we demonstrated that knockdown of brg1 , an atpase driving the chromatin remodeling activity of mammalian swi / snf enzymes , globally alters long - range genomic interactions and results in a reduction of tad boundary strength . we provided evidence suggesting that this effect may be due to brg1 affecting nucleosome occupancy around ctcf sites present at tad boundaries . in this review , we elaborate on our findings and speculate that brg1 may contribute to the regulation of the structural and functional properties of chromatin at tad boundaries by affecting the function or the recruitment of ctcf and dna topoisomerase complexes .

pubmed

when the objective of the study is investigation of count data using some variables , statistical modeling is used . in statistics , poisson distribution is used for investigating the count data if the mean and variance of the distribution are the same . if these two measures are not the same ( variance is larger than the mean = over dispersion ) , then negative binomial distribution is preferred . statistical modeling is from suitable methods to investigate the relationship between various phenomena , especially in medicine and health . poisson regression is of the models which is used in count variables such as the number of blood donations , the number of stopping addiction , the number of failed courses or semester etc . and the poisson regression is a subset of a large set of statistical modeling which is called generalized linear model ( glm ) . sometimes the count variables which are used to build a statistical modeling , have an inflation in zero which are divided into poisson zero inflated or negative binomial zero inflated distributions according to the nature of the variables . there are various researches that used statistical modeling on count data which applied negative binomial or poisson regressions ; zero inflated poisson or negative binomial regressions . rafiee ( 1 ) used negative binomial distribution for modeling of the period of hospitalization of mothers after child birth as the best model . wong and lam ( 2 ) applied poisson regression with zero inflated for modeling of dmf for the students health situation . barondess et al ( 3 ) used poisson regression with zero inflated to model the estimated number of cigarettes which is used by new smokers of different races in the usa in 2010 . mohammadfam et al ( 4 ) applied a model for the number of work accidents in 2009 and showed the best model is a poisson regression with zero inflation . one of the applications of this method is in the modeling of educational count data such as the number of failed courses or the number of failed semesters in university students . this application which is considered as a part of the students academic performance has a great efficient . the academic performance has relationship with some educational and demographic factors of the students as well . mlambo ( 5 ) showed that gender , age and entry qualifications have a relationship with the academic performance . foster ( 6 ) proved that gender , age , motivation , prior academic performance are variables which have relationship with the academic performance . kooi ( 7 ) showed that age and academic background have strong relationship with the academic performance . garkaz et al ( 8) showed that the type of diploma , students interest , employment status and gender are factors which have relationship with the academic performance . trockel et al ( 9 ) showed that exercise , eating and sleep habit , time management , religious habit , period of work in a week , gender and age are from the variables which affect the academic performance of the students . since there are many demographic , educational , andeconomicalfactors which affect the academic performance of the students , in each research only some of them are presented . also , in most recent research , descriptive statistics or ordinary or logistic regressions are used to analyze or model the educational data and there is very few work on academic performance of the university students using poisson of negative binomial regression with zero inflation . unfortunately , there is nothing in the literature about the application of zero inflated poisson or negative binomial for modeling of the failed courses and semesters in university students . this studyintend to apply statistical modeling , especially poisson and negative binomial regressions with zero inflated to model the number of failed courses and semesters in the students of iran university of medical sciences . in these models some educational and demographic factors which affect the academic performance of the students were used as the predictor variables . this cross - sectional study was performed in 2009 and 2010 in iran university of medical sciences . the target population was all current students of the university in 2009 which were almost 6000 in different educational levels . the sample of 670 students was selected using stratified random sampling . to choose the sample from the target population , simple random sampling in each stratum the demographic and educational data was collected from the university education file and the information regarding to attendance of the student in university accommodation was collected from the deputy head of the university in students affairs . the data consist of the first and surnames of the students , the student i d , discipline , faculty ( one for each faculty and zero for others ) , the educational level ( one for each level and zero for others ) , gender ( one for males and zero for females ) , the year of entrance , marital status according to year of entrance ( single=1 and married=0 ) , using university accommodation , being native , quota system ( no quota=1 , others=0 ) , university average , the number of failed courses and semesters . the students score are from zero to 20 in all iranian university systems and if a student receives a score of less than 10 in undergraduate or less than 14 in postgraduate courses , he fails the course . also , the mean of less than 12 in undergraduate and less than 14 in postgraduate semesters are considered as the failed semester . the students can not pursue their study if they have more than four failed semesters . the students demographic and educational data were combined to the file of the number of failed courses and semesters to produce the final data set . for data analysis and modeling , the descriptive statistics and zero inflated poisson regression and zero inflated negative binomial regression were used to analyze the final data set . the number of students with no failed courses were 478 ( 71.3% ) and 192 ( 28.7% ) students had one or more failed courses . the students with one , two , three , four or more failed courses were 92 ( 13.7% ) , 37 ( 5.5% ) , 22 ( 3.3% ) and 41 ( 6.2% ) , respectively . the number of students with one or more failed semester were 24 ( 3.6% ) and 31 ( 4.6% ) , respectively . the results of zero inflated ( zi ) poisson regression fit are shown in table 1 . in poisson regression for the number of failed courses ( table 1 ) , one unit increase in total university average resulted in 0.64 decreases in logarithm of the number of failed courses . also , one unit increase in the number of failed semesters caused 2.73 decreases in the logarithm of the odd of inflated zero . furthermore , the change of educational level from other levels to bachelor or master levels resulted in 0.27 and 0.96 decrease of the expected logarithm of the numbers of failed courses . finally , the change of gender from male to female caused 0.24 decrease of expected logarithm of the numbers of failed courses , and the change of marital status from married to single increased 0.05 of the expected logarithm of the numbers of failed courses . in the regression of failed semesters , the university average had negative relationship with the numbers of failed semesters and a unit increase in university average resulted in 0.24 decrease in expected logarithm of the numbers of failed semesters . also , with the change of quota system from other quota system to free quota system , the expected logarithm of the numbers of failed semester decreased by 0.47 . furthermore , a unit increase in the number of failed courses caused the logarithm odd of inflated zero to decrease by 1.56 . * - using robust method as one can see , in regression of the number of failed courses , a unit increase in university average resulted in 0.83 decreases in logarithm of the number of failed courses . also , with a unit increase in the number of failed semesters , the logarithm of odd of the zero inflated decreased by 35.88 . the change of faculties from other to faculty of management and rehabilitation caused 1.24 and 1.07 increase in expected logarithm of the number of failed courses , respectively . the change of educational level from other levels to bachelor , certificate and master levels , the expected logarithm of the number of failed courses decreased by 0.81 , 0.8847 and1.45 , respectively . in regression of failed semesters , the university average has negative relationship with the number of failed semesters and with a unit increase in university average , the expected logarithm of the number of failed semesters decreased by 0.42 . also , with the change of quota system from other system to free quota system , the expected logarithm of the numbers of failed semesters decreased by 0.47 . furthermore , with a unit increase of the numbers of failed courses , the logarithm odd of inflated zero decreased by 1.56 . all of these results are the same as table 1 on the regression with the response variables of the number of failed semesters . the research results showed that in poisson and negative binomial regressions with zero inflated using the number of failed semesters as the response variable , the variables of the university average and quota system have inverse relationship with the response variable , so the increase of the university average and the change from other quota system to free quota system caused a decrease in failed semesters . in a research ( 10 ) it was shown that in the students with free quota system , the number of failed semesters is less than other students which is the same as this study . also , we have to consider the fact that failing is less in clever students and in these students the failure is a rare outcome . furthermore , the students with free system are almost clever and have very less failure . therefore , quota system is from the factors which decrease the failed semesters . on the other hand , a unit increase in the number of failed courses in both models , decrease the logarithm odd of inflated zero . thus , the increase in the number of failed courses resulted in the number of failed semesters . this is in the same direction due to the high correlations ( 0.81 ) of the failed courses and semesters . therefore , with increase of the number of failed courses , the chance of having failed semesters increase . in both regression models with the response variable of the number of failed courses , the university average and the bachelor and master educational levels have negative relationship . in both models , the increase of university average resulteda decrease of the number of failed courses , and the decrease in negative binomial regression is more than poisson regression . in a research it was shown that there is a negative relationship between the university average and the number of failed courses which is the same as this research ( 10 ) . also , the change from other educational levels to bachelor and master levels caused a decrease of the number of failed courses in these levels . the decrease in the number of failed courses in bachelor and master levels is logical due to the little failure courses in these levels . in poisson regression with the failed courses as the response variable , the variables of genderandmarital status have inverse and positive relationship to the number of failed courses , respectively , and the failed courses are more in males than females and in singles than married . in different research ( 5,6,8,10,11 ) , it was shown that gender and marital status have effect to the failed courses or semesters . research it is shown that the singles are more successful in their educations than married and have less failed courses which is opposite of this study ( 12 ) . in the negative binomial regression , with the number of failed courses , the variables of bachelor level and the faculties of management and rehabilitation caused the increase in the number of failed courses . since the number of failed courses in bachelor level is more than master level , so the result makes sense . also , rehabilitation faculty has the most failure in the university which is the same as the model result . finally , in seems that the entrance of the management faculty to the model , with many failures , is for adjustment of the effects of other variables in the model . on the other hand , a unit increase in the failed semester , in both models , increases the logarithm odd of inflated zero and this reduction in negative binomial model is 13 times of the poisson model , and this result was expected due to the high correlation between the failed courses and semesters . it is concluded from all results that in the failed courses regression model , the university average and quota system have the most roles and the increase in university average resulted in the reduction of failed semesters . also , the change from other quota system to free quota system resulted in the decrease of failed semesters . furthermore , in these models , the increase in the number of failed courses cause a decrease in the logarithm odd of zero inflated . therefore , with the increase of the university average and choosing free quota system it is possible to reduce the failed semesters . in the regressions of the number of failed courses , the university average has an important role and its increase causes the decrease of the number of failed courses . other affected variables to the number of failed courses are different in both models . on the other hand , the increases in the number of failed semesters , decrease the logarithm of zero inflated in both models and the amounts of these changes are different . therefore , the increase in university average and choosing more bachelor and master students can reduce the failed courses . this work was supported by iran university of medical sciences under the grant number of 370 .

background : the number of failed courses and semesters in students are indicators of their performance . these amounts have zero inflated ( zi ) distributions . using zi poisson and negative binomial distributions we can model these count data to find the associated factors and estimate the parameters . this study aims at to investigate the important factors related to the educational performance of students . methods : this cross - sectional study performed in 2008 - 2009 at iran university of medical sciences ( iums ) with a population of almost 6000 students , 670 students selected using stratified random sampling . the educational and demographical data were collected using the university records . the study design was approved at iums and the students data kept confidential . the descriptive statistics and zi poisson and negative binomial regressions were used to analyze the data . the data were analyzed using stata . results : in the number of failed semesters , poisson and negative binomial distributions with zi , students total average and quota system had the most roles . for the number of failed courses , total average , and being in undergraduate or master levels had the most effect in both models . conclusion : in all models the total average have the most effect on the number of failed courses or semesters . the next important factor is quota system in failed semester and undergraduate and master levels in failed courses . therefore , average has an important inverse effect on the numbers of failed courses and semester .

pubmed

electrostatic interactions have been shown to play a vital role in enzyme catalysis . these interactions assist in the binding of ligands and facilitate the enzyme - catalyzed chemical reactions by keeping the reacting species positioned properly as well as stabilizing the transition state in some cases . moreover , these electrostatic interactions are expected to be significantly modulated by the conformational changes that occur over the catalytic cycle . in particular , the electrostatic environment in the active site is likely to change as the ligands bind , the chemical reaction occurs , and the ligands are released . quantifying the electrostatic contributions to the individual catalytic steps , as well as the impact of conformational changes on the electrostatic interactions , is a challenging yet important goal in the field of enzyme catalysis . to gain a better understanding of the catalytic role of electrostatics and conformational motions , we probed the changes in the active site electrostatic environment resulting from structural rearrangements that occur along the catalytic cycle of escherichia coli dihydrofolate reductase ( ecdhfr ) . this enzyme catalyzes the nadph - dependent conversion of 7,8-dihydrofolate ( dhf ) to 5,6,7,8-tetrahydrofolate ( thf ) . x - ray crystallographic , nuclear magnetic resonance ( nmr ) , theoretical , and photophysical studies have shown that ecdhfr exhibits significant structural rearrangements along the catalytic cycle . thus , ecdhfr is a highly suitable system for studying how conformational changes can affect the active site electrostatic environment , which can then modulate enzyme catalysis . recently , thiocyanate and other vibrational probes have been incorporated into a number of biological systems to investigate the local electrostatic environments . boxer and co - workers have explored the use of nitrile ( cn ) probes to detect differences in the electric field projected along the cn axis in proteins upon ligand binding , protein folding , and protein protein association . using this method , they showed that the electrostatic environment in the active site of -3-ketosteroid isomerase ( ksi ) exhibits limited rearrangement upon photoexcitation of a ligand , where this excitation was proposed to mimic the movement of charge during the catalyzed chemical transformation . the authors concluded that the ksi active site is electrostatically preorganized for catalysis in its tertiary protein structure and that major rearrangement is not necessary for catalysis . this observation is not unexpected due to the relatively rigid structure of ksi that limits substantial conformational mobility , thereby limiting electrostatic rearrangement . however , calculations have shown that further active site reorganization , manifesting as small conformational changes that position the reacting species for chemistry , is still required in ksi to enable the catalyzed proton transfer reactions . in contrast to ksi , which exhibits limited protein motion along the catalytic cycle , ecdhfr is known to undergo significant conformational changes along the catalytic cycle , suggesting that larger changes in electrostatics could play a more significant role in all steps of the catalytic cycle . to examine the electrostatic interactions within the active site of ecdhfr , we incorporated thiocyanate probes at site - specific locations . using vibrational and c nmr spectroscopic techniques , as well as computer simulations , we analyzed the electrostatics and degree of hydration of the microenvironments surrounding the probes . the electrostatic interactions arising from the active site microenvironments surrounding the small , catalytically nonintrusive probes led to different observed cn vibrational stretching frequencies and c nmr chemical shifts along the catalytic cycle . experimental data clearly show changes in the active site electrostatic environment due to structural rearrangements . further insights were obtained through mixed quantum mechanical and molecular mechanical ( qm / mm ) analyses of classical molecular dynamics ( md ) simulations . these calculations were able to reproduce the experimentally measured vibrational frequency shifts reported by the probes across the enzyme - catalyzed hydride transfer step of the catalytic cycle . moreover , the electrostatic contributions from the active site microenvironments surrounding the probe were decomposed into the major components ( individual residues and ligands ) . in addition , the experimentally validated theoretical model allowed us to examine the electric field along the hydride transfer pathway between the substrate and cofactor , a property that is catalytically important yet unattainable experimentally . this integrated approach provides valuable insights into the interplay between electrostatic and conformational changes along the catalytic cycle of ecdhfr . 7,8-dihydrofolate ( dhf ) and [ ( 4r)-h]nadph ( nadpd ) were prepared according to previously described procedures . -nicotinamide adenine dinucleotide phosphate reduced tetra(cyclohexylammonium ) salt ( nadph ) , -nicotinamide adenine dinucleotide phosphate hydrate ( nadp ) , potassium cyanide - c ( 99 atom % c ) , methotrexate , methotrexate - agarose , dithiothreitol ( dtt ) , folic acid ( 97% ; fol ) , sodium phosphate monobasic monohydrate ( 98102% ) , and 5,5-dithiobis(2-nitrobenzoic acid ) ( ellman s reagent ) were purchased from sigma - aldrich and used without further purification . sodium phosphate dibasic heptahydrate and 2-nitro-5-thiocyanatobenzoic acid ( 97% ; ntcb ) were purchased from emd and tci america , respectively . ph values were measured using an accumet model 13 - 620 - 300 standard combination electrode calibrated with vwr certified standard aqueous buffers ( ph = 4 , 7 , and 10 ) . all of the experiments , including kinetics and spectroscopic measurements , were done in ph 7.0 50 mm sodium phosphate buffer unless otherwise specified . first , the two native cysteines ( c85 and c152 ) in wild - type ( wt ) ecdhfr were mutated to ala and ser , respectively , to generate cys ecdhfr using the stratagene quikchange site - directed mutagenesis kit and the wt ecdhfr template as described elsewhere . primer sequences were 5-c gaa gcc atc gcg gcg gct ggt gac gta cca g-3 ( c85a ) and 5-c tcg cat agc tat tca ttc gaa atc ctc g-3 ( c152s ) . the choice of amino acid substitution was based on a previous report showing that the c85a / c152s substitution on ecdhfr induced minimal perturbation to the enzymatic activity . selective incorporation of cysteine was achieved through subsequent mutations using the following primers : 5-ca atc ggt agg cct tgc ccc ggc cgc aaa aat att atc-3 ( l54c ) and 5-g att atg ggg cgc cat tgc tgg gaa tca atc g-3 ( t46c ) . plasmid construction , protein expression , and purification of mutant dhfrs were performed according to published protocols . labeling of the single - cysteine ecdhfr constructs ( cys l54c and cys t46c , denoted as l54c - cn and t46c - cn , respectively ) was achieved using a modified version of a published procedure . inside an amicon ultra-4 10k nominal molecular weight limit ( nwml ) centrifugal filter tube , approximately 400 m of each ecdhfr construct was mixed with four times excess of ntcb in 4 ml of 0.1 m sodium phosphate buffer at ph 7.4 . the mixture was allowed to react at room temperature for 10 min , and reaction progress was monitored by the appearance of the phenolic product using a uv vis spectrophotometer . the protein mixture was then concentrated by spinning at 5k rpm in a centrifuge at 4 c . the protein sample was then washed twice with 12 mm kcn in ph 7.0 50 mm sodium phosphate buffer , with a concentration step after each wash . this was followed by washing the protein sample with 50 mm ph 7.0 sodium phosphate buffer two more times before concentrating the protein solution down to 0.8 ml , which was then applied onto a size exclusion column ( econo - pac 10dg ; bio - rad ) that was pre - equilibrated with ph 7.0 50 mm sodium phosphate buffer . the isolated labeled proteins were concentrated in an amicon ultra-4 10k nmwl centrifugal filter tube . the intact masses of scn labeled and unmodified samples were confirmed by esi - ms , which yield deconvoluted m / z values of 17965 ( l54c)/17978 ( t46c ) and 17940 ( l54c)/17953 ( t46c ) da , respectively . the theoretical molecular weights for the unmodified samples are 17941 ( l54c)/17953 ( t46c ) da . similar preparation procedures were used for the scn labeled samples . a mixture containing 10 mm ellman s reagent and 0.5 m kcn in ph 7.0 0.1 m sodium phosphate buffer was prepared at room temperature for 15 min . this mixture was then allowed to react with a sample of the unlabeled protein in an amicon ultra-4 10k nmwl for 10 min at room temperature . the final concentrations of a typical reaction mixture were 500 m protein , 5 mm ellman s reagent , and 0.25 m kcn . the subsequent purification and isolation steps were similar to those for scn labeling , except that kcn was used for washes rather than kcn . successful labeling was confirmed spectroscopically by the release of one equivalent of the phenolic product as well as c nmr comparison with the unlabeled proteins . the pre - steady - state hydride transfer reaction was monitored on an applied photophysics stopped - flow spectrophotometer thermostated at 25 c . all of the enzymatic reactions were performed in 50 mm sodium phosphate buffer ph 7.0 . one of the syringes in the stopped - flow analyzer was loaded with 20 m enzyme , 250 m nadph , and 2 mm dtt . after combining dhfr and nadph as described above , the mixtures were prepared on ice for 5 min prior to the onset of the chemical reaction . upon mixing , the final concentrations of the individual species in the reaction chamber were halved . the conversion of dhf to thf was followed by the loss of fluorescence resonance energy transfer from the enzyme to nadph . the reaction mixture was excited at 290 nm , and the emission was measured using a 400 nm cutoff output filter . the measure of absorbance vs time trace ( of burst phase ) was fit to standard single exponential decay to obtain the hydride transfer rate ( khyd ) . the kinetic isotope effect ( kie ) for the hydride transfer rate was also determined under the same experimental conditions , with the exception that nadpd was used instead of nadph in comparative experiments . steady - state enzyme turnover kinetics ( kcat ) were studied following similar experimental conditions , with the exception that the reaction progress was monitored at 340 nm . under each condition , at least 5 independent kinetic runs were conducted , and the average rate constants were used for analysis . ftir spectra were obtained at room temperature using a mattson research series ftir spectrometer ( madison instruments , inc . ) equipped with a liquid - nitrogen cooled hgcdte ( mct ) detector ( infrared associates , inc . ) . cyanylated dhfr samples ( [ protein ] = 1.72.5 mm ) were mixed with appropriate ligands ( 1:10 mol equiv ) in aqueous buffer and placed in a liquid cell with two caf2 windows separated by a 56 m teflon spacer . all nitrile ( cn ) stretch absorbance spectra for cyanylated dhfr samples consisted of 2000 scans and the data were collected with 1.0 cm resolution . the cn stretch absorbance spectra for cyanylated dhfr samples were obtained by subtracting a spectrum of the aqueous buffer solution from the protein spectrum . the baseline for each spectrum was corrected by fitting a fifth - order polynomial fit to the data , as done previously by fafarman et al . and mcmahon et al . the fifth - order polynomial local fit functions were obtained by defining roots at least 15 cm from the peak maximum , meaning the experimental data and fits were forced to be equal at points more than 15 cm from the central frequency . all published spectra are the averages of two independently prepared protein samples after the baseline has been corrected and the peak height has been normalized to one . the peak positions obtained from independently prepared protein samples typically differed by < 0.5 cm , which is substantially less than the variation among peak positions due to the protein binding or releasing the ligands . detailed experimental setup and data analysis are provided in the supporting information . c nmr spectra were collected on a bruker av - iii-600 ( 150.9 mhz c frequency ) at 25 c . samples were prepared with 1.6 mm of the scn labeled protein , 8 mm ligands , and 50 mm potassium phosphate ( ph 7.0 ) in water . a sealed capillary tube containing 1 m acetone in d2o was inserted into the nmr tube that holds the protein sample . the 1d c nmr spectral data obtained for each sample were compared with its corresponding c dept 45 ( distortionless enhancement by polarization transfer ) data to confirm the identity of the assigned peaks . under the same experimental conditions , we were unable to detect the c nmr signals corresponding to the scn probes for samples containing either the nonisotopically enriched scn label or the unlabeled proteins . crystals of e. coli t46c - cn and l54c - cn dhfr variants were obtained by the hanging - drop vapor diffusion method using conditions similar to those previously described . detailed methods , data collection , and refinement statistics are provided in the supporting information . we calculated the ir spectra for the cn stretching mode of the incorporated thiocyanate probes using a two - step method . first we propagated classical md trajectories for the entire solvated enzyme with bound ligands . the configurations sampled from the md trajectory were then analyzed using a series of qm / mm single point energy calculations to determine the cn stretching potential energy curve . the qm region was treated with a semiempirical method that was reparameterized for the calculation of nitrile vibrational frequencies , and the mm region was represented as a collection of atomic point charges . the instantaneous vibrational wave functions and associated frequencies from the energy level splittings were obtained from the cn stretching potential energy curve using a grid - based numerical approach . the simulated ir spectra were calculated using the fluctuating frequency approximation , which includes the dynamical contributions of the protein motion to the simulated lineshapes . a full description of the methodology can be found in our previous application to ksi , with some minor adjustments that will be discussed below . we propagated classical md trajectories for the cysteine - free t46c - cn and l54c - cn ecdhfr mutants using the gromacs program . the first state has folate ( fol ) bound in the substrate pocket and the nicotinamide adenine dinucleotide phosphate cofactor bound in the oxidized form ( nadp ) . in this state , the met20 loop is in the closed conformation , which is thought to facilitate the hydride transfer reaction by positioning the ligand and cofactor while excluding solvent molecules . this state has been shown to structurally mimic the reactive michaelis complex ( e : dhf : nadph ) prior to hydride transfer . although we can simulate the reactive michaelis complex without hydride transfer occurring , we simulated the e : fol : nadp ternary complex to compare directly with the experimentally measured ir spectra . the second state that we simulated has the product thf and nadp bound . in this state , the met20 loop is in the occluded conformation , and the nicotinamide ring of nadp is excluded from the active site . these two states are used to model the closed to occluded transition that occurs across the hydride transfer step of the ecdhfr catalytic cycle . the initial structures for the md simulations were obtained from crystal structures solved with the nitrile probes included for both the l54c - cn and t46c - cn variants ( pdb accession codes 4p68 and 4p66 , respectively ) . the crystal structures have methotrexate bound in the substrate binding pocket and nadph bound in the active site . in both of these structures , this state is thought to mimic the transition state for the hydride transfer reaction . for the simulations in the closed conformation ( nadp / fol ) , the folate molecule was incorporated by aligning the folate substrate with the corresponding heavy atoms of the methotrexate ligand from the crystal structure . for the simulations in the occluded conformation ( nadp / thf ) , we used the initial coordinates of a wt crystal structure of ecdhfr complexed with nadph and 5,10-dideazatetrahydrofolate ( ddthf ) ( pdb 1rx6 ) . the thiocyanate probes were incorporated into the occluded structure by aligning the backbone atoms of the five previous and five subsequent residues to the cyanylated cysteine residue in the new crystal structure that contains the probe to the corresponding backbone atoms of the 1rx6 structure . we then replaced the specific residue with the aligned cyanalated cysteine and optimized the probe residue geometry and position using a steepest - descent algorithm in gromacs , while the ligands , the crystallographic water molecules , and the rest of the enzyme were held fixed . the substrate ligand ( ddthf ) was replaced by thf by aligning the corresponding heavy atoms in ddthf and thf , and the oxidized cofactor was modeled by aligning the heavy atoms of nadp with the nadph from the crystal structure . the modeled ligands were also optimized using the steepest - descent algorithm with the enzyme and the crystallographic water molecules held fixed . for all of the md simulations , we used the amber99sb force field to compute the potential energy and forces for the protein . the bonded and nonbonded parameters for the nadp cofactor were consistent with those from our previous work . the fol and thf charges were calculated using the restrained electrostatic potential ( resp ) method , and the bonded and van der waals parameters were obtained from the generalized amber force field ( gaff ) . for methyl thiocyanate and the cyanylated cysteine residue , the bonded parameters were obtained from cho et al . , and the nonbonded parameters were obtained from lindquist and corcelli , as used in our previously published work on ksi . each of the enzymatic systems was solvated with tip3p water in a truncated octahedral box , where the sides of the box were at least 10.0 from every atom in the enzymatic complex , including the crystallographic waters . after solvation , we added 12 sodium atoms using the gromacs genion utility to neutralize the system before simulation . all bonds involving a hydrogen atom were constrained to their equilibrium bond lengths using the lincs algorithm as implemented in gromacs . nonbonded van der waals interactions were calculated with a cutoff of 1.0 nm , and electrostatic interactions were calculated using the particle - mesh ewald method . after the initial preparation of each system , we followed a well - defined equilibration procedure . the solvent configurations were minimized using the steepest - descent algorithm in gromacs with the enzyme and ligands held fixed to mitigate any possible bad contacts between the solvent and solute molecules . the solvent molecules and counterions were then equilibrated for 50 ps at 300 k in the canonical ( constant nvt ) ensemble with the protein and ligands still fixed . after solvent equilibration , the entire system was then minimized using the steepest - descent algorithm . subsequently , the system was equilibrated using a simulated annealing procedure in which the initial velocities were generated according to a boltzmann distribution at 50 k. the system was equilibrated in the isothermal isobaric ensemble ( constant npt ) at 50 k for 50 ps , and then the temperature was increased up to 300 k in increments of 50 k with 50 ps of equilibration at each temperature . after this simulated annealing procedure , an additional 1 ns of equilibration in the constant npt ensemble was performed , and finally 1 ns of equilibration in the constant nvt ensemble was performed . during the simulations , the nos hoover thermostat was used to maintain the temperature , and the parrinello the production trajectories were propagated in the canonical ensemble at 300 k with a time step of 1 fs and a total simulation time of 1 ns for each independent trajectory . configurations were saved at every time step and were analyzed using the qm / mm methodology described below . for each mutant and catalytic state , we propagated at least two independent trajectories with different initial configurations . the data for a single trajectory are presented in the main paper , while data from additional trajectories are provided in the supporting information . the results for independent trajectories associated with the same system were found to be consistent within the numerical errors of the methodology . the methodology used to calculate the vibrational frequency of the nitrile group is similar to that used in our previous work . here we highlight the differences between the methods used in our previous work and the methods employed in the present work . as in our previous work , we calculated the vibrational frequency by generating the one - dimensional potential energy curve along the cn stretching mode using a qm / mm method . the potential energy is calculated along the cn vibrational coordinate by freezing all of the atomic centers in the system and moving only the nitrogen atom to stretch the cn distance from 1.0 to1.4 with a step size of 0.05 . the qm calculations were performed with the mm region treated as atomic point charges that polarize the qm region . these atomic point charges were the same as those used in the md simulations and were obtained from the amber99sb force field for the standard amino acid residues , the tip3p force field for the water molecules , and the resp calculations for the ligands . the choice of the qm region for each probe studied in this work will be discussed below . in our previous work , the one - dimensional potential energy curve for the cn stretching mode was fit to a morse function , and the energy levels were calculated analytically from the morse parameters . in the present work , the potential energy curve was fit to a third - order b - spline potential , and the vibrational wave functions and energy levels were calculated numerically using the fourier grid hamiltonian ( fgh ) method . an advantage of the fgh method is that it does not assume any functional form of the potential energy curve and therefore avoids the bias associated with fitting to the morse function , which depends on only three parameters . in principle , any electronic structure method can be used to calculate the vibrational stretching potential , but the level of theory must be computationally tractable for the millions of configurations that are required to obtain statistically converged ir spectra . as in our previous work , we used a reparameterized version of the semiempirical pm3 method . in this semiempirical method , some of the computationally expensive integrals in hartree these empirical parameters can be varied to reproduce properties from either electronic structure theory calculations or experimental measurements . because we altered our approach to use the fgh numerical method to calculate the vibrational frequencies , we reoptimized the pm3 parameters . the reparameterization procedure is similar to that from our previous studies , and a full description is provided in the supporting information . we also confirmed that the new set of parameters reproduces the results for ksi from our previous work when the fgh numerical method is used for calculating the vibrational frequencies . table s6 provides the calculated frequencies for the two different vibrational probes in ksi that were studied previously . to assist in the analysis of the vibrational frequency calculations , we calculated the average electric field at the midpoint of the cn bond in the thiocyanate group from our md simulations . the vibrational stark effect between two states is described by1here h is planck s constant , cn is the shift in vibrational frequency between the two states , eenv is the difference in the electric field vector at the nitrile bond as a result of the change in the environment , cn is the difference dipole moment for the vibrational mode that lies along the cn bond axis , and is the angle between these two vectors . the magnitude of cn is the stark tuning rate , which is a measure of the susceptibility of the probe to changes in the local electric field . boxer and co - workers have measured the stark tuning rate for thiocyanate probes in various environments , including in proteins , to be 0.7 cm/(mv / cm ) . we emphasize that this equation accounts only for the classical electrostatic effects of the environment and does not account for the quantum mechanical aspects of effects such as specific hydrogen - bonding interactions and polarization . while these phenomena have significant classical components , they also have nonclassical aspects that can be incorporated into the calculations by treating interacting residues quantum mechanically , as will be discussed below . recent work explores the role of a local field correction factor that scales the externally applied electric field to obtain the local field experienced by the probe , which is typically larger because of the polarization of the local environment induced by the applied electric field . this local field correction factor has been estimated to be between 1.1 and 1.3 for protein environments , but recent investigations have suggested that it may be as large as 1.8 . we have not considered the role of the local field correction factor in our analysis of the electric fields in dhfr due to the lack of consensus about its value for a protein environment . moreover , applying the local field corrections would result in changes that are within the error bars of our calculations . to calculate the electric field at the nitrile bond , a virtual particle was positioned at the midpoint of the cn axis during the md trajectory . this virtual particle had no electrostatic or van der waals interactions with other particles , and the bond , angle , and dihedral parameters were set to zero . the configurations from the md trajectory were saved , and subsequently the forces acting on all particles were recalculated for these configurations when the charge on the virtual particle was set to + 1 . the electric field , evp , at the virtual particle location was calculated as2where fvp is the force acting on the virtual particle and qvp is the charge of the virtual particle . the total electric field vector , as well as the component along the cn axis , was calculated for each configuration along the md trajectory . calculation of the component of the electric field along the cn axis allows us to calculate the change in the vibrational frequency due to classical electrostatics using eq 1 . the remaining frequency change can be attributed to the quantum mechanical aspects of specific hydrogen - bonding interactions and polarization effects . these effects can be investigated by using a classical polarizable force field or by treating the interacting residues quantum mechanically . note that the experimentally measured and theoretically calculated frequency shifts can be compared directly , but the breakdown into the classical electrostatics and quantum mechanical aspects is available only from the theoretical calculations . we also analyzed the contributions of individual residues and ligands to the electric field at the midpoint of the cn bond . because the classical electric field is the sum of the individual contributions from each atom , the electric field analysis of the contributions from specific residues provides insight into the impact of conformational changes occurring during the closed to occluded transition associated with hydride transfer . we generated the t46c - cn and l54c - cn mutants by site - specifically incorporating a thiocyanate probe at each position ( figure 1 ) . the t46c - cn probe is situated in close proximity to the junction between the two reacting ligands , dhf and nadph , allowing us to monitor the microenvironment close to the reaction center . the l54c - cn probe is located 9 from the center of the hydride donor acceptor ( d a ) axis . this probe monitors the folate binding pocket , and its spectral character is expected to respond to the formation of the ternary michaelis menten complex upon folate binding , as well as the release of thf in the enzyme turnover step . ( a ) superposition of the t46c - cn and l54c - cn ecdhfr mutants in the closed conformation with folate and nadp bound , where only the thiocyanate residue is shown for the l54c - cn mutant . ( b ) ecdhfr in the closed ( red ) and the occluded ( blue ) conformations exhibited by the ( c ) five major complexes in its catalytic cycle . the enzyme activity of the two ecdhfr variants was evaluated by their ability to catalyze the nadph - dependent reduction of dhf and the enzyme turnover rates at ph 7.0 and 25 c . the catalytic efficiencies of the t46c - cn ( khyd = 120 10 s ; kcat = 6.5 0.5 s ) and l54c - cn ( khyd = 250 20 s ; kcat = 28 3 s ) variants were comparable to that of wt ecdhfr ( khyd = 220 s ; kcat = 12 s ) . we also found the cys ecdhfr ( c85a / c152s ; khyd = 215 8 s ; kcat = 10.3 0.8 s ) variant to exhibit activity similar to the wt enzyme . furthermore , figure s1 shows that the crystal structures of the ternary complexes of the t46c - cn and l54c - cn mutants in the closed conformation overlap well with the wt crystal structure . these observations suggest that the incorporation of the scn probes into the selected locations of the active site does not significantly alter the structure or activity of the enzyme . it should be noted that we also incorporated the scn probe at the i50 position ( cys - i50c - cn ) . however , such a modification decreased the hydride transfer rate by 10 times at ph 7.0 and 25 c . we felt that the degree of enzyme activity reduction in cys - i50c - cn might result in a less accurate representation of the electrostatic environment in the active site of the wt enzyme . thus , further investigations were not performed for the cys - i50c - cn construct . it is expected that even small modifications to the crowded active site can have large effects on enzyme activity . the ecdhfr catalytic cycle contains the five major species ( figure 1c ) that reflect the significant conformational changes in the active site met20 loop ( figure 1b ) . menten complex ( e : nadph : dhf ) to an occluded conformation in the initial ternary product complex ( e : nadp : thf ) . the enzyme remains in the occluded conformation in the two subsequent product - containing species ( e : thf and e : nadph : thf ) , until the met20 loop reverts back to the closed conformation upon the rate - limiting release of thf to generate e : nadph ( figure 1c ) . nmr relaxation dispersion experiments have found that the rates of the met20 loop conformational change are temporally similar to their corresponding enzymatic processes . in other words , the hydride transfer and enzyme turnover rates are similar to the values determined from the nmr experiments for the conformational transitions that connect the relevant enzyme complexes . while the major change in the met20 loop conformation appears to be induced by the formation of the product thf , theoretical simulations have shown that minor structural rearrangements of the ligands and protein , not restricted to the met20 loop , are necessary to progressively optimize the substrate orientations and the active site environment along the reaction coordinate of the hydride transfer reaction . herein , we utilized the scn probes to experimentally monitor the changes in the local microenvironments in two locations in the active site over the five major catalytic species . the five complexes that constitute the catalytic cycle of ecdhfr ( figure 2 ) were generated using 1.52.5 mm of the enzyme and at least five times excess of the ligands in ph 7.0 phosphate buffer . these concentrations should be more than sufficient to push the equilibria toward full complexation , thus allowing us to obtain vibrational ftir spectra of the five major complexes that constitute the ecdhfr catalytic cycle , as well as for the apoenzyme and the e : nadph : fol complex . note that the e : nadph : dhf state is modeled by e : nadp : fol , as discussed in the literature . for each enzyme complex , the ir spectrum was obtained in duplicate / triplicate using separately prepared samples , and these spectra were found to be very reproducible ( data in supporting information ) . after correcting the ir data for baseline and subsequently normalizing the amplitude of the peak to 1.0 , we determined the maximum frequency ( max ) from the first derivative of the corrected ir peak . the peak position is a measure of the transition energy between the vibrational ground state and first excited state of the thiocyanate probe . the transition energy is sensitive to the electric field along the cn bond because the vibrational potential energy function is perturbed by the local electrostatic environment ( surrounding residues , ligands , and solvent molecules ) which modulates the energy difference between the vibrational quantum states . color coding the frequency of maximum absorption detected in each complex illustrates the changes in the local electrostatic interactions experienced by the cn probe over the catalytic cycle ( figure 2 ) . ( b ) nmr ( scn ) measurements along the catalytic cycle of ecdhfr . the color scales for the ftir and nmr data have units of cm and ppm , respectively . the cofactor is orange , and the substrate / product is purple in all complexes ( the coloring of the cofactor and substrate / product does not correspond to the color scales that represent the nmr shift or ir frequency ) . enzyme complex labels in blue are in the occluded conformation , and those in red are in the closed conformation . the measured values for ir frequency and nmr chemical shift are provided in table s4 . figure 2 indicates that the local environment of each cn probe changes significantly over several key steps in the catalytic cycle : formation of the ternary michaelis menten model ground state complex , the hydride transfer step , and the rate - limiting release of thf . the largest frequency shifts observed for the t46c - cn probe occurred across the two most important steps in the catalytic cycle : ( 1 ) upon the conversion between the model michaelis menten e : nadp : fol complex and the initial e : nadp : thf product complex ( 4.1 cm ) , which is analogous to the hydride transfer step ; ( 2 ) across the enzyme turnover step , which is the release of thf ( 5.5 cm ) . for the l54c - cn probe , the most significant change occurred upon the formation of the ternary e : nadp : fol complex ( 2.4 cm ) . crystal structures of the wt enzyme have shown that the t46 and l54 residues retain their positions and orientations in the five catalytic complexes , suggesting that the inserted cn probes likely experience limited freedom of movement in the active site . md simulations confirm that there are no major conformational changes in the probe orientation during 5 ns trajectories . therefore , the observed changes in the microenvironments as the system moves through the various complexes in the catalytic cycle are likely due to protein conformational rearrangements that induce changes in the electrostatic interactions between the probes and their surroundings . note also that the redox state of the cofactor ( nadph / nadp ) has very little effect on both the ir and c nmr data ( table s4 ) . in many cases , we also observed changes in the peak width [ full - width at half - maximum ( fwhm ) ; table s4 ] , which usually reflects the variation of chemical environments sampled by the probe as well as the strength of intermolecular interactions such as hydrogen bonding . for example , the l54c - cn probe , which is located in the folate binding site , reported peak widths that are approximately two times smaller in complexes with bound fol or thf . this observation is consistent with the probe sampling a less fluctuating and more well - defined electrostatic environment resulting from the various interactions that organize the fol / thf to bind in the active site . there is also some evidence indicating that the e : nadph complex and the apoenzyme exhibit a greater degree of hydrogen bonding between a solvent water molecule and the l54c - cn probe . one indication of this phenomenon is obtained from inspection of the c nmr - ir relationship , which shows that the data for the above two complexes deviate from the rest of the data in a manner that implicates greater specific solvent hydrogen - bonding interactions ( figure s4 ) . md simulations support the presence of additional solvent hydrogen - bonding interactions in these two complexes for the l54c - cn probe , with a water molecule hydrogen bonded to the nitrile probe in nearly half of the configurations . we also observed a slightly broader ir peak in the e : nadph : fol complex , which can be attributed to the slow reduction of fol over the course of the ir measurement . this is not unexpected since the l54c - cn probe is located in the folate binding pocket . these trends are less obvious for the t46c - cn probe , which seems to be located in a more fluctuating environment , as indicated by the relative ir peak widths . we also prepared the isotopically enriched cys - cn variants ( t46c - cn and l54c - cn ) to obtain the complementary c nmr data for all of the studied complexes . while the magnitude of the observed changes are different because the c nmr chemical shift is less sensitive to specific solvent hydrogen - bonding interactions than is the cn vibrational stretching frequency , the c nmr data illustrate similar trends to those shown by the corresponding ir data . in particular , we observed a changing microenvironment around the scn probes as the enzyme evolves through the catalytic cycle ( figure 2b ) . similar to the ir data , the microenvironment detected at the t46c - cn probe exhibits larger changes along the catalytic cycle than that at the l54c - cn probe . this trend is expected due to the proximity of the t46c - cn site to the regions of the substrates that undergo the hydride transfer reaction . overall , the experimental data summarized as figure 2 clearly demonstrate a dynamic electrostatic environment in the active site of ecdhfr across its catalytic cycle . the differences in the microenvironments of the two probes and the way they change among the different complexes , especially between the closed and occluded conformations , in the ecdhfr catalytic cycle also provide an indication of the highly heterogeneous nature of the enzyme active site . to identify the various environmental contributions that give rise to the observed cn stretching vibrational frequency shifts along the catalytic cycle , we conducted qm / mm simulations to monitor the electrostatic interactions experienced by the probes for the complexes along the catalytic cycle . these data provide information about how the microenvironments of the probes reorganize across individual steps of the catalytic cycle . we simulated the ir spectra of the t46c - cn and l54c - cn mutants of ecdhfr for two different complexes along the catalytic cycle : ( 1 ) the ternary complex with the reactant analog fol and nadp bound , which mimics the e : nadph : dhf state prior to hydride transfer ; ( 2 ) the ternary complex with the product thf and nadp bound , which is the e : nadp : thf state following hydride transfer . first we examined the local environment around the t46c - cn probe , which is located near the midpoint between the two bound ligands ( figure 1a ) . the qm region includes the t46c - cn residue , the substrate ( either fol or thf ) , and the nearest water molecule to the nitrile nitrogen , as depicted in figure 3a . the qm region was determined by calculating the cn frequency including the entire probe microenvironment in the qm region for a subset of the configurations sampled and comparing these results to those obtained with smaller qm regions . the details of the procedure used to determine the qm region are provided in the supporting information . configurations from md simulations for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr mutants . the residues with thicker lines are included in the qm region for the qm / mm calculations of vibrational frequencies . figure 4a presents the calculated and experimentally measured ir spectra for the t46c - cn mutant for both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . the calculated ir spectrum for the closed conformation ( solid black curve ) has a maximum at 2165.8 cm , which is within 1.8 cm of the measured peak in the experiments . the agreement between the calculated and experimental absolute frequencies is potentially fortuitous , but the level of agreement is similar to that calculated in our previous studies of ksi . the fwhm of the calculated ir spectrum for the closed conformation is 11 cm , which is in good agreement with the experimentally measured line width of 14 cm . the calculated ir spectrum for the occluded product state ( solid red curve ) has a maximum at 2162.1 cm . the calculated shift between these two states is 3.7 cm , which is in excellent agreement with the experimentally measured shift of 4.1 cm . note that our md simulations and cn frequency calculations are performed on the nanosecond time scale . the probe could experience multiple additional conformations on the longer time scale associated with the ir measurements . during our md simulations of 5 ns , no major structural rearrangements of the probe orientation occurred , indicating that we are only sampling a single conformational state . since the starting configurations for our simulations were based on crystal structures that include the probe , we expect to be sampling the most stable and therefore the dominant configuration of the probe . moreover , the agreement between the calculated and experimental cn frequency shifts provides further validation of the methodology . calculated and experimentally measured ir spectra of the cn vibrational stretching frequency for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound ( closed conformation ; black line ) and with nadp / thf bound ( occluded conformation ; red line ) . the solid lines are the simulated spectra from the qm / mm calculations , and the dotted lines are the measured spectra from the ftir experiments . in addition to the ir spectra , we calculated the electric field at the midpoint of the cn bond for configurations sampled by the md trajectory using the methodology described above . from these calculations , we can correlate the measured vibrational frequency shifts to changes in electrostatics from the protein environment . figure 5a depicts the calculated electric field along the cn axis for the two states . the average projected electric field for the closed ( nadp / fol ) and occluded ( nadp / thf ) states are 15.9 and 19.0 mv / cm , respectively . using the experimental stark tuning rate of 0.7 cm/(mv / cm ) in conjunction with eq 1 , the difference of 3.1 mv / cm corresponds to a shift in the vibrational frequency of 2.2 cm . the difference between this frequency shift arising from only classical electrostatics and the actual shift of ca . 4.0 cm obtained from both calculations and experiments is attributed to the quantum mechanical aspects of hydrogen - bonding interactions and polarization effects that are not included in the linear relation given by eq 1 . calculated electric field along the cn bond at the midpoint of this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound ( closed conformation ; black line ) and with nadp / thf bound ( occluded conformation ; red line ) . previously it has been shown experimentally and computationally that significant deviations of 10 cm from the vibrational frequency shifts predicted by eq 1 can occur when the nitrile probe participates in specific hydrogen - bonding interactions . according to our md simulations , the t46c - cn thiocyanate probe is solvent accessible in both the closed and occluded conformations and directly hydrogen bonds to a water molecule , as depicted in figure 3a . in both protein conformations , the nitrile probe is also able to act as a hydrogen - bond acceptor from the amine group in the substrate ( fol / thf ) backbone . note that the observation of hydrogen - bonding interactions with the probe for these two states is consistent with the experimental ir - nmr correlation data in figure s4 . thus , the difference between the frequency shift of 2.2 cm predicted from eq 1 , which includes only classical electrostatic effects , and the shift of 3.7 cm obtained from the qm / mm calculations of the full system can be attributed to hydrogen - bonding interactions with the water molecule and polarization effects from the water molecule and the substrate ( fol / thf ) . to confirm this hypothesis , we performed the qm / mm frequency analysis with only the probe residue ( t46c - cn ) in the qm region ( i.e. , switching the water molecule and the substrate from the qm region to the mm region ) . in this case , the calculated frequency shift between the two states was 2.6 cm , which agrees well with the 2.2 cm shift obtained from eq 1 . beyond the calculation of the total electric field at the probes , we can decompose the field along the cn axis into contributions from individual residues in the enzymatic complexes . in essence , this methodology allows us to decompose the microenvironment surrounding the cn probe into the constituent parts , which then allows us to identify the major contributing components . for the t46c - cn probe ( figure 3a ) , the sum of the contributions from the main contributors given in table 1 corresponds to a change in electric field along the cn axis of 2.9 mv / cm , which is consistent with the change of 3.1 mv / cm calculated with the entire enzyme . the contributions from the residues in the closed conformation are shown pictorially in figure 6a , where the residues that are colored blue contribute positively and those that are colored red contribute negatively to the total field along the cn bond . for the t46c - cn probe , the nadp cofactor and the nearest water molecule are the dominant contributors to the change in the electric field along the cn axis , although asn18 and ser49 also make significant contributions ( table 1 ) . the presence of a positive charge on the nicotinamide ring and the short 4 distance between the nitrile nitrogen and the aromatic nitrogen in the nicotinamide ring lead to a strong contribution to the electric field in the closed conformation . after the transition to the occluded conformation , the cofactor is excluded from the active site , and the magnitude of the contribution to the field from the nicotinamide ring is much smaller due to the larger distance of 14 between the two nitrogen atoms . this effect of the movement of nadp on the electric field at the probe demonstrates how atomic motions can significantly alter the local electrostatics in the active site . moreover , the 3.2 mv / cm change in the electric field contributed by asn18 and ser49 is consistent with the structural rearrangement from the closed to the occluded conformation . specifically , the amide nitrogen of asn18 is 1.5 closer to the nitrile probe in the closed conformation than in the occluded conformation , and the hydroxyl group of ser49 is 1.0 closer to the nitrile probe in the closed conformation . contributions to the calculated electric field along the cn bond at the midpoint of this bond for the ( a ) t46c - cn and ( b ) l54c - cn ecdhfr systems with nadp / fol bound and the met20 loop in the closed conformation . the color for each residue corresponds to the calculated values in tables 1 and 2 , respectively , using the color scale provided , although residues contributing a magnitude > 4.0 mv / cm are depicted in the darkest color . the residues that contribute negatively to the field are colored red , the residues that contribute positively to the field are colored blue , and the residues that have no net contribution to the field along the cn bond are colored white . changes in the electric field along the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . additionally , following the closed to occluded transition , the cn probe becomes more accessible to the solvent . the contribution to the electric field along the cn axis from the closest water molecule changes by 7.46 mv / cm during this transition . moreover , the sign of the average electric field along the cn axis due to this water molecule is different for the closed and occluded conformations , with a value of 2.14 mv / cm for the closed conformation and 5.32 mv / cm for the occluded conformation . analysis of the md trajectories indicates that a water molecule is hydrogen bonded to the nitrile probe 26% of the time in the closed ( nadp / fol ) conformation and 52% of the time in the occluded ( nadp / thf ) conformation . the positive contribution to the electric field along the cn axis from the nearest water molecule in the closed conformation is due to configurations in which the nearest water molecule has its negatively charged oxygen atom pointing toward the nitrile . these configurations are stabilized by the hydrogen - bonding interaction of the water molecule with the hydroxyl group of ser49 , as shown in figure s5 . the negative contribution to the field in the occluded conformation arises from configurations in which a hydrogen atom of the nearest water molecule is oriented toward the probe , forming a hydrogen bond between the water molecule and the cn group . thus , this computational approach is also able to probe the detailed active site hydration status , which is not easily accessible with experimental means . we performed the same analysis for the l54c - cn mutant to examine the microenvironment near the folate binding pocket . figure 4b depicts the calculated and experimentally measured vibrational spectra for the l54c - cn mutant in both the closed ( nadp / fol ) and occluded ( nadp / thf ) states . the l54c - cn probe is distal from the active site , and it is located in a relatively hydrophobic pocket near the carboxylate tail of the substrate , as shown in figure 1 . the qm region used for this mutant includes the l54c - cn residue , phe31 , and arg57 , as depicted in figure 3b . the qm region was determined by calculating the cn frequency including the entire probe microenvironment in the qm region for a subset of the configurations sampled and comparing these results to those obtained with smaller qm regions . the details of the procedure used to determine the qm region are provided in the supporting information . the calculated frequency shift between the two states for the l54c - cn probe is 2.5 cm , which is in reasonable agreement with the shift of 1.0 cm measured experimentally . figure 5b depicts the calculated electric fields along the cn bond for the l54c - cn mutant in the two states . the calculated average electric fields along the cn probe in the closed and occluded conformations are 8.4 and 13.8 mv / cm , respectively . using the experimental stark tuning rate of 0.7 cm/(mv / cm ) in conjunction with eq 1 , this shift of 5.4 mv / cm corresponds to a shift in vibrational frequency of 3.8 cm due to only classical electrostatic interactions . to model the situation with only classical electrostatic interactions , we calculated the shift in the vibrational frequency when only the residue containing the thiocyanate group ( l54c - cn ) is treated quantum mechanically and the remainder of the system , including phe31 and arg57 , the calculated frequency shift when only the probe residue is treated quantum mechanically is 3.5 cm , which is consistent with the value of 3.8 cm obtained from eq 1 . however , this frequency shift is greater than the shift of 2.5 cm calculated with the larger qm region . the greater shift predicted by the purely classical electrostatic model indicates that the polarization effects due to the phe31 and arg57 residues decrease the vibrational frequency shift relative to that predicted by classical electrostatics . table 2 provides the contribution to the electric field along the cn axis for several key residues near the l54c - cn probe . the sum of these contributions is 7.3 mv / cm , which is slightly higher than the change of 5.4 mv / cm calculated with the entire enzyme , indicating that other residues are also contributing to this field . in a similar manner to the analysis for the t46c - cn mutant , the arg57 residue exhibits the largest change between the two states with a net shift of 6.58 mv / cm . in the closed conformation , the line connecting the midpoint of the cn bond to the protonation site of the positively charged arg57 is nearly perpendicular to the cn axis , resulting in almost zero contribution to the electric field along the cn axis . the change in the electric field contribution by arg57 is likely due to a geometrical shift that moved the positive charge on arg57 away from this orthogonal orientation with respect to the probe in the occluded conformation . interestingly , while the substrate exerts a large contribution to the electric field along the cn axis , this contribution remains nearly constant between the two states . the stable orientation of the cn axis relative to the negatively charged carboxylate moiety in the bound substrate results in negligible change in the contribution of the substrate to the electric field at the cn probe . changes in the electric field along the cn bond in going from the closed conformation ( nadp / fol ) to the occluded conformation ( nadp / thf ) . while the calculated electric fields along the cn axes of the probes provide a measure of the electrostatic microenvironments within the active site , the electric field along the hydride transfer d encouraged by the good agreement between the experimental and the simulated data described above , we applied the same methodology to evaluate the total electric field at the midpoint of the d a axis and the field projected along the d a axis in the closed ( e : nadp : fol ) state . the negative value of the field indicates that it points from the substrate to the cofactor and that this field will facilitate the transfer of a negatively charged hydride from the cofactor to the substrate . in other words , the projection and the magnitude of the field along the d a axis in the ternary michaelis complex is highly favorable for the hydride transfer reaction to occur . the substrate and cofactor contribute a total of 32.4 mv / cm to the field along the d a axis . the rest of the protein contributes approximately an additional 31% ( 15.4 mv / cm ) of the total field . also , the solvent molecules and ions contribute an additional 1.1 mv / cm to the field along the d a axis . this analysis suggests that a major function of the enzyme is to bring the substrate and cofactor into a configuration that realizes the highly favorable field for facilitating the hydride transfer reaction ( figure 7 ) . in the absence of the enzyme , it would be less probable for the reacting species , nadph and dhf , to achieve the same relative orientation in solution . moreover , this analysis provides a new perspective on the role of electrostatic catalytic contributions in dhfr . it is currently impossible to experimentally measure the electric field along the d a axis , but the calculations provide reasonable estimates . a favorable electric field of 15.4 mv / cm from the protein and 32.4 mv / cm from the ligands along the d a axis could potentially reduce the free energy barrier by several kcal / mol . while these values suggest a substantial catalytic contribution generated from the active site electrostatic environment , it is important to acknowledge that the full mechanism of catalysis is far more complex than a simplistic evaluation based solely on the electric field along the d a axis . depiction of the component of the electric field along the hydride transfer d a axis calculated from an md simulation of wt ecdhfr with nadp / fol bound and the met20 loop in the closed conformation . the color for each residue corresponds to the calculated electric field values using the color scale provided , although residues contributing a magnitude > 4.0 mv / cm are depicted in the darkest color . the three arrows in the red oval represent the total electric field of 48.9 mv / cm ( purple ) , the field of 32.4 mv / cm resulting from the ligands ( yellow ) , and the field of 16.5 mv / cm resulting from the rest of the system ( green ) projected along the d a axis . we also decomposed this field into the contributions from all of the protein residues , and our analysis identified many residues that contribute to the electric field along the d a axis . figure 7 depicts the contributions from each of the residues to the field along the d a axis . the residues that are colored blue contribute positively and those that are colored red contribute negatively to the field pointing from the substrate to the cofactor along the d a axis . the residues that are shown with side chains contribute to the field with a magnitude > 1 mv / cm . interestingly , there is a strong degree of overlap between the residues that strongly contribute to the field along the d a axis and the network of coupled motions that has been identified for dhfr catalysis . the residues ile14 , tyr100 , and asp122 contribute 1.2 , 4.2 , and 2.4 mv / cm , respectively , and were all implicated in the network of coupled motions . additional residues that strongly contribute to the electric field along the d a axis are lys32 ( 3.2 mv / cm ) , arg52 ( 3.9 mv / cm ) , and arg57 ( 4.2 mv / cm ) . the residues that contribute strongly against the field along the d a axis include asp27 ( 5.4 mv / cm ) , arg98 ( 3.4 mv / cm ) , and his124 ( 3.0 mv / cm ) . all of these residues represent potential targets to alter the electrostatic contribution to catalysis . we investigated the changing electrostatic environment at two different thiocyanate probes inserted into the active site of ecdhfr for five complexes along the catalytic cycle . our results illustrate a changing electrostatic environment near the hydride transfer site ( t46c - cn ) as well as a smaller change in key regions of the folate binding pocket ( l54c - cn ) . the changes in electrostatic environment are associated with conformational changes occurring along the catalytic cycle of ecdhfr . experimental evidence for the changing electrostatic environment includes both ir frequency and c nmr measurements , which interrogate the electric field along the cn axis and the total electric field at the probe . these experimental data demonstrate fundamental aspects of the electrostatic landscape for catalysis in ecdhfr . molecular dynamics simulations provided an atomic - level picture of the local environment experienced by the thiocyanate probes in complexes that model the states prior to and subsequent to the hydride transfer reaction . the qm / mm vibrational frequency calculations reproduced the experimentally measured frequency shifts for these two states , which span the closed to occluded transition associated with hydride transfer . analysis of the md trajectories provided insight into the conformational changes occurring between these two states . the factors leading to the vibrational frequency shifts were broken down into classical electrostatic effects , specific hydrogen - bonding interactions , and polarization effects . thus , the vibrational frequency shifts are not simply a manifestation of the classical electric fields but rather include additional contributions from the environment . moreover , the electric fields along the cn axis of the probes were decomposed into contributions from specific residues , ligands , and solvent molecules . the electric field along the hydride d a axis was also calculated with the experimentally validated methodology . the calculations illustrate that the cofactor and substrate , as well as the enzyme , impose a substantial electric field along the d a axis that facilitates hydride transfer . overall , experimental and theoretical data provide evidence for significant electrostatic changes in the microenvironments that constitute the heterogeneous ecdhfr active site as the enzyme progresses along the catalytic cycle . the electrostatic interactions between the protein and the ligands assist in orienting the reacting species in a geometry that maintains a large electric field favoring hydride transfer from the cofactor to the substrate . the enzyme active site environment also provides a significant portion ( 1/3 ) of the total electric field that facilitates the hydride transfer reaction , suggesting the catalytic importance of enzymatic conformational changes that alter the active site electrostatics . future work will investigate the five trapped states along the catalytic cycle for the two probes studied in this work as well as additional probe sites in the ecdhfr system to provide a comprehensive picture of the electrostatic landscape for the entire catalytic cycle .

electrostatic interactions play an important role in enzyme catalysis by guiding ligand binding and facilitating chemical reactions . these electrostatic interactions are modulated by conformational changes occurring over the catalytic cycle . herein , the changes in active site electrostatic microenvironments are examined for all enzyme complexes along the catalytic cycle of escherichia coli dihydrofolate reductase ( ecdhfr ) by incorporation of thiocyanate probes at two site - specific locations in the active site . the electrostatics and degree of hydration of the microenvironments surrounding the probes are investigated with spectroscopic techniques and mixed quantum mechanical / molecular mechanical ( qm / mm ) calculations . changes in the electrostatic microenvironments along the catalytic environment lead to different nitrile ( cn ) vibrational stretching frequencies and 13c nmr chemical shifts . these environmental changes arise from protein conformational rearrangements during catalysis . the qm / mm calculations reproduce the experimentally measured vibrational frequency shifts of the thiocyanate probes across the catalyzed hydride transfer step , which spans the closed and occluded conformations of the enzyme . analysis of the molecular dynamics trajectories provides insight into the conformational changes occurring between these two states and the resulting changes in classical electrostatics and specific hydrogen - bonding interactions . the electric fields along the cn axes of the probes are decomposed into contributions from specific residues , ligands , and solvent molecules that make up the microenvironments around the probes . moreover , calculation of the electric field along the hydride donor acceptor axis , along with decomposition of this field into specific contributions , indicates that the cofactor and substrate , as well as the enzyme , impose a substantial electric field that facilitates hydride transfer . overall , experimental and theoretical data provide evidence for significant electrostatic changes in the active site microenvironments due to conformational motion occurring over the catalytic cycle of ecdhfr .

pubmed

magnetic particle imaging ( mpi ) is a new three - dimensional imaging method using static and dynamic magnetic fields to visualize the distribution of superparamagnetic iron oxide nanoparticles ( spios).1 imaging relies thereby on three characteristics of the spio , ie ; their superparamagnetic nature , their nonlinear magnetization curve , and their ability to be magnetically saturated.2 because of these characteristics , mpi can visualize and quantify the spatial distribution of spio , providing very high temporal and spatial resolution combined with high signal - to - noise ratios . possible application scenarios for mpi might include cardiovascular imaging and even guidance of cardiovascular interventions.3,4 in principle , mpi would allow visualization and quantification of vascular pathologies while also being able to determine additional functional parameters in one examination . further , mpi could be used as an imaging method for percutaneous transluminal coronary angioplasty , if indicated . apart from the need for suitable mpi scanners , this diagnostic and therapeutic pathway relies on appropriate tracer material . ideally , long circulating spio are desirable , and should be able to provide a sufficient signal within the vessels after a single tracer injection at the beginning of the procedure , so a diagnostic scan and intervention can be done consecutively . the circulation time of spio in blood is mainly determined by their overall so - called hydrodynamic diameter . although spio with a greater dimension of the iron oxide core , and consequently a larger hydrodynamic particle size , improve the superparamagnetic behavior of contrast agents in general , the coatings play a major role in the biodistribution and lesional uptake of spio . previous reports demonstrate that spio with a hydrodynamic size greater than 50 nm have in common rapid and specific uptake by the reticuloendothelial system , and thus are cleared rapidly from the blood stream . compared with magnetic resonance imaging ( mri ) , the properties of spio not only influence pharmacokinetics but also greatly influence the quality and sensitivity of the entire mpi system . to achieve high performance in mpi , an ideal tracer should exhibit spio with a homogeneously distributed iron core diameter of 30 nm , resulting in a relatively high hydrodynamic diameter and a shorter half - life in blood.1 although the characteristics of an ideal mpi tracer are well understood in theory , and this issue has been addressed by many working groups , no mpi - specific tracer has reached the marketplace so far . since the advent of mpi , many spio agents have been evaluated for their usefulness in mpi.5 resovist , an approved t2 * contrast agent for mri , is the most important benchmark material , and remains the best performing commercial spio for mpi today and the standard of reference for evaluation of mpi hardware and application , although it has known drawbacks , ie , a hydrodynamic diameter greater than 50 nm and therefore , a limited in vivo distribution.6 however , in the context of cardiovascular mpi , knowledge about the pharmacokinetics of resovist , especially its half - life in blood , is important . naturally , this issue was investigated about 20 years ago , when resovist was evaluated as a contrast agent for mri.7 however , because the imaging principle is fundamentally different in mpi , results from mri can not be easily extrapolated to mpi , as not all resovist particles contribute equally to the mpi signal and larger particles are cleared more rapidly by the reticuloendothelial system ( res ) . thus , the aim of this study was to evaluate the blood half - life of mpi using magnetic particle spectroscopy ( mps ) , given that it may differ significantly from the blood half - life in mri . marketing of resovist by bayer pharma ag ( berlin , germany ) has been discontinued , and it is only available now from the japanese distributor , irom pharmaceutical co ltd ( tokyo , japan ) . however , until now , most mpi experiments were done using resovist from bayer pharma ag , whereas most working groups now use resovist from irom pharmaceutical co ltd . the experimental protocol was approved by the state regulations governing the performance of animal studies . all experiments were conducted under strict adherence to the guidelines for the care and use of laboratory animals as described by the national institutes of health . two different versions of resovist were studied , ie , that distributed by bayer pharma ag in europe and that distributed by irom pharmaceutical co ltd . it was approved for clinical use in 2001 in europe and in 2002 in japan.8,9 marketing of the bayer pharma ag product has now been discontinued in europe . resovist ( 50 mol / kg ) was administered into the ear artery of ten female new zealand white rabbits after drawing of a control blood sample . five animals received resovist by irom pharmaceutical co ltd and five by bayer pharma ag , respectively . each rabbit weighed approximately 4 kg . directly ( about 30 seconds ) after administration of resovist , a second blood sample was collected as a baseline sample ( t0 ) from the contralateral ear artery to avoid contamination with residual resovist . this was repeated after 5 , 10 , 15 , 30 , 45 , 60 , 75 , 90 , 105 , and 120 minutes . during the experiments , the animals were kept under full anesthesia by bodyweight - adapted subcutaneous injection of ketamine hydrochloride ( 0.7 ml / kg bodyweight ; ceva animal health , dsseldorf , germany ) and xylazine hydrochloride ( 0.3 ml / kg bodyweight ; ceva animal health ) . blood samples were collected in accordance with the recommendations of the german society for laboratory animal science.10 the mpi signal was evaluated using mps . mps can be described as a zero - dimensional system that measures the remagnetization spectrum of spio . in mps , only the oscillating magnetic field that excites the spio is used . the magnetic gradient field , which is used for spatial encoding in mpi , is omitted.11 using mps it is possible to evaluate the quality of the mpi signal of particles , so the applicability of the particles for mpi can be characterized . in an ideal superparamagnetic particle , the spectra consist only of odd harmonics due to the symmetry of the particle magnetization curve . in an experimental setup , even harmonics are added by the earth s magnetic field , but can be neglected in evaluation of the particles.12 thus , the signal strength can be determined by evaluating the odd harmonics of spio above noise level . the magnitude of the signal is proportional to the concentration of the spio . as in previous experiments,5 the mps excitation frequency was 25 khz ( as is most common in mpi systems ) , the magnetic field amplitude was 20 mt , and the acquisition time was 5 seconds ( corresponding to 125,000 repetitions ) to provide a high signal - to - noise ratio . ten milliliters of each blood sample were transferred into individual eppendorf tubes directly after sampling , and then measured using mps . the signal strength of the odd harmonics from the third to the 49th harmonic was measured at each time point and related to the signal strength of the baseline sample ( t0 ) . thus , the decline of the mpi signal in blood could be described in relation to the signal maximum at baseline directly after administration of resovist . for each group , ie , resovist distributed by bayer pharma ag and irom pharmaceutical co ltd , respectively , the average ( standard deviation ) decline in signal strength was calculated for each time point . ten microliters of pure resovist ( 500 mol fe / ml ) were drawn from each package unit and diluted using ringer s solution to 400 , 300 , 200 , 100 , 80 , 60 , 40 , 20 , 10 , 8 , 6 , 4 , 2 , and 1 mol fe / ml . the hydrodynamic diameter and size distribution of the spio for the two different types of resovist were measured using photon cross correlation spectroscopy ( nanophox , sympatec gmbh , clausthal - zellerfeld , germany ) . in photon cross correlation spectroscopy , the particle size is measured by three - dimensional cross correlation of the scattered light of two intersecting focused laser beams.13 the statistical significance of the difference in the signal decay in blood between the two types of resovist was calculated using the mann further , the statistical significance of the difference between the two package units in the dilution series was calculated in the same way . in both cases , two different versions of resovist were studied , ie , that distributed by bayer pharma ag in europe and that distributed by irom pharmaceutical co ltd . it was approved for clinical use in 2001 in europe and in 2002 in japan.8,9 marketing of the bayer pharma ag product has now been discontinued in europe . resovist ( 50 mol / kg ) was administered into the ear artery of ten female new zealand white rabbits after drawing of a control blood sample . five animals received resovist by irom pharmaceutical co ltd and five by bayer pharma ag , respectively . each rabbit weighed approximately 4 kg . directly ( about 30 seconds ) after administration of resovist , a second blood sample was collected as a baseline sample ( t0 ) from the contralateral ear artery to avoid contamination with residual resovist . this was repeated after 5 , 10 , 15 , 30 , 45 , 60 , 75 , 90 , 105 , and 120 minutes . during the experiments , the animals were kept under full anesthesia by bodyweight - adapted subcutaneous injection of ketamine hydrochloride ( 0.7 ml / kg bodyweight ; ceva animal health , dsseldorf , germany ) and xylazine hydrochloride ( 0.3 ml / kg bodyweight ; ceva animal health ) . blood samples were collected in accordance with the recommendations of the german society for laboratory animal science.10 mps can be described as a zero - dimensional system that measures the remagnetization spectrum of spio . in mps , only the oscillating magnetic field that excites the spio is used . the magnetic gradient field , which is used for spatial encoding in mpi , is omitted.11 using mps it is possible to evaluate the quality of the mpi signal of particles , so the applicability of the particles for mpi can be characterized . in an ideal superparamagnetic particle , the spectra consist only of odd harmonics due to the symmetry of the particle magnetization curve . in an experimental setup , even harmonics are added by the earth s magnetic field , but can be neglected in evaluation of the particles.12 thus , the signal strength can be determined by evaluating the odd harmonics of spio above noise level . the magnitude of the signal is proportional to the concentration of the spio . as in previous experiments,5 the mps excitation frequency was 25 khz ( as is most common in mpi systems ) , the magnetic field amplitude was 20 mt , and the acquisition time was 5 seconds ( corresponding to 125,000 repetitions ) to provide a high signal - to - noise ratio . ten milliliters of each blood sample were transferred into individual eppendorf tubes directly after sampling , and then measured using mps . the signal strength of the odd harmonics from the third to the 49th harmonic was measured at each time point and related to the signal strength of the baseline sample ( t0 ) . thus , the decline of the mpi signal in blood could be described in relation to the signal maximum at baseline directly after administration of resovist . for each group , ie , resovist distributed by bayer pharma ag and irom pharmaceutical co ltd , respectively , the average ( standard deviation ) decline in signal strength was calculated for each time point . ten microliters of pure resovist ( 500 mol fe / ml ) were drawn from each package unit and diluted using ringer s solution to 400 , 300 , 200 , 100 , 80 , 60 , 40 , 20 , 10 , 8 , 6 , 4 , 2 , and 1 mol fe / ml . the hydrodynamic diameter and size distribution of the spio for the two different types of resovist were measured using photon cross correlation spectroscopy ( nanophox , sympatec gmbh , clausthal - zellerfeld , germany ) . in photon cross correlation spectroscopy , the particle size is measured by three - dimensional cross correlation of the scattered light of two intersecting focused laser beams.13 the statistical significance of the difference in the signal decay in blood between the two types of resovist was calculated using the mann further , the statistical significance of the difference between the two package units in the dilution series was calculated in the same way . in both cases , the average mpi signal in blood declined to 30% of the baseline signal 5 minutes after injection . ten and 15 minutes after injection , the average mpi signal was 14.8% and 7% , respectively , of the baseline signal . within 30 minutes , the mpi signal was below the noise level of the mps ( see table 1 ) . regardless of this result , the mpi signal was measured consecutively until 120 minutes after application ; however , it was still not possible to detect any signal above the noise level . for further evaluation , the signal decline for both types of resovist was compared . the signal for resovist from bayer pharma ag decreased to 39.7% of baseline after 5 minutes , to 20.5% after 10 minutes , and to 12.1% after 15 minutes . the signal for resovist from irom pharmaceutical co ltd showed a rapid decrease to 20.4% of the baseline signal after 5 minutes and to 7.8% after 10 minutes ( see table 1 and figure 1 ) ; in contrast with resovist from bayer pharma ag , the signal dropped below noise level even after 15 minutes . this difference was statistically significant ( mann whitney u - test , p=0.0056 ) . in contrast with the signal decay , the absolute signal strength of the baseline blood mpi signal was not significantly different between the two resovist products ( 1.60e-88 , 09e-9 amhz for resovist from bayer pharma ag and j1.98e-84 , 13e-9 amhz for resovist from irom pharmaceutical co ltd , p=0.3953 , mann in addition , the dilution series showed no statistical significant difference in mpi signal decay between the two compounds ( mann whitney u - test , p=0.6241 , see figure 3 ) . photon cross correlation spectroscopy showed a median hydrodynamic diameter ( x50 ) of 70.54 nm for resovist from bayer pharma ag and 61.86 nm for resovist from irom pharmaceutical co ltd . the size distribution was x1060.41 nm and x9082.07 nm ( x10/x90=1.36 ) for resovist from bayer pharma ag and x10 and x9051.16 nm and 73.08 nm ( x10/x90=1.4 ) for resovist from irom pharmaceutical co ltd . first , resovist is not suitable as an intravascular tracer in terms of prolonged circulation . given the current lack of alternatives in mpi , it may be used as a bolus tracer either intravenously for a diagnostic scan or be applied during a cardiovascular intervention using an intravascular catheter . the present study underlines once more the need for a dedicated mpi tracer with a prolonged blood half - life of its mpi signal . second , it highlights the fact that the blood half - life of the mpi tracer can not be equalized to the blood half - life time of the mpi signal . the concordance of these two values depends on the fraction of particles contributing to the mpi signal . third , the difference in blood half - life between resovist sourced from bayer pharma ag and that from irom pharmaceutical co ltd has to be taken into account when comparing different studies . further , due to the inhomogeneous size distribution from batch to batch , it is even possible that tracers of the same type show differences in blood half - life . the rapid decay of the mpi signal is in concordance with the initial phase of elimination of resovist in the studies conducted in humans using mri . in their mri studies , hamm et al14 observed two phases of elimination of the contrast agent . in the first phase , about 80% of the resovist concentration was cleared , with a blood half - life of 3.95.8 minutes , whereas that of the second phase was 2.43.6 hours . the blood half - life was therefore independent of the concentration administered.14 the package insert for resovist states that the first phase is 15.611.4 minutes and the second phase is 4.360.75 hours.8 in contrast , no second phase of tracer elimination could be detected using mps in our study . this difference can be explained by the different principles of signal generation in mpi and mri , and the broad size distribution of the spio in resovist . in mri , the effect of the spio is a shortening of the t2 time in the surrounding tissues , leading to a signal loss , especially in t2 * weighted images . thus , in mri , spio work as an indirect contrast agent influencing the signal characteristics of the surrounding tissue . this occurs independently of the size of the spio , as long as they still have superparamagnetic characteristics . in mpi , the spio are visualized directly by their change of magnetization in an oscillating magnetic field . thus , they work like a tracer and not like a contrast agent . as mentioned above , the diameter of the iron core should be around 30 nm for an ideal mpi signal.1 in resovist , theoretically only 3% of particles meet this criterion and contribute substantially to the mpi signal.1 recently , eberbeck et al6 demonstrated by analysis of the m(h ) curve for resovist that there are clusters of smaller spio in resovist that behave like single domain particles and have an effective mean iron core diameter of 241 nm . in their data , this fraction of particle clusters amounted to 30%3% of the nanoparticles in resovist and explain its good mpi performance.6 the other fraction of spio in resovist has an effective mean iron core diameter of 5.53 nm , and does not contribute substantially to the mpi signal.6 it is important to note that the physical mean iron core diameter as measured by , eg , transmission electron microscopy and vibrating sample magnetometry , is between 5 nm and 15 nm , as described by many working groups , and the clusters contributing most of resovist s mpi - signal as described in eberbeck et al6 contain these smaller particles , as can also be shown by transmission electron microscopy.6,1519 these clusters may also account for a fraction with a larger hydrodynamic diameter than the other particle fractions in resovist . these would be cleared more rapidly from the blood by the reticuloendothelial system and the mpi signal would decrease more rapidly than the actual concentration of the tracer measured , eg , on mri . this explanation is supported by a study reported by khandhar et al.19 the authors evaluated resovist and a self - designed tracer for their mpi performance in mice and showed that the decay of the mpi signal from resovist in blood was considerably faster than the decay of the magnetization curve measured by vibrating sample magnetometry . this also indicates that there are resovist particles left in the blood that do not contribute to the mpi signal.19 in contrast with our results , they observed an even faster decrease in the mpi signal to below noise level within 10 minutes . this is probably due to the differing sensitivities of the respective mps systems , as there are no international standardized systems on the market as yet . given that khandhar et al19 did not reveal the dose of resovist related to body weight of the mice , differences in the dose of resovist initially applied might explain the shorter time above noise level as well . further , khandhar et al described an initial slow increase of mps signal from 0 to 5 minutes after administration.19 this also seems to be in contrast with the findings of weizenecker et al,20 who were the first group to present in vivo mpi images , which visualized the beating heart of a mouse after application of a bolus of resovist from bayer pharma ag . an approximate equilibrium was achieved in their study about 15 seconds after intravenous injection , and a rapid decay of the tracer concentration determined by the mpi demonstrator in the vena cava from about 3.8 mmol fe / l at first pass to 0.28 mmol fe / l at third pass 10.2 seconds later was described.20 in summary , it seems that the resovist particles contributing most to the mpi signal are cleared most rapidly from the blood . interestingly , we found a significant difference in blood half - life between resovist manufactured by bayer pharma ag and that manufactured by irom pharmaceutical co ltd . however , our measurements do not indicate significant differences in hydrodynamic diameter or size distribution , which would be the most likely explanation for these differences in blood half - life . resovist from bayer pharma ag showed a slightly larger hydrodynamic diameter than that from irom pharmaceutical co ltd . a difference in formulation of the polymeric coating is improbable , given that the japanese company that developed resovist in cooperation with schering ag ( berlin , germany ) still produces the drug substance for resovist , ie , ferucarbotran ( meito sangyo co ltd , nagoya , japan ) . further , there is no discrepancy in the baseline signal strength or signal loss in the dilution series between the two types of resovist , ie , the mpi performance of the tracers . resovist from irom pharmaceutical co ltd performed slightly , albeit not significantly , better than resovist from bayer pharma ag ( figure 3 ) . all mpi - specific research to date regarding particle characterization and dynamics has been conducted using resovist from bayer pharma ag , and there are no such data for resovist from irom pharmaceutical co ltd . tsuchiya et al21 indicate that the iron core diameter of resovist from irom pharmaceutical co ltd is 5 nm , but do not provide a reference for this or a method for how the iron core diameter was measured . given that the mpi performance of resovist sourced from the two distributors is almost the same , it is highly probable that the mechanism of clustering of resovist particles as described by eberbeck et al6 for resovist by bayer pharma ag holds true , otherwise , the mpi performance would be well below that of resovist distributed by bayer pharma ag . thus , for resovist to be used further in the setting of mpi , two issues have to be considered . firstly , the distribution of resovist by bayer pharma ag in europe has been discontinued and it is no longer commercially available . therefore , resovist distributed by irom pharmaceutical co ltd may be the standard of reference until a dedicated spio tracer is marketed , whereas the first in vivo experiments in mpi were conducted using resovist distributed by bayer pharma ag.20 this should be considered when evaluating experimental results . secondly , the difference in blood half - life between the two compounds is significant , but not as high as to indicate a change of application . as we showed , in accordance with other investigators , the mpi signal of resovist in blood lacks the second , slower elimination phase observed in mri and rapidly decays below noise level , so is not ideal for applications requiring long circulating or even blood pool tracers . in this study , the mpi signal was measured using mps , which is known to be the most sensitive method for signal detection ; thus , the time frame in which resovist can be visualized with current and upcoming mpi scanners will probably be even shorter , and resovist will most likely be suitable only as a bolus tracer for vascular applications . in addition to the challenges concerning scanner design , the design of tracers will be a key aspect in future development of mpi . spio formulations originally designed for mri do not exhibit ideal mpi characteristics , so research is now focused on development of a dedicated mpi tracer , ie , an spio tracer with a homogenously distributed iron core diameter of about 30 nm.6,18,19,22 here , resovist serves as an international standard of reference for the performance of mpi using new tracers . mps is the most important tool for evaluation , but magnetorelaxometry , vibrating sample magnetometry , and m(h ) curves are also important tools , particularly for description , and explain the sometimes complex mpi performance of spio.6,18,19 apart from the need for ideal mpi characteristics , mostly determined by the iron core and the homogenous size distribution of the particles , pharmacokinetics have a crucial role in the use of the tracer in vivo . for long circulating tracers , a small hydrodynamic diameter is desirable . because the iron core diameter should be 30 nm , the minimum hydrodynamic diameter is limited , even if the coating is made of , eg , thin monomer layers recently , rahmer et al reported a proof of principle study in which red blood cells in mice were loaded with resovist , enabling mpi of the vascular system for several hours after application.23 another option might be to modify the coating and charge on the spio in order to enhance circulation time.24 outside the mpi community , a lot of research has already been conducted with regard to different spio particle coatings and surface modifications for specific areas of application , eg , for targeted imaging or to prolong the blood circulation time , and mpi can benefit from this knowledge . in order to enable cooperation and dissemination of knowledge , a standardized physicochemical testing protocol as a part of particle development is desirable , as already proposed.25,26 these standards may even simplify the long way from bench to bedside . however , resovist remains the only industrially produced ( in japan ) and approved spio formulation applicable for mpi . feraheme ( ferumoxytol , amag pharmaceuticals , waltham , ma , usa ) is a spio formulation approved by the us food and drug administration for the treatment of iron deficiency anemia in adult patients with chronic kidney disease , but is not approved for mpi due to its inadequate mpi signal characteristics . additionally , the diverging performance of the two types of resovist could not be fully elucidated . therefore , studies of different batches of resovist in larger groups of animals may be necessary . further , analysis of resovist distributed by irom pharmaceutical co ltd corresponding to , ie , eberbeck et al , should be executed in the future.6 ideally , the pharmacological formulation of the two distributions should be analyzed in detail , because this may be a reason for the different half - life times . finally , the results need to be confirmed by visualization of vessels in larger animals in real time to determine the effect on mpi in vivo . in conclusion , our study shows that resovist is not a suitable tracer for applications requiring a long circulation time of the mpi tracer in blood . more importantly , our study highlights a difference in a tracer s blood half - life with regard to the mpi signal on the one hand , and the mri signal and iron concentration on the other . interestingly , there are even distinctions between different distributions of the same pharmaceutical agent , in this case resovist by bayer pharma ag and irom pharmaceutical co ltd . consequentially , all spio formulations have to be evaluated for the half - life of the mpi signal in blood before use in mpi .

backgroundmagnetic particle imaging ( mpi ) uses magnetic fields to visualize superparamagnetic iron oxide nanoparticles ( spio ) . today , resovist is still the reference spio for mpi . the objective of this study was to evaluate the in vivo blood half - life of two different types of resovist ( one from bayer pharma ag , and one from irom pharmaceutical co ltd ) in mpi.methodsa resovist concentration of 50 mol / kg was injected into the ear artery of ten new zealand white rabbits . five animals received resovist distributed by irom pharmaceutical co ltd and five received resovist by bayer pharma ag . blood samples were drawn before and directly after injection of resovist , at 5 , 10 , and 15 minutes , and then every 15 minutes until 120 minutes after the injection . the mpi signal of the blood samples was evaluated using magnetic particle spectroscopy.resultsthe average decline of the blood mpi signal from the two distributions differed significantly ( p=0.0056 ) . resovist distributed by bayer pharma ag showed a slower decline of the mpi signal ( 39.7% after 5 minutes , 20.5% after 10 minutes , and 12.1% after 15 minutes ) compared with resovist produced by irom pharmaceutical co ltd ( 20.4% after 5 minutes , 7.8% after 10 minutes , no signal above noise level after 15 minutes).conclusionin mpi , the blood half - life of an spio tracer can not be equalized to the blood half - life of its mpi signal . resovist shows a very rapid decline of blood mpi signal and is thus not suitable as a long circulating tracer . for cardiovascular applications in mpi , it may be used as a bolus tracer .

pubmed

intracellular pathogens have evolved over many millions of years to coexist in the face of a wide array of host defense mechanisms . in vitro studies of the host - pathogen interaction exclude the inflammatory milieu , but a major challenge in studying the host response in vivo is dissecting pathogen - specific responses from the general inflammatory or common host response ( jenner and young , 2005 ) that occurs as a result of tissue insult during the infectious process . this latter response is in part driven by activation of components of the complement cascade ( ricklin et al . , 2010 ) , the coagulation pathway ( oikonomopoulou et al . , 2012 ) , and the acute phase response ( suffredini et al . , 1999 ) , which all come into play within minutes of infection , and is compounded by rapid changes to the inflammatory cytokine and chemokine environment ( oghumu et al . , 2010 ; sadik and luster , 2012 ; soehnlein and lindbom , 2010 ) . each of these stimuli may have profound effects on phagocyte function ( galvan et al . , 2012 ; hoffmann et al . , 2010 ) and reflect , in the broader sense , the normal and/or essential response of phagocytes to inflammatory insult . hence , responses that directly reflect intracellular infection are difficult to distinguish from dominant inflammatory signatures by conventional experimental approaches . we hypothesized that it might be possible to use the transcriptomic response of uninfected cells exposed to inflammation as a measure of the baseline or normal in vivo response to insult . following this argument , transcriptomic networks specifically observed in infected cells , but not in neighboring uninfected cells , would represent cell - intrinsic pathogen - induced changes . in contrast , transcriptomic networks restricted to uninfected cells would serve to identify responses potentially beneficial to the host , the lack of which ( in infected cells ) might favor pathogen survival . we tested the above hypothesis in a tractable experimental model of leishmania infection , a pathogen associated with a significant global health burden ( alvar et al . , 2012 ) . leishmania infect a variety of myeloid cells ( including tissue - resident macrophages , inflammatory monocytes , dendritic cells [ dcs ] , and neutrophils ) , but in models of experimental visceral leishmaniasis , leishmania donovani amastigotes have a predilection for resident tissue macrophages , including kupffer cells ( kcs ) , the resident tissue macrophage lining the hepatic sinusoids ( beattie et al . , 2010 ; murray , 2001 ) . in this study , we discovered that expression of an rxra - centered transcriptomic pathway , which is downregulated in kc in response to inflammation , is aberrantly maintained in infected cells . pharmacological manipulation of rxr activity perturbed the transcriptomic network of infected kcs , leading to enhanced leishmanicidal activity . this study demonstrates that aberrant expression of the rxra - centered transcriptional pathway in leishmania - infected kcs favors parasite survival and validates the use of cells exposed to inflammation as a means to discover pathogen - regulated transcriptomic pathways in vivo . in the livers of uninfected ( lysm - cre mt / mg)f1 mice , kcs were obvious from their large size , expression of membrane gfp , and their continued and intimate contact with liver sinusoidal endothelial cells , as has been previously described ( lee et al . four - dimensional ( 4d ) intravital two - photon imaging further allowed the fine membrane structure and lamellipodia dynamics of steady - state kcs to be observed in exceptional detail ( figure 1a ; movie s1 ) . the continued extension and retraction of kc lamellipodia manifested itself as small fluctuations of cellular volume ( figure 1b ) and surface area over time ( figure 1c ) . in contrast , monocytes and/or neutrophils were identified as moving rapidly through the sinusoidal spaces , whereas liver sinusoidal endothelial cells ( lsecs ) lined the sinusoidal lumen ( figures 1f and 1 g ) . shape factor , a quantitative measure of how close a cell is to a perfect sphere ( shape factor = 1 ) , showed small fluctuations over time for kcs ( figure 1d ) but was always lower than that for either lsecs or monocytes and/or neutrophils ( figure 1h ) . the effects of kc membrane fluctuation can also be represented by a velocity , which was calculated by measuring the movement around the centroid point of the cell in three dimensions . thus , although kcs do not actively migrate along the sinusoids , velocity as measured here provides an indication of the extent of membrane activity . in contrast , nonmotile lsecs have an even lower velocity ( limited membrane activity ) . many of the monocytes and/or neutrophils that were examined actively migrated along the sinusoids within the time frame of observation , and their velocity was not surprisingly high ( figure 1i ) . as the early response of kcs to infection with l. donovani amastigotes in vivo is largely uncharacterized , we examined kc behavior before and after infection in live mice . we injected ( lysm - cre mt / mg)f1 mice with transgenic td - tomato - expressing l. donovani amastigotes ( beattie et al . , 2010 ) at a dose that resulted in approximately 20% of kcs becoming infected over a 2 hr time period ( a time by which most parasites have been cleared from the blood by kcs and other tissue phagocytes ; figures 2a and 2b ; gorak et al . , 1998 ) . the binding of amastigotes to the kc membrane ( figure 2a ) was followed by complete phagocytosis within 20 s ( movie s2 ) . neither injection of live , nor of heat - killed , amastigotes resulted in detectable changes in kc volume ( figure 2c ) or surface area ( figure 2d ) , irrespective of whether or not the kcs contained visible amastigotes . in contrast , injection of live , but not dead , amastigotes induced a rapid increase in the average shape factor that was significant for kcs that contained live parasites and showed a trend in this direction for kcs that did not contain amastigotes ( figure 2e ) . when velocity was measured as an indicator of membrane activity ( figure 2f ) , a significant decrease was observed in both infected and uninfected kcs following injection of live amastigotes , suggesting that both populations of cells were responding to the inoculated parasites through rapid activation . such activation was not observed following injection of dead amastigotes ( figure 2f ) . collectively , these data were indicative of a cellular response to active infection , rather than a response merely to nonspecific debris that may be present within the amastigote preparations . the observation that uninfected kcs in the immediate vicinity of infected cells also responded to the infection by loss of membrane activity ( figure 2f ) suggested that the above changes in kc behavior were the result of either signals provided from infected kcs and operating rapidly in trans or from the activation of serum - derived mediators of inflammation , rather than being a direct consequence of intracellular parasitism per se . in either case , the capacity to activate this response was absent from heat - killed amastigote preparations . as we wished to examine the transcriptional events that accompanied the observed change in kc behavior , we developed a method to identify and isolate kcs based on whether they contained parasites ( herein termed infected ) or not ( herein termed inflamed ) . flow cytometry analysis of hepatic mononuclear cell preparations with gating on forward and side scatter ( figure 3a ) , complement receptor of the immunologlobulin superfamily ( crig ) ( helmy et al . , 2006 ) , and gr-1 ( figure 3b ) demonstrated that both infected and inflamed populations of kcs could be observed and discriminated by the presence of fluorescent amastigotes ( figure 3c ) . both infected and inflamed kcs showed heterogeneous expression of cd11b but relatively uniform expression of f4/80 ( figure 3d ) . backgating on the infected and inflamed kcs showed that both populations contained both crig gr-1 cells and crig gr-1 cells ( figure 3e ) . to confirm that a proportion of kcs express gr-1 in situ , we labeled liver sections with f4/80 , crig , and gr-1 ( figures 3f3i ) and observed variable expression of gr-1 on kcs , in addition to gr-1 neutrophils that did not express crig or f4/80 and were readily identifiable from their irregular - shaped nuclei ( figures 3h and 3i ) . we then used fluorescence - activated high - speed cell sorting to isolate kcs and separate them into either infected or uninfected cell populations . we identified kcs from infected mice at 2 hr and 12 hr postinfection ( p.i . ) , as described above , based on forward and side scatter ( size and granularity ) ( figure 3j ) and expression of crig , gr-1 ( figure 3k ) , and f4/80 ( figure 3l ) and then further sorted these based on whether or not they contained parasites ( figures 3m3o ) . cytospins of the sorted populations demonstrated that the cells had macrophage - like morphology , that neutrophils were not included in the analysis , and the presence of amastigotes within the infected population ( figure 3q ) , but not in the inflamed population ( figure 3p ) . kcs were also isolated from sham - infected mice to serve as a control population that exhibited steady - state activity . to determine whether there were underlying transcriptional events that differed between kcs infected with amastigotes in an inflammatory setting and those induced in kcs exposed to inflammatory stimuli alone , transcriptomic profiles for each population , in addition to kcs from sham - infected controls , were generated by microarray analysis . instead of performing a direct comparison of the differences in gene expression between the infected and inflamed kcs , we generated a list of genes differentially expressed between infected kcs and sham - infected kcs ( using a cutoff of > 3-fold difference ) . this approach eliminated the possibility that any of the regulated genes were induced or suppressed due to the isolation procedure , as the sham - infected kcs were subjected to the same digestion and sort process . the same analysis was then performed for the inflamed kcs . as a result of this analysis , we therefore generated two gene lists ( infected and inflamed kcs ) for 2 hr p.i . and two gene lists for 12 hr p.i . at 2 hr p.i . , the expression of 1,188 genes ( 909 up and 279 down ) was differentially regulated between sham - treated and infected kcs , whereas only 353 genes ( 327 up and 26 down ) were differentially regulated between sham - treated and inflamed kcs . by 12 hr p.i . , the number of genes showing changes in expression was substantially increased ( 2,135 between sham - treated and infected kcs , of which 1,337 had increased , and 798 had decreased , expression ) . string analysis of these genes showed that although there were some interactions between these genes , there were a large number of genes that were outside of the central network ( figure s1 ) . although substantially fewer genes ( 1,288 ) were differentially expressed at a messenger rna ( mrna ) level between the sham - treated and inflamed kcs overall ( 713 upregulated and 575 downregulated ) , string analysis for these genes showed a higher degree of connectivity between the genes within this list and a substantial network connection that did not appear to be present in the infected cells ( figure s1 ) . the top 20 genes ( based on fold change compared to sham - infected kcs for each comparison , or according to relative expression ) are provided in table s1 . we interpret these data as indicating that there are genes that are normally regulated as part of the inflammatory response that are not regulated in infected cells . to determine the identity of genes and gene pathways that were common between the infected and inflamed cells , and those that were unique to either population , we performed a subtractive analysis of the gene lists ( figure 4a ) . from this analysis , we could readily identify genes that were either commonly regulated in both infected and inflamed cells or uniquely regulated in either infected or inflamed kcs at both 2 hr and 12 hr p.i . for all genes identified as being selectively or commonly expressed in infected kcs and inflamed kcs , we have provided the top 20 genes ranked by expression level ( table s2 ) . the gene lists were then subjected to pathway and gene ontology ( go ) analysis ( see experimental procedures for detail ) . a total of 1,088 genes were uniquely regulated in their expression at 12 hr p.i . genes that showed differentially regulated expression in both infected and inflamed cells were enriched for go terms suggestive of responsiveness to inflammation , including host defense and inflammatory response and cytokine - cytokine receptor interaction and chemokine activity . significantly , we identified 241 genes that were differentially regulated in their expression in inflamed cells , but not in cells that contained intracellular amastigotes , in spite of exposure of these cells to the same inflammatory insult . ingenuity pathway analysis ( ipa ) was then used to identify genes that fell within ipa - generated pathways at a higher frequency than would normally be expected to occur for a randomly selected set of genes . this analysis identified 8 interacting networks within the inflamed gene list , in which genes involved in cell - to - cell signaling and the inflammatory response ( ipa network score of 38 ) , cell death ( ipa network score of 37 ) , antigen presentation ( ipa network score of 35 ) , cancer ( ipa network score of 26 ) , immune cell trafficking and cell cycle ( ipa network score of 23 ) , and cell morphology and cellular compromise ( ipa network score of 10 ) were all overrepresented . the network that showed the highest level of gene enrichment was that of the lipid metabolism and small - molecule biochemistry pathway ( figure 4c ) , which had an ipa network score of 40 . the fold change and the expression values for this pathway are shown in table 1 . as the above networks were not regulated in infected cells , we hypothesized that they might represent genes and gene pathways for which homeostasis was favorable to parasite survival and , inter alia , that they represented genes potentially associated with antimicrobial defense regulated by inflammation . to test this hypothesis , we selected the most substantially represented network for further proof - of - concept studies ( figure 4c ) . ipa identified that , 11% ( 27/241 genes ) of genes selectively regulated in their expression in inflamed kcs were found within the lipid metabolism and small - molecule biochemistry pathway ( figure 4c ) , which had retinoid x receptor ( rxra ) as a key hub ( with rxra itself being downregulated 3.5-fold in inflamed cells ) . we then sought to confirm the level of mrna accumulation in infected and inflamed kcs in independent samples by quantitative real - time pcr ( figure 5 ) . although rxra mrna expression was not significantly different in infected and inflamed cells ( figure 5b ) , other genes within this network were validated as being differentially expressed between infected and inflamed cells , including cebpa ( figure 5c ) , vamp4 ( figure 5d ) , lxr/nr1h3 ( figure 5f ) , and lxr/nr1h2 ( figure 5 g ) . as infection with leishmania resulted in maintenance or upregulation of expression of genes within the rxra - associated gene network , we reasoned that if this was associated with parasite survival , then forced down modulation of rxr activity might sufficiently perturb the entire rxra - related gene network within infected cells to generate an enhanced antimicrobial effect . we therefore tested this approach by targeted pharmacological intervention , using a small - molecule antagonist hx531 , specific for rxr ( ebisawa et al . , 1999 ; yamauchi et al . , 2001 ) . to first establish that hx531 treatment had a direct effect on the gene network in infected kcs , we treated mice with hx531 or saline , infected the mice with l.donovani , and isolated infected or inflamed kcs to examine gene expression by quantitative real - time pcr ( figure 5a ) . treatment had no significant effect on the expression of rxra at an mrna level ( figure 5b ) , but treatment resulted in a significant modulation of the expression of a number of network - associated genes , including cebpa ( figure 5c ) , vamp4 ( figure 5d ) , vdr ( figure 5e ) , lxr ( but not lxr ) ( figures 5f and 5 g ) , and rara ( figure 5h ) . peroxisome proliferator - activated receptor ( ppar ) was not regulated in infected cells ( figure 5i ) and showed no change in expression following hx531 treatment . we also examined the effect of hx531 treatment on a number of rxr - related genes that were not represented within the identified network ( figure 4c ) , including abca1 ( figure 5j ) , abcg1 ( figure 5k ) , apoe ( figure 5l ) , cyp27a1 ( figure 5 m ) , and cyp27b1 ( figure 5n ) . treatment had no measurable effect on the expression of these genes in kcs , with the exception of abca1 , where treatment appeared to block upregulation in uninfected cells ( figure 5j ) . hx531 treatment blocked the upregulation of il-1 in infected kcs ( figure 5o ) but had no effect on inducible nitric oxide synthase ( inos ) mrna levels in infected or inflamed kcs ( figure 5p ) . to confirm that genes within this network were not subject to regulation merely by phagocytosis , we performed rt - pcr for a subset of genes within this network on kc isolated from mice injected with latex beads . as shown in figure s2 , uptake of latex did not affect mrna levels for rxra ( figure s2a ) , lxr ( figure s2b ) , cebpa ( figure s2c ) , or vamp4 ( figure s2d ) , relative to kc isolated from control - injected mice . to determine the effect that the perturbation of the rxr pathway would have on infection , and hence to examine the potential of the expression profiling approach that we have used for the discovery of important host response pathways , we treated mice with hx351 prior to and for 4 days following infection with td - tomato l. donovani amastigotes ( figure 6a ) . using a flow cytometric approach to quantify the number of parasites in the tissues , we first assessed splenic and hepatic parasite burden at 2 hr p.i . , when there was no difference in the presence or absence of treatment , and at 96 hr p.i . treatment significantly reduced the number of parasites present in both the liver and the spleen at 4 days postinfection relative to the saline - treated control animals . to examine whether perturbation in this pathway could also be achieved by stimulating , rather than inhibiting , rxr , we treated mice with lg268 ( an rxr agonist ) . lg268 also reduced the number of parasites present in the spleen but not the liver . to determine whether rxr perturbation could be operating through effects on lxr , we treated mice with gw3965 ( an lxr agonist ) . gw9395 treatment had no effect on parasite burden in either tissue ( figures 6b and 6c ) , suggesting that lxr is not involved in control of parasite load mediated through the rxr network . in order to examine if the therapeutic effect observed reflected leishmanistatic or leishmanicidal activity , we also calculated the fold change in parasite numbers in the 2 organs at 4 days p.i . , parasite numbers increased over a 96 hr period , and this growth was inhibited after drug treatment . in contrast , in liver , parasite numbers did not increase significantly over 96 hr in nontreated mice and were reduced to below the level seen at 2 hr p.i . in drug - treated mice , suggestive of leishmanicidal activity ( figure 6e ) . to confirm this observation , we used a histological approach in the liver and further showed that the percentage of infected kcs was significantly reduced in the lg268-treated animals and showed a trend in this direction in hx531-treated mice but not in gw3965-treated mice ( figures 6f and 6 g ) . although not significant in the single experiment shown in figure 6 , replicate experiments with mice treated with hx531 confirmed that this drug enhanced the leishmanicidal activity of kcs ( p = 0.0028 ; n = 11 mice for saline controls and 8 mice for hx531 treatment ) . in conclusion , these data indicate that pharmacological manipulation of rxr function was sufficient to perturb the gene network that we identified as being aberrantly expressed in infected kcs . furthermore , it was unlikely that the effects of hx531 or lg268 were due to direct effects on lxr , as stimulation of lxr had no measurable biological effect . we have examined the early in vivo response of liver macrophages to infection with leishmania , independently studying the activation of infected and uninfected cells exposed to inflammatory signals within the infected tissue microenvironment . our approach provides a number of unique insights into the transcriptomic response induced by l. donovani infection in vivo and provides proof of concept that the use of cells exposed to inflammation is a powerful method for the discovery of pathogen - regulated gene pathways . first , the isolation of kc from infected mice revealed a transcriptional response in infected kcs that is somewhat distinct from that observed by others using different types of mononuclear host cells ( including bone marrow - derived macrophages , monocytes , and dcs ) . it remains to be determined whether this reflects cell specificity in host response or differences due to the analysis being performed on cells infected in vitro versus in vivo . in most previous in vitro experiments , the possible influence of uninfected cells on gene expression profile , e.g. , by dilution of the signal associated with infected cells , was not taken into account ( buates and matlashewski , 2001 ; chaussabel et al . , 2003 ; , 2009 ; rodriguez et al . , 2004 ) . in one series of studies , dcs infected in vitro with l. amazonensis were sorted to allow microarrays to be conducted only on infected cells ( giraud et al . , 2012 ; lecoeur et al . , while these studies revealed unique signatures not evident from bulk population analysis , data were not provided to show whether the uninfected dc within the culture system had also made bystander responses ( e.g. , mediated by cytokine release from infected dcs in culture ) . furthermore , the transcriptomic signatures generated by microarray in this and indeed most other studies ( including those cited above ) have not been validated fully at the protein level . second , our data demonstrate significant quantitative and qualitative differences in transcriptomic responses compared to previous in vitro gene expression studies . in vitro experiments have usually been interpreted as indicating that visceralizing species of leishmania result in a relatively silent infection of macrophages ( zhang et al . , 2010 ) , significant suppression of gene expression ( buates and matlashewski , 2001 ) , or a signature distinct from that associated with interferon ( ifn)- activation ( rodriguez et al . , 2004 ) . the tendency of l. donovani to induce a weak inflammatory response in vitro may be species specific , with l. donovani metacyclics inducing lower fold changes in inflammatory gene expression compared to l. major metacyclics in one study ( chaussabel et al . , 2003 ) . alternatively , there may be life cycle stage - specific determinants of gene expression , as shown for l. mexicana ( aebischer et al . , 2005 ) . in an additional study , l. amazonensis amastigotes were also shown to induce significant gene expression changes in vitro , including changes to the sterol biosynthesis pathway ( osorio y forta et al . , 2009 ) . importantly , the impact of l. chagasi on host macrophage response was significantly modulated by the addition of t cells to the culture in vitro ( ettinger and wilson , 2008 ) , suggesting that factors external to the infected macrophage impact the response of the infected cell . our data support and extend these observations by adding the full in vivo complexity of the microenvironment created in the infected liver . third , we show that uninfected kcs also undergo significant biological and gene transcriptional changes associated with the inoculation into mice of live l. donovani amastigotes . the activation of uninfected kcs at early time points , with further downstream transcriptional consequences seen at 12 hr , suggests that soluble mediators or signals operating in trans facilitate global activation of macrophages within the infected tissue . although this activation shows both qualitative and quantitative differences between infected and inflamed kcs , many of the genes that have been associated with cell - autonomous defense against intracellular protozoa stimulated by interferons ( macmicking , 2012 ) are induced to similar levels in infected and uninfected kcs at 12 hr postinfection . this would place such genes in the category of commonly - regulated genes within the framework of our analysis . such genes include the guanylate - binding proteins gbp2 and gbp5 , indoleamine 2,3-dioxygenase 1 ( ido1 ) , immunity - related gtpase family m members 1 and 2 ( irgm1 and irgm2 ) , and immunoresponsive gene 1 ( irg1 ) . these data are somewhat intuitive , given that ifn is a soluble mediator , capable of trans - activating neighboring cells . collectively , our comparative analysis of the gene expression profiles of infected and inflamed kcs taken from the livers of infected mice suggests that a degree of caution should be exercised when interpreting whether genes observed to alter their expression in response to infection truly represent autonomously regulated genes associated with the presence of intracellular pathogens . fourth , we have identified and validated the importance of a gene network centered around rxra that is associated with the survival of amastigotes within the liver of l. donovani - infected mice . within this rxra network , although lipid metabolism centered around rxr has not previously been implicated in l. donovani pathogenesis in mice , other studies have observed changes in lipid metabolism genes associated with leishmania infection . for example , changes in components of the sterol biosynthesis pathway were induced by macrophage infection with l. amazonensis ( osorio y forta et al . previous studies have characterized a role for two heterodimeric partners of rxr in the control of leishmania major infection , the vitamin d receptor ( vdr ) ( ehrchen et al . , 2007 ; whitcomb et al . , 2012 ) and the liver x receptor ( lxr ) ( bruhn et al . , in addition , upregulation of vdr was previously reported following in vitro infection of macrophages with l. chagasi ( rodriguez et al . , 2004 ) as well as l. donovani and l. major ( chaussabel et al . , 2003 ) , suggesting that vdr is an important component of the host response to leishmania infection . we believe that by targeting rxr as the hub of the identified network , we have successfully perturbed an entire gene network that controls l. donovani survival in kcs . perturbation of rxr in either direction via activation with the agonist lg268 or suppression with the antagonist hx531 had similar effects on the growth of l. donovani in vivo . the mechanism by which this network controls parasite survival is currently subject to further investigation . however , we speculate that the gene network may regulate the usage of lipids and cholesterol by the parasite while it is present in the phagolysosome of the host cell ( mcconville and naderer , 2011 ) . alternatively , macrophage sensing of leishmania - derived lipids may be having profound effects on the nature of the innate immune response that is initiated ( nagy et al . , 2012 ) . finally , and of broader significance , we have described an approach to study host - pathogen interactions , which is capable of revealing important host response pathways that would be invisible in studies of the interaction between macrophages and pathogens in vitro . importantly , we show that manipulation of such pathways in vivo can impact parasite survival , reducing parasite load at early stages after liver infection . targeting of nuclear receptors for therapy is a burgeoning field of medicine , with rxr therapeutics showing efficacy in clinical settings of cutaneous t cell lymphoma ( duvic et al . , 2001 ; khuri et al . , 2001 ) and a preclinical model of alzheimer s disease ( cramer et al . , 2012 ) . rxr antagonists , including hx531 , have also shown efficacy in preclinical models of diet - induced obesity and type 2 diabetes ( yamauchi et al . , 2001 ) . in this respect , while it is tempting to suggest that our findings may also set the foundation for the use of rxr - mediated therapies for leishmaniasis , detailed investigations into the mechanism of parasite growth arrest and/or killing are now required . mt / mg ( muzumdar et al . , 2007 ) and lysmcre ( clausen et al . , 1999 ) mice were bred and housed under specific pathogen - free conditions and used at 612 weeks of age . the ethiopian strain of leishmania donovani ( lv9 ) and tandem tomato fluorescent protein expressing lv9 ( tdtom.lv9 ) ( beattie et al . , 2010 ) were maintained by serial passage in rag-1 mice . amastigotes were isolated from infected spleens ( smelt et al . , 1997 ) , and mice were infected with 3 10 l. donovani amastigotes intravenously ( i.v . ) via the tail vein in 200 l of rpmi 1640 ( gibco ) . parasites were killed via heat treatment at 56c for 15 min , followed by labeling with pkh26 red fluorescent cell linker kit ( sigma ) . for latex bead experiments , 1 m fluoresbrite polychromatic red microbeads ( polysciences ) were washed twice in saline , and 3 10 beads per mouse were injected i.v . all experiments were approved by the university of york animal procedures and ethics committee and performed under uk home office license ( immunity and immunopathology of leishmaniasis ref # ppl 60/3708 ) . mice were anesthetized , and surgery was performed similar to that previously described ( beattie et al . , 2010 ) , except that anesthesia was maintained by inhalation of 4% isofluorane ( abbott laboratories ) . images were acquired on an inverted lsm 780 multiphoton microscope ( zeiss ) maintained at 36c by a blacked - out environmental chamber ( solent scientific ) . images were acquired with a 40 1.1 water immersion objective and fluorescence excitation provided by a chameleon xr ti : sapphire laser ( coherent ) tuned to 870 nm . for 4d analysis , 2035 m z stacks were acquired with a z distance of 2 m approximately every 1530 s. real - time injection was performed via tail vein catheter ( strategic applications incorporated , infusion technologies ) . in addition to collecting baseline data for animals before infection , individual kcs were imaged prior to and for 2 hr directly following infection of mice with l. donovani . data were rendered and analyzed using volocity software ( perkinelmer ) , and cell analysis was performed automatically with manual checking . confocal microscopy was performed on 20 m frozen sections as previously described ( beattie et al . , 2011 ) . slides were blinded before imaging on a zeiss lsm 510 axioplan microscope ( zeiss ) . hepatic mononuclear cells were prepared from the livers of tdtom.lv9- or rpmi - injected c57bl/6 mice . we conducted four independent infection experiments to generate samples for analysis . in each experiment , n = 16 mice , and at each time point studied ( 2 hr and 12 hr postinfection ) , we isolated kcs from the livers of four mice treated with rpmi and four mice infected with l. donovani . liver mononuclear cells were pooled prior to sorting . for real - time pcr analysis , replicates represent pooled samples from independently sorted samples . livers were perfused with 0.4 mg / ml of warmed liberase tl ( roche ) then digested at 37c for 45 min . digested livers were passed over 100 m cell strainers , washed in pbs containing 2% fetal calf serum ( fcs ) , and resuspended in 33% percoll for centrifugation at 693 g for 12 min . isolated cells were blocked with anti cd16/32 then labeled with f4/80-alexa 488 , gr-1 pecy7 ( ebioscience ) , and crig ( helmy et al . , 2006 ) that was conjugated to atto 647h via lightning - link conjugation ( innova biosciences ) . a small fraction of cells ( approximately 3,000 sorted cells ) were spun onto glass slides , fixed in methanol , and stained with giemsa for morphological analysis . rna was isolated from purified kc and amplified via agilent low input quick amp labeling kit ( agilent technologies ) . amplified rna was then assayed with agilent sureprint g3 mouse gene expression 860k microarray chips that were scanned with an agilent c scanner with surescan high - resolution technology ( agilent ) . comparison of the gene expression data between the rpmi controls and the infected or inflamed samples was performed using the benjamini and hochberg false discovery rate ( fdr ) correction ( pawitan et al . , 2005a ) , albeit with the caveat that this approach may have low sensitivity with the sample size used ( pawitan et al . , 2005b ) . this analysis was performed with genespring software ( version 9 ; agilent ) as a standard 5% fdr , with the variances assessed by the software for each t test performed . all data for long intergenic noncoding rna ( lincrna ) probe sets were removed from the analysis at this point . gene ontology analysis was performed using the david ( huang et al . , 2009 ) and ingenuity pathway analysis software packages ( ingenuity systems ) , and gene maps generated with string ( www.string-db.org ) . mice were pretreated with saline , 100 mg / kg hx531 ( yamauchi et al . , 2001 ) , 30 mg / kg lg268 ( shen et al . , 2004 ) , or 20 mg / kg gw3965 ( lei et al . , 2013 ) 26 hr prior to infection and then every 24 hr until 72 hr for hx531 and lg268 or every 12 hr for gw3965 . at 96 hr postinfection , the livers and spleens were collected , and the parasite burden was determined both by flow cytometry and in fixed liver slices . data were normalized to the baseline 2 hr time point to determine the survival of parasites relative to the initial dose of infection . liver and spleens were collected , homogenized , and resuspended in a specific volume of rpmi 1640 ( gibco ) . a total of 200 l of each sample was aliquoted in duplicate to a 96-well plate , centrifuged at 2,000 g for 10 min , and the pellets were resuspended in 2 mg / ml saponin for 5 min at room temperature ( rt ) . the centrifugation was repeated and followed by resuspension in 1 mg / ml saponin . the remaining pellets were washed a further two times , and the wells were resuspended in 200 l then spiked with 2 l of 6 m polystyrene microspheres ( polysciences , inc . ) at 4.6 10 particles/l . samples were acquired on a cyan adp analyzer ( beckman coulter ) in which both forward scatter detector neutral density filters had been removed to allow for small - particle detection . the number of parasites was quantitated by determining the ratio of beads to parasites and then multiplying to calculate the total number of parasites in the starting volume . rna was reverse transcribed into complementary dna ( cdna ) with a superscript iii first - strand synthesis kit ( invitrogen ) . oligonucleotides used for the specific amplification of target genes are described in the supplemental experimental procedures . real - time quantitative pcr was performed with fast sybr green pcr master mix in a steponeplus real - time pcr system ( applied biosystems ) . accumulation of target genes was normalized to hprt , -actin ( shih et al . , 2012 ) , or ribosomal protein l32 ( reis et al . , 2013 ) and expressed as relative expression via the change in cycle threshold ( ct ) analysis method ( relative expression in test sample versus rpmi controls ) .

summaryintracellular pathogens modulate host cell function to promote their survival . however , in vitro infection studies do not account for the impact of host - derived inflammatory signals . examining the response of liver - resident macrophages ( kupffer cells ) in mice infected with the parasite leishmania donovani , we identified a transcriptomic network operating in uninfected kupffer cells exposed to inflammation but absent from kupffer cells from the same animal that contained intracellular leishmania . to test the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival , we pharmacologically perturbed the activity of retinoid x receptor alpha ( rxr ) , a key hub within this network , and showed that this intervention enhanced the innate resistance of kupffer cells to leishmania infection . our results illustrate a broadly applicable strategy for understanding the host response to infection in vivo and identify rxra as the hub of a gene network controlling antileishmanial resistance .

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the control of appetite is complex and involves the coordination of inputs from both physiological and environmental sources . early theoretical approaches were based on the idea that the control mechanism was dedicated exclusively to signals from glucose metabolism , amino acids or proteins , or adipose tissue . respectively , these formed the glucostatic ( mayer , 1955 ) , aminostatic ( mellinkoff et al . , 1956 ) and lipostatic ( kennedy , 1953 ) hypotheses . more recent central nervous system models have emphasised inputs from adipose tissue and the gastrointestinal ( gi ) tract [ along with input from sensory features of food ( e.g. schwartz and morton , 2001 ; keesey and powley , 2008 ) ] . the major signals include leptin from adipose tissue , and hormones such as ghrelin , pyy , glucagon - like peptide 1 ( glp-1 ) , cholecystokinin ( cck ) , amylin and insulin from specialised cells in the gi tract or associated organs . such models are said to account for the regulation of appetite and , in turn , contribute to the regulation of body weight , through the identification of specific molecular signals carrying information from the periphery to a network of pathways centred on the hypothalamus . interest in this type of formulation strengthened during the period when molecular biology began to take over from integrative physiology as an explanatory approach in biological sciences . the major task facing any model of human appetite regulation is to account for the ongoing and recurring drive to eat , together with the intermittent suppression of eating via episodic satiety signalling and tonic inhibition ( halford and blundell , 2000 ) . in this commentary , we outline the basis of a new formulation that explains how aspects of body composition and energy expenditure ( ee ) regulate hunger and appetite . this formulation is based on 15 years of psychobiological research with overweight and obese human subjects , and should help to contribute to understanding the relative impact of biological and environmental cues on body weight . an alternative to the molecular approach to appetite control has arisen from the study of energy balance : the inter - relationships between ee and energy intake ( ei ) . in part , this view stems from the statement that any increase in ee will be met with an equivalent increase in ei ( mayer et al . , 1956 ) . indeed , it was proposed that the differences between the intakes of food [ of individuals ] must originate in differences in the expenditure of energy edholm s experimental studies , however , revealed no correlation between ee and ei in any one single day , but there was a good correlation over 2 weeks . the lack of any relation in a day is perhaps not surprising , because any episode of ee ( such as work or exercise ) happening towards the end of the day would not leave time for a balancing effect of eating . moreover , daily ee can fluctuate markedly from day to day according to individual behavioural tendencies . by contrast , resting metabolic rate ( rmr ) is the largest component of total daily ee and is quite uniform across the day and between days ( johnstone et al . , 2005 ; johnstone et al . , 2006 ; ravussin et al , 1982 ) , and it is plausible that ee from rmr could provide a tonic signal of energy demand that could act as a driver of daily ei . by contrast , physical activity ee ( being of greater intensity and sporadic ) would be expected to exert a mechanistically different type of control over appetite . exercise can raise resting values of ee several fold depending on the volume , mode and intensity . in addition , the effect of exercise on various body systems means that it will have numerous physiological effects in addition to an increase in ee . such effects include an overall increase in heart rate , and changes in the distribution of blood flow , sympathetic nervous system activity , gut hormone activity and in the absorption of nutrients . therefore , it might be anticipated that there could be a number of separate effects on mechanisms influencing appetite . in addition , it can be presumed that a single acute bout of exercise would have different effects from continued daily sessions of exercise that would exert an increasing cumulative challenge to energy balance . a comprehensive picture therefore requires a comparison between various intensities of exercise over varying periods of time with measurement , wherever possible , of all variables in the energy budget . a central issue underlying the investigation of exercise is to determine [ as mayer proposed ( mayer et al . , 1956 ) ] whether or not exercise inevitably induces a compensatory response characterised by an increase in hunger and in turn food intake that would lead to an increase in ei that is sufficient to balance the increase in ee generated by exercise . over the last 15 years , we have developed an experimental platform based on a psychobiological approach and incorporating a multi - level system ( caudwell et al . this has allowed the disclosure of relationships between variables in the domains of body composition [ fat free mass ( ffm ) , fat mass ( fm ) ] , metabolism ( rmr , respiratory quotient , respiratory exchange ratio ) , gi hormones , food composition , psychological sensations , food behaviour , psychometric traits , nutritional composition and allelic variation in a single cohort of overweight and obese subjects . in summary , we find that acute ( single ) bouts of exercise do not lead to a consistent increase in the average food intake in the immediate post - exercise period or in the following 24 hours ( for reviews see blundell and king , 1998 ; martins et al . , 2007 ) . however , there is considerable variability associated with appetite variables ( finlayson et al . , 2009 ) . as exercise sessions are repeated over periods varying from 9 to 16 days , a partial compensatory response begins to appear ( stubbs et al . there seems to be little or no compensation through a decrease in non - exercise - related activity ( turner et al . when exercise was performed daily over a period of 16 days and total ee was measured using the doubly labelled water method , it was calculated that exercise raised average daily ee by 3.5 mj and generated a 30% compensation in ei ( whybrow et al . , 2008 ) . several other studies have measured the impact of different exercise prescriptions over periods of 12 to 32 weeks ( church et al . a typical 12-week study using overweight and obese men and women showed that exercise caused an average weight loss of 3.3 kg , indicating that the exercise ee was not fully compensated ( king et al . , 2008 ) , but individual variability was very large [ also observed in other studies ( church et al . a key feature of this study was that exercise sessions ( five times per week for 12 weeks ) was fully supervised and the ee measured so as to rule out the possibility of the outcome being influenced by lack of compliance . subsequent investigations indicated a dual action of exercise on appetite control that partly explained the large variation in body weight change . the exercise caused a significant increase in fasting levels of hunger ( a compensatory response ) , but this was highly variable between subjects . however , there was an associated increase in the post - prandial satiety signalling from meals , indicated by a change in the satiety quotient ( green et al . , 1997 ; king et al . consequently , the effect of exercise on appetite comprises two components : one generating a tonic stimulation of appetite and the other a counteractive meal - related episodic inhibition . the second response seems to be related to changes in either the release of , or sensitivity to , gi peptides ( broom et al . , 2007 ; martins et al . , 2010 ) . these effects of exercise - related ee on appetite are likely to be quite different from any effect related to the lower intensity and more uniform tonic action of rmr . using the multi - level experimental platform described above ( caudwell et al . , 2011 ) , it has been possible to observe the relationships between variables in separate domains ( biological , nutritional , behavioural , metabolic ) , all measured objectively and to similar degrees of accuracy . importantly , the measurements have been made simultaneously in the same group of overweight and obese people . this has enabled the disclosure of unexpected associations between body composition and appetite . a little more than 10 years ago , the discovery of leptin was decisive in focussing attention on adipose tissue as a major controller of energy balance ( ei and ee ) . several models of appetite regulation have described the role of leptin ( as a signal from adipose tissue ) in influencing hypothalamic neuropeptide pathways that control the stimulation and inhibition of food intake ( morton and schwartz , 2001 ; badman and flier , 2005 ) . with most attention directed to the role of adipose tissue ( or fm ) , it can be considered that ffm has become the forgotten variable , even though it is clearly important under some conditions ( dulloo et al . , 1997 ) . it is therefore interesting that detailed analysis of body composition and appetite variables using the multi - level platform have demonstrated that ffm , but not fm or body mass index ( bmi ) , is strongly correlated with meal size and daily ei ( blundell et al . the strong implication of this relationship is that some privileged molecules arising from ffm , or some physiological consequences that reflect the activity of ffm , act as a signal to drive food intake . considering total daily ee , it is noticeable that rmr is its largest component ( 5070% ) , of which ffm is the major contributor , accounting for about 6070% , whereas fm accounts for as little as 57% , with gender and age being minor components ( johnstone et al . , 2005 ) . consequently , it is plausible that rmr could act as a mediating variable to reflect the influence of ffm on appetite control . accordingly , in a series of analyses based on the multilevel platform described earlier we have demonstrated that rmr is positively associated with meal size and with daily ee ( note that the relationship between rmr and total daily ei mirrors the relationship between ffm and total daily ei , which is shown in fig . in addition , rmr is also a predictor of fasting levels of hunger and influences the profile of hunger across the day ( illustrated by fig . the strong implication of these relationships is that rmr as a measure of ee reflects a physiological demand for energy that acts as a driver of food intake . this is plausible and constitutes a demonstration of edholm s proposal that differences in food intake must originate in the differences in energy expenditure ( edholm et al . , 1955 ) . although edholm did not demonstrate a relationship between ei and the ( highly variable ) total daily ee ( within any single day ) , we have demonstrated a clear relationship with the more stable and uniform factor of rmr . the existence of a relationship ( within a single day ) between rmr and daily ee would depend on the total volume and intensity of the discretionary physical activity component of ee , and upon the relative strengths of the dual processes of appetite control generated by exercise ( see above ) . most recent models of appetite regulation have drawn attention to the roles for adipose tissue and gi peptides . however , these models are better able to account for the inhibition of eating rather than its initiation . the models embody the view that tonic and episodic inhibitory factors modulate an intrinsic orexigenic drive to eat . this endogenous drive is not usually well defined . however , in early theorising about appetite control , equal emphasis was given to the inhibitory and excitatory ( drive ) features of appetite . the latter was conceptualised as morgan s central motive state ( morgan , 1943 ) , and in stellar s embodiment of this in an excitatory centre in the hypothalamus ( stellar , 1954 ) . one major issue was to explain what gave animals ( including humans ) the energy and direction that motivated food seeking . in light of knowledge about the physiology of homeostasis , it is plausible that a drive for food arises from the energy used to maintain physiological and metabolic functioning of the body . this resonates with the proposal of edholm ( edholm et al . , 1955 ) , and represents a merging of parallel thinking from psychobiologists working on animal behaviour and physiologists working on human energy balance . together with modern ( post - leptin ) discoveries , it can be suggested that the physiology of appetite regulation comprises three components : a tonic drive for food arising from the physiological demand for energy ; a tonic inhibition arising from signals of energy storage ( mainly adipose tissue ) ; and episodic signals arising from the mouth and gi tract in response to the periodic consumption of food . this last category is primarily inhibitory from the classical satiety signals but also excitatory ( signals registering food palatability ) . ( a ) scatter plot shows the relationship between ffm and daily ei in a group of 41 overweight or obese men and women . the values represent the average values from objective measures taken at specific probe days at weeks 0 , 6 and 12 . ( b ) daily hunger profiles for individuals with the highest ffms ( eight women and five men ) and those with the lowest ffms ( eight women and five men ) . the groups were formed by dividing the whole sample into tertiles of ffm and comparing the upper and lower tertiles ( but keeping similar numbers of men and women in each tertile ) . the difference between the area under the curve for the two profiles is significant ( p<0.01 ) ; asterisks indicate time points at which the profiles are significantly different . the profiles indicate that hunger in the high ffm group is greater before the onset of a meal and in the period leading up to the meal , but not at the end of the meal . this is consistent with the fact that the high ffm individuals eat a larger meal , which would return hunger to a level appropriate for full satiation . together , data displayed in a and b indicate that ffm is positively associated with daily ei and with daily hunger levels . additionally , because rmr is highly and significantly correlated with ffm , the relationships between rmr , ei and hunger very closely match those for ffm shown in the figure . hunger is measured in units on a 100 unit scale on the screen of a hand - held pda . the investigations reviewed here have provided evidence for the physiological drive for food by demonstrating a specific association of ffm , but not fm , with objectively measured food intake variables ( blundell et al . , 2012 ) . in turn , this means that the energy required to maintain the body s lean tissue mass thus preventing wasting creates a physiological demand that determines a minimal level of ei at meals and over the day . it is worth noting that the relationship between ffm and ei is consistent with the amino - static hypothesis put forward more than 50 years ago by mellinkoff et al . ( mellinkoff et al . , 1956 ) , and with the more detailed proposal of a role for the protein - stat mechanism described by millward ( millward , 1995 ) . a new formulation for appetite control . a proposed tonic signal for the drive to eat that reflects the body s demand for energy arises ( mainly ) from ffm and rmr in turn , this drive is under tonic inhibition from leptin , whose action reflects the amount of stored energy reserves in the body . as the amount of adipose tissue increases , leptin insensitivity occurs and this tonic inhibition is reduced . the drive to eat is periodically interrupted and suppressed by episodic signals in the form of peptides that are released from the gi tract in response to food consumption . the resulting pattern of eating is a consequence of the interactions between tonic and episodic physiological signals . prolonged exercise displays a dual - process action by stimulating hunger ( an effect that is highly variable between individuals ) but also by increasing post - prandial satiety signalling ( king et al . , 2009 ) through an effect on gi peptides ( martins et al . , 2010 ) . a further step in the formulation comes from the demonstration that rmr is also strongly correlated with objectively measured self - determined ei ( caudwell et al . , this could be expected from the recognition that ffm is the largest contributor to rmr ( accounting for approximately 60% in our studies ) . it is worth noting that ee arising from ffm receives contributions from the liver ( 20% ) , brain ( 20% ) , heart ( 11% ) , gi tract ( 9% ) and skeletal muscle ( 20% ) ( elia , 1992 ) . therefore , the physiological demand is created by the need to provide energy to maintain the functioning of the body s vital systems . moreover , the ee from these organ systems is maintained across the day and is reflected in the relative stability and uniformity of rmr within and between days . consequently , the proposal that rmr acts as a physiological drive ( or is a marker of such a drive ) underlying appetite is both parsimonious and plausible . this formulation is quite consistent with those models of appetite regulation that assign major roles to adipose tissue and the gi tract . however , it also draws attention to other aspects of body composition as determinants of appetite . combined with other findings , this interpretation provides mechanistic explanations for the link between ffm , rmr and eating behaviour . this formulation can incorporate an extended role for leptin and is in keeping with a widening ergostatic function of additional adipokinetic hormones in energy homeostasis , as envisaged by frhbeck and gmez - ambrosi ( frhbeck and gmez - ambrosi , 2001 ) . the idea of ffm creating an orexigenic or ergostatic drive has also been proposed in other descriptions of the appetite regulatory system ( halford and blundell , 2000 ) . adipokines exert effects not only on adipose tissue but also on skeletal muscle ( sinz et al . , 2009 ) , and that differential expression of genes and pathways that are intrinsic to skeletal muscle might also be involved ( wu et al . , 2011 ) . consequently , it can be envisaged that leptin could act as a major tonic inhibitory signal ( to dampen the physiological drive ) and also as an energostatic factor through its influence on ffm . the proposal that ffm and rmr contribute to a physiological demand for energy that influences appetite is plausible and has implications . first , it is one further reason to be dissatisfied with the use of measures of body weight ( or bmi ) in the research and management of obesity . the recognition that ffm and fm have different functional properties in relation to appetite is a strong reason to use body composition ( rather than the coarse variable of body weight ) in both the research and management of obesity . for example , two individuals with similar bmis ( or body mass ) might have quite different proportions of ffm and fm , and this would confer different properties on their physiological and behavioural responses . those people with a high ffm should have a proportionately higher orexigenic drive to maintain a greater minimal meal intake ( i.e. they should eat more ) than people with less lean tissues and organs . this means that obese people ( with a greater lean mass in support of a large amount of adipose tissue ) and people carrying a large muscle mass ( field athletes , rugby players , swimmers , etc . ) should have a stronger tendency to consume larger meals than smaller people . it follows that such people would have greater difficulty in tolerating dietary restriction ( because the more energetically active lean mass would sustain a drive for a minimal amount of food ) . moreover , in elderly people subject to sarcopenia , the reduced lean mass would result in a diminished appetite . however , it should not be inferred that the influence of ffm and rmr upon food intake is a cause of weight gain or obesity . this mechanism is a physiological way of achieving energy balance ( ensuring that ei does not fall below the energy demand of the body ) . as such , the mechanism influences the strength of the drive to eat ( feeding behaviour ) and determines the level of hunger at the beginning of a meal ( fig . the amount of energy that is actually consumed is strongly modulated by the energy density of the food available ( ello - martin et al . , 2005 ; rolls , 2000 ) . high energy density of food results in passive overconsumption ( blundell and macdiarmid , 1997 ) , which has been identified as a major component of the obesogenic environment ( swinburn et al . , 2011 ) . consequently , a high rmr could influence weight gain by maintaining a high level of hunger , but a positive energy balance would depend on the energy density and palatability of the diet . this is an example of a physiological regulatory process being undermined by the nature of the modern diet in many technologically advanced countries . in turn , the formulation proposed here can help to promote research to clarify the relative strength of biological and environmental variables that contribute to ei and to changes in body weight ( and body composition ) . moreover , we envisage that the system would adapt to progressive changes that occur in body composition over time and could influence energy balance ( e.g. hall et al . , the most likely scenario is that any increase in ffm ( and rmr ) would increase the drive to eat . this amplifying effect on ei would mean that weight gain becomes part of a positive , rather than a negative , feedback system . increasing body weight therefore could facilitate further weight gain and increase the difficulty of weight loss or maintenance .

a long - running issue in appetite research concerns the influence of energy expenditure on energy intake . more than 50 years ago , otto g. edholm proposed that the differences between the intakes of food [ of individuals ] must originate in differences in the expenditure of energy . however , a relationship between energy expenditure and energy intake within any one day could not be found , although there was a correlation over 2 weeks . this issue was never resolved before interest in integrative biology was replaced by molecular biochemistry . using a psychobiological approach , we have studied appetite control in an energy balance framework using a multi - level experimental system on a single cohort of overweight and obese human subjects . this has disclosed relationships between variables in the domains of body composition [ fat - free mass ( ffm ) , fat mass ( fm ) ] , metabolism , gastrointestinal hormones , hunger and energy intake . in this commentary , we review our own and other data , and discuss a new formulation whereby appetite control and energy intake are regulated by energy expenditure . specifically , we propose that ffm ( the largest contributor to resting metabolic rate ) , but not body mass index or fm , is closely associated with self - determined meal size and daily energy intake . this formulation has implications for understanding weight regulation and the management of obesity .

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one of the most widely available materials in prosthetic dentistry is polymethyl methacrylate ( pmma ) , a material that has been used as a denture base since 1937 . heat - polymerized pmma resin is used as a denture base material because of its excellent esthetics , low water sorption and solubility , relative lack of toxicity , repair ability , and simple processing technique . a conventional method for curing resin advantages of this method include ease of processing , its familiarity for dentists and technicians , and the lack of need for any sophisticated or expensive equipment . disadvantages of this technique are dimensional changes and inaccuracies in the fit of the denture base . therefore , the popularity and relative simplicity of the compression molding technique are usually overshadowed by the high processing stresses induced in the resin during polymerization . chemically activated resins are much less frequently used for denture base fabrication than heat - activated resins . these materials have a higher solubility and inferior color stability due to oxidation of the amine accelerator . chemically activated resins , most often when compression molded , display less polymerization shrinkage than their heat - activated counterparts , which leads to greater dimensional accuracy . this could be attributed to a reduction in residual stresses that have been induced during the processing cycle . the injection molding technique when compared with the compression molding technique has a reduced processing time , lower cost , lower skin sensitivity to the evaporated monomer , and availability of the resin reservoir to compensate for acrylic resin shrinkage . the type of processing could affect the mechanical properties of the resins such as the bond strength between the denture teeth and the denture base resins . debonding of denture teeth from denture bases is a major problem in the prosthodontic practice . previous surveys reported that 26 - 33% of denture repairs are the result of debonded teeth which cause distress and increased cost for patients . this may be related to the basic properties of the materials ( teeth or denture base materials ) , processing factors ( contaminations or curing cycle duration ) , and the available monomer during processing . attempts to increase the bond strength between acrylic resin teeth and denture base resins include grinding the glossy ground surface , painting the ground surface of the teeth with monomer , and cutting mechanical retention features in the ground surface of the teeth . clancy and boyer compared the bond strengths of light- and heat - polymerized , and autopolymerized denture base resins to the denture teeth . in their study , the strongest bonding occurred between heat - polymerized resin and plastic teeth . evaluated the bonding of two resin denture teeth to microwave and heat - processed denture bases . they obtained higher bond strength of denture teeth with the conventional heat - polymerized than the microwave - polymerized acrylic resins . the purpose of this investigation was to evaluate the effect of surface treatments on shear bond strength of denture teeth to heat - polymerized and autopolymerized denture base resins . the null hypotheses were : 1 ) there is no significant difference between the shear bond strength of the denture teeth to the two denture base resins and 2 ) surface treatments of the denture teeth have no effect on their bond strengths to the denture base resins . in this experimental in vitro study , 60 maxillary central incisor acrylic teeth ( ivoclar , vivadent , naturno , italy ) were divided into two groups which were processed with heat - polymerized acrylic resin ( meliodent , heraeus - kulzer gmbh , wehrheim , germany ) ( group m , n = 30 ) and autopolymerized acrylic resin ( futura gen , shutz - dental , rosbach , germany ) ( group f , n = 30 ) , respectively . denture teeth were further subdivided into the following three subgroups with n = 10 per group : 1 ) m1 , f1 : untreated ground surface ; 2 ) m2 , f2 : treated with monomer on ground surface ; and 3 ) m3 , f3 : treated with airborne particle abrasion . the methyl methacrylate monomer was applied to the teeth bonding surfaces using a cotton bud for 180 s. the airborne particle abrasion was done using 50 m aluminum oxide particles for 30 s from a distance of 10 mm under 2 bar pressure . the denture teeth were mounted on two sides of six triangular shaped wax models [ figure 1 ] and covered with silicone putty ( optosil , heraeus - kulzer , leverkusen , germany ) to facilitate deflasking . before processing , all teeth denture teeth in triangular shaped wax model each model was flasked and the wax removed by boiling water . six molds were made in this procedure for groups ( m1 , m2 , m3 , f1 , f2 , f3 ) . three molds were packed with meliodent denture base resin and cured in a curing machine by an automatic curing device ( ewl , type 5518 , kavo , leutkirch , germany ) in accordance with the manufacturer 's instructions . the three other molds were injected with futura gen resin and processed using unipress machine ( shutz - dental ) . after completion of the curing cycle and deflasking , the meliodent and futura gen specimens were stored in distilled water at 37c for 7 days . then , the shear bond strengths of the specimens were evaluated by universal testing machine ( instron corp . , canton , ma , usa ) . force was applied by a stainless steel pin , 1 mm in diameter , at an angle of 130 to the long axis of the tooth at a crosshead speed of 5 mm / min until fracture occurred . in a class i occlusion , this stimulates the average angle of contact between the maxillary and mandibular anterior teeth . the mean shear bond strength values were subjected to two - way analysis of variance ( anova ) followed by tukey 's test . for all statistical analyses , two - way anova demonstrated that the shear bond strength was significantly affected by the type of acrylic resin ( f = 4.71 , p = 0.03 ) and the surface treatment ( f = 21.09 , p < 0.001 ) , but the interaction between these two factors was not statistically significant ( f = 0.004 , p = 1.00 ) [ table 2 ] . mean and standard deviation of the studied groups tukey 's honestly significant difference ( hsd ) multiple comparison test revealed that the control groups had significantly lower bond strength than the treated groups ( p < 0.001 ) , but there was no significant difference between the bond strengths of groups treated by monomer or airborne particle abrasion ( p = 0.29 ) [ table 1 ] . failure mode was predominantly mixed in the meliodent groups , whereas adhesive failure was reported for futura gen specimens [ figure 2 ] . according to the results of the present study , there was significant difference between the shear bond strengths of the denture teeth to the two denture base resins and surface treatment of the denture teeth affected the bond strength significantly . some studies have reported that the bond strength of acrylic teeth to denture base resin is due to the diffusion and polymerization of monomer across the teeth base interface to form an interpenetrating polymer network . the efficiency of the attainment of such bond depends primarily on the rate at which the monomer diffuses from the base resin mixture . higher diffusion rate of the monomer of a denture base polymer mixture into the acrylic resin polymer teeth is achieved with increasing polymerization temperature . injection molding does not have any heat conduction as with the conventional water bath technique , thus the bond strength of the denture teeth in the meliodent group was higher than that in the futura gen group . development of alternative materials such as futura gen ( pmma ) is a breakthrough in denture base materials used for prosthetic dentistry . the advantages of futura gen include less shrinkage , outstanding denture adhesion characteristics , less processing time , and ease of polishing . the manufacturer claims that the quality of this material has been improved because of modification and binding ability which has resulted in higher bond strength . in addition , the lack of a monomer results in higher strength compared to that of heat - cured resin . according to the manufacturer , due to changing of the initiator system and replacement of changed copper and barbituric acid ions , instead of the tertiary amine , the amount of residual monomer in futura gen is similar to that of heat - polymerized resin . in similar studies on the bond strengths of denture teeth to different types of denture base resins , heat - polymerized acrylic resins showed the highest bonding values in comparison to autopolymerized or light - polymerized acrylic resins , which is in agreement with this study . on the contrary , vallitue suggested that heat - polymerized pmma did not adhere to the acrylic resin tooth better than the autopolymerizing pmma . the results of this study showed that the surface treatment with monomer is an effective method for improving the bond strength between denture teeth and denture base resins . acrylic teeth treated with monomer failed predominantly in the cohesive and mixed modes in meliodent groups , which is in agreement with the findings of previous studies on the benefits of chemical locks . the monomer that was applied to the tooth surface before packing provided a solvent effect on the tooth surface which favored a more effective diffusion of the monomer of the denture base polymer across the tooth denture base interface and formed interpenetrating polymer networks . one of the technical factors that contribute to the bond strength of denture tooth to denture base resin is ridge lap surface contamination with wax or tinfoil substitute residues , which can be cleaned with monomer application before packing pmma into the mold . the airborne particle abrasion of the denture teeth ground surface improved significantly the shear bond strength compared to untreated samples . although these results are in agreement with some studies , however , there are other studies which have shown that airborne particle abrasion or grinding decreases the bond strength of the treated teeth to denture bases . other studies have reported that teeth base roughening with cutting or abrasive rotary instruments or aluminum oxide air abrasion provides slightly higher bond strength values than those achieved without surface modification , or with cavity preparation or monomer etching . it has been shown that the use of mechanical retention is a primary means of securing the bond strength between acrylic resin teeth and the denture base resins . the airborne particle abrasion increases the surface area of the ridge lap portion of the denture teeth and , hence , improves their bond strengths to denture base resins . in this study , most of the futura gen specimens ( f1 > f2 > f3 ) showed adhesive failure , but failure mode of the meliodent specimens was predominantly mixed ( adhesive and cohesive ) ( m1 > m2 and m3 ) [ figure 2 ] . hatim and hasan demonstrated that the failure modes are mostly adhesive in all types of selected acrylic teeth with heat - polymerized resins and mixed for microwave acrylic denture bases . fletcher - stark et al . showed adhesive debonding between the acrylic denture teeth and light - polymerized resin without a bonding agent . but when a bonding agent was used with light- and heat - polymerized denture base resins , mixed and cohesive failures were observed . mechanical fatigue , thermal cycling , and chewing simulation were not considered in the present study ; therefore , the results of the present study should be interpreted with caution . within the limitations of this study , these conclusions can be drawn : treatment of the ridge lap surface of denture teeth by monomer or by airborne particle abrasion significantly improved their bond strengths to the denture base resins.there was no significant difference between applying monomer or airborne particle abrasion in improvement of the bond strength.the bond strength of the denture teeth to the studied denture base resins was significantly different.the failure mode was predominantly adhesive with futura gen and mixed in meliodent . treatment of the ridge lap surface of denture teeth by monomer or by airborne particle abrasion significantly improved their bond strengths to the denture base resins . there was no significant difference between applying monomer or airborne particle abrasion in improvement of the bond strength . the bond strength of the denture teeth to the studied denture base resins was significantly different .

background : debonding of denture teeth from denture bases is the most common failure in removable dentures . the purpose of this study was to evaluate the effect of surface treatments on shear bond strength of denture teeth to heat - polymerized and autopolymerized denture base resins.materials and methods : in this experimental in vitro study , 60 maxillary central incisor acrylic teeth were divided into two groups . group m was polymerized with heat - polymerized acrylic resin ( meliodent ) by compression molding technique and group f was processed by autopolymerized acrylic resin ( futura gen ) by injection molding technique . within each group , specimens were divided into three subgroups according to the teeth surface treatments ( n = 10 ) : ( 1 ) ground surface as the control group ( m1 and f1 ) , ( 2 ) ground surface combined with monomer application ( m2 and f2 ) , and ( 3 ) airborne particle abrasion by 50 m al2o3 ( m3 and f3 ) . the shear bond strengths of the specimens were tested by universal testing machine with crosshead speed of 5 mm / min . data were analyzed by two - way analysis of variance ( anova ) and tukey 's honestly significant difference ( hsd ) tests ( p < 0.05).results : the mean shear bond strengths of the studied groups were 96.40 14.01 , 124.70 15.64 , and 118 16.38 n for m1 , m2 , and m3 and 87.90 13.48 , 117 13.88 , and 109.70 13.78 n for f1 , f2 , and f3 , respectively . the surface treatment of the denture teeth significantly affected their shear bond strengths to the both the denture base resins ( p < 0.001 ) . however , there were no significant differences between the groups treated by monomer or airborne particle abrasion ( p = 0.29 ) . the highest percentage of failure mode was mixed in meliodent and adhesive in futura gen.conclusion:monomer application and airborne particle abrasion of the ridge lap area of the denture teeth improved their shear bond strengths to the denture base resins regardless of the type of polymerization .

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the light emitted during arc welding contains intense ultraviolet radiation ( uvr ) . in the absence of a barrier , this radiation is emitted into the surrounding environment , and as a result , extremely large number of workers at workplaces where arc welding is performed are exposed to uvr . this includes not only expert arc - welding professionals , whose numbers are estimated at 350,000 in japan , but also welders who perform arc welding occasionally and workers engaged in tasks other than arc welding . however , a precise border between uvr and visible light can not be defined because visual sensations from very bright sources are experienced at wavelengths below 400 nm . therefore , the borders necessarily vary from situation to situation . uvr below approximately 190 nm is known as vacuum uvr because it is strongly absorbed by oxygen molecules and therefore can not propagate through air . uvr is emitted during arc welding over its entire wavelength range excluding vacuum uvr , although the wavelength distribution of uv differs depending on the welding conditions . uvr from the arc welding of steel consists mostly of a large number of spectral lines of iron scattered over this wavelength range . for example , because it is strongly absorbed by proteins and water , uvr incident on a living organism is primarily absorbed at the surface , causing damage confined to surface regions . well - known examples of acute health effects include keratoconjunctivitis and erythema , whereas delayed health effects include cataracts and skin cancer . in practice , such acute health effects because of uvr occur frequently at workplaces where arc welding is performed . the japan welding engineering society surveyed incidences of uv keratoconjunctivitis among 1667 workers at 47 workplaces where arc welding is performed ; the survey included workers who performed arc welding as well as workers who did not . the results indicated that as many as 86% of the workers reported past experience with symptoms of uv keratoconjunctivitis , including foreign body sensation , ophthalmalgia , lacrimation , and photophobia , whereas 45% reported ongoing experience with this ailment with one or more recurrences per month even though the majority of arc welders who experienced uv keratoconjunctivitis wore welding face shields . possible causes for this include ( a ) cases in which workers mistakenly put on their face shields after starting the arc instead of immediately before thus exposing themselves to uvr and ( b ) cases in which workers were exposed to uvr by co - workers performing arc welding nearby . in addition , in a survey by emmett et al . , 92% of welders had suffered one or more flash burns ( keratoconjunctivitis ) , and 40% were afflicted with erythema in the neck . these findings demonstrate the need to introduce protective measures at workplaces that use arc welding to protect all workers from uvr . as the basis for designing such measures , it is desirable to obtain a quantitative understanding of the hazard of uvr emitted during arc welding . in an actual work site , arc welding is performed under a variety of conditions , and the degree of hazard of the emitted uvr is thought to differ for each condition . therefore , it is necessary to examine the hazards of the uvr emitted during arc welding under various conditions . arc welding of metallic materials is conducted primarily on mild steel , aluminum alloys , and stainless steel alloys . gas metal arc welding ( gmaw ) , which uses a continuously fed consumable electrode and a shielding gas , is most often used for mild steel . gmaw is conducted while covering the welding point and the arc by the shielding gas . the shielding gas prevents a decrease in welding quality , such as the formation of an oxide or nitride because of exposure of the weld metal to air . the gmaw electrode is a coiled consumable wire that is automatically supplied throughout the welding . a few previous studies have measured the uvr emitted during arc welding processes , such as the gmaw of mild steel , and assessed their acute health effects . however , in these studies , detailed information on the actual welding was not provided . therefore , the available data about the level of uvr emitted by gmaw is unreliable and insufficient . in particular , the effects of specific conditions have not been examined systematically . one of the authors of this paper performed gmaw using 100% co2 in experiments and examined in detail the uvr emitted under these conditions . it was clearly found that the uvr hazard tends to increase with welding current and is dependent on the type of welding wire . in this study , we conducted a survey of the uvr hazard present during the gmaw of mild steel with a shielding gas of 80% ar + 20% co2 or 100% co2 . the uvr emitted during gmaw performed with the recently popular digital inverter - type pulsed arc welding machine was measured , and its acute health effects were assessed in accordance with the american conference of governmental industrial hygienists ( acgih ) guidelines . in particular , we studied the influence of ( i ) the type of shielding gas and the magnitude of the welding current , and ( ii ) using pulsed vs. non - pulsed welding current . according to the acgih guidelines , the degree of urv hazard as a cause of acute health effects is measured by the effective irradiance . the effective irradiance is defined by where eeff is the effective irradiance ( mw / cm ) , e is the spectral irradiance at wavelength ( mw/ ( cmnm ) ) , s ( ) is the relative spectral effectiveness at wavelength , and is the bandwidth ( nm ) . for the uvr measurements , we used an x13 hazard lightmeter and an xd-45-huv uv - hazard detector head ( both from gigahertz - optik ) , which are designed for measuring effective irradiance . the relative spectral responsivity of the detector head agrees well with the relative spectral effectiveness around 270 nm . some discrepancy between the relative spectral responsivity and the relative spectral effectiveness is visible from 310 to 320 nm . however , because the relative spectral effectiveness in this wavelength regime is small ( 0.015 - 0.0010 ) , we consider the influence of this discrepancy to be too small to cause issues in practice . thus , we conclude that this detector head is well suited to measure the effective irradiance . in actual experiments , the value measured by the devices is the effective radiant exposure ( j / m ) . the measurement device was calibrated by the manufacturer and used within the one - year validity of the calibration . the position of the welding torch was fixed to produce arcs in the same position , and the base metal was fixed on a movable table , which moved the metal for welding . the distance between the arc and the detector head was set to 500 mm to mimic the actual distances to welders . in addition , the detector head was positioned at an angle of 40 from the surface of the base metal and at an angle of 90 from the welding direction . the measurement time was set to 40 s. to exclude the time required for the arc to stabilize immediately after welding begins and the time required for the movable table to accelerate , measurements did not begin until 5 s after the start of welding . in this study , no local exhaust ventilation system was used during the measurement of uvr because local exhaust ventilation is usually not used in welding workplaces ( local exhaust ventilation may disturb the airflow around the arc and cause welding defects ) . experimental setup for measuring effective irradiance ( schematic diagram ) measurements furthermore , following the acgih guidelines , we divided 3 mj / cm by our measured values of effective irradiance to determine the maximum daily exposure time allowable at that irradiance ( equation ( 2 ) ) . in this equation , tmax is the maximum daily exposure time ( s ) and eeff is the effective irradiance . we measured the effective irradiance of the uvr emitted during three types of welding : 100% co2 welding using non - pulsed welding current , non - pulsed 80% ar + 20% co2 welding , and pulsed 80% ar + co2 welding . the welding apparatus was a digital inverter - type pulsed arc welding machine ( dp350 , daihen welding and mechatronics systems co. , ltd . ) , which has recently become popular . the inclination of the welding torch was fixed at 110. the type of welding was bead - on - plate welding in which the base metal is melted while the welding wire is added . the base metal was rolled ss400 , which is a mild structural steel specified by the japanese industrial standards ( jis ) . for the welding wire , we used solid wire ygw12 of diameter 1.2 mm specified by jis . the usable range of welding current for this wire was 80 - 350 a for flat position welding . the shielding gas was 80% ar + 20% co2 or 100% co2 , and the shielding gas flow rate was 15 l / min . the distance between the base metal and the contact tip was 17 mm , and the wire extension length before the start of welding was 12 mm . the welding voltages were preset values corresponding to the welding current determined by the manufacturer of the welding equipment . welding parameters of 100% co2 , non - pulsed 80% ar+20% co2 welding and pulsed 80% ar+20% co2 welding to investigate the influence of the type of shielding gas , we conducted bead - on - plate welding and measured the resulting uvr when using 80% ar + 20% co2 or 100% co2 as the shielding gas and non - pulsed welding current . the range of the welding current was 100 - 350 a as shown in table 1 . because it is less expensive , 100% co2 is used more commonly as shielding gas in the gmaw of mild steel in japan , whereas mixed gas is usually used when manufacturing high - quality products efficiently . to investigate the influence of pulsed current , we performed pulsed current gmaw with an 80% ar + 20% co2 shielding gas and measured the resulting uvr . we then compared it with the results for non - pulsed 80% ar + 20% co2 welding . incidentally , the digital inverter - type pulsed arc welding machine provides excellent stability of the arc in the low current range , highly effective reduction of sputter , and is mainly used under 250 a. therefore , the range of welding current used was 100 - 250 a as shown in table 1 . pulsed current is now commonly used in the welding of thin plates of automobile bodies . to investigate the influence of the type of shielding gas , we conducted bead - on - plate welding and measured the resulting uvr when using 80% ar + 20% co2 or 100% co2 as the shielding gas and non - pulsed welding current . the range of the welding current was 100 - 350 a as shown in table 1 . because it is less expensive , 100% co2 is used more commonly as shielding gas in the gmaw of mild steel in japan , whereas mixed gas is usually used when manufacturing high - quality products efficiently . to investigate the influence of pulsed current , we performed pulsed current gmaw with an 80% ar + 20% co2 shielding gas and measured the resulting uvr . we then compared it with the results for non - pulsed 80% ar + 20% co2 welding . incidentally , the digital inverter - type pulsed arc welding machine provides excellent stability of the arc in the low current range , highly effective reduction of sputter , and is mainly used under 250 a. therefore , the range of welding current used was 100 - 250 a as shown in table 1 . pulsed current is now commonly used in the welding of thin plates of automobile bodies . figure 2 and 3 display the effective irradiance for various welding conditions in bead - on - plate welding . the effective irradiance measured in this study at a distance of 500 mm from the arc was in the range of 0.51 - 12.9 mw / cm . the allowable daily exposure times corresponding to these values are 0.23 - 5.9 s. effective irradiance for 100% co2 welding and non - pulsed 80%ar+20%co2 welding . 2 shows that the effective irradiance in bead - on - plate welding of mild steel was influenced by the welding current and the shielding gas composition . the effective irradiance increased with increasing welding current for each type of shielding gas . for the case of non - pulsed 80% ar + 20% co2 welding , the effective irradiance measured with welding currents of 100 - 350 a was in the range of 0.51 - 12.9 mw / cm . the increase in effective irradiance between current intervals is very large , starting from 250 a. for 100% co2 welding , the effective irradiance measured with welding currents of 100 - 350 a was in the range of 0.69 - 7.4 mw / cm . the effective irradiances measured with non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less were approximately equal . however , at 300 a and higher , the effective irradiance for non - pulsed 80% ar + 20% co2 welding increased beyond that for 100% co2 welding . 3 , the effective irradiance of uvr emitted during mild steel arc welding was strongly influenced by the pulsed current . the effective irradiance measured for pulsed 80% ar + 20% co2 welding with welding currents of 100 - 250 a was in the range of 3.4 - 11.6 mw / cm . in addition , the effective irradiance of uvr occurring during pulsed 80% ar + 20% co2 welding was very high in comparison with that during non - pulsed 80% ar + 20% co2 welding . the effective irradiance observed at a distance of 500 mm from the arc was in the range of 0.51 - 12.9 mw / cm . at these irradiances , the allowable daily exposure times are just 0.23 - 5.9 s , which are extremely short times . these results indicate that exposure to the uvr emitted by the gmaw of mild steel is quite hazardous . it is thought that workers are often exposed to uvr when the arc is started . although the exposure is brief for each start of an arc , this may occur often because workers usually start an arc many times in a day . therefore , the actual total exposure time may easily exceed the allowable daily exposure times determined in this study . thus , we conclude that if workers engage in the gmaw of mild steel without adequate protection , they are exposed to hazardous quantities of uvr for short periods of time . one of the authors previously measured the uvr emitted during the gmaw of mild steel when using 100% co2 as a shielding gas and obtained an effective irradiance of 0.028 - 0.785 mw / cm at a distance of 1 m for welding currents of 120 - 500 a. if we assume that the effective irradiance decreases as the inverse square distance from the arc , the effective irradiance would be 0.106 - 3.14 mw / cm at 0.5 m from the arc , which is roughly half the effective irradiance obtained in the present study for the same type of welding . this difference is considered to be because of differences in welding conditions such as the welding device , welding wire , and ventilation conditions . in this study , no local exhaust ventilation system was used during the measurement of uvr because local exhaust ventilation is usually not used in the welding workplace . local exhaust ventilation removes the welding fume , which strongly mitigates uvr by scatter and absorption . therefore , if local exhaust ventilation had been used during the measurement , the effective irradiance would have been higher . if we assume that the effective irradiance of uvr decreases as the inverse square distance from the arc , the allowable daily exposure times at a distance of 5 m from the arc will be in the range of 23 - 590 s. thus , even at a distance of 5 m from the arc , exposure to uvr is hazardous in cases in which the emitted uvr is intense . moreover , even in cases in which the emitted uvr is weak , we believe that prolonged exposure is hazardous . thus , in cases where the gmaw of mild steel is performed , it is necessary to take precautions to ensure that surrounding workers are not exposed to the uvr emitted by the arc . the effective irradiance measured in this study , regardless of the pulse current and the type of shielding gas , increased with increasing welding current ( figure 2 , 3 ) . this trend was also previously observed for the gmaw of mild steel using 100% co2 and 5% o2 + 95% ar as the shield gas . more recently , we also observed the trend for the gmaw and the gtaw of aluminum and magnesium alloys . thus , the welding current is an important factor influencing the degree of hazard of the uvr emitted during the welding process ; the uvr hazard can be understood to be a rapidly increasing function of the welding current . the effective irradiance of the uvr generated during non - pulsed 80% ar + 20% co2 welding was in the range of 0.51 - 12.9 mw / cm and increased with increasing welding current . in particular , a significant difference in the increase in effective irradiance for welding currents from 100 to 250 a and from 250 a upward was observed . to investigate the reason for this difference , we observed the arc during welding . 4 , short - circuit transfer was observed at 100 a. in short - circuit transfer , the tip of the welding wire is short - circuited with the base metal ( molten pool ) , and the molten metal is shifted to the base material . in addition , globular transfer was observed at 250 a. in globular transfer , the welding wire tip melted by the arc is transferred to the base metal in which the grain size grows larger than the wire diameter . as also shown in fig . 4 , it was not possible at 300 a to observe the droplets , which were blocked by the arc light . however , the wire tip was pointed , and because no change appeared in the shape of the wire tip with the passage of time , we assumed this was spray transfer . in spray transfer , the welding wire tip melted by the arc is transferred to the base material with a grain smaller than the wire diameter . these results were consistent with the research of takeuchi et al .. the effective irradiance of the uvr emitted by non - pulsed 80% ar + 20% co2 welding increased with increasing welding current from 100 to 250 a , and no change was seen in this increasing trend ( fig . , the effect of changing the metal transfer mode from short - circuiting to globular on the effective irradiance was considered to be small . however , the effective irradiance increased rapidly between 250 a and 300 a. the metal transfer mode changed to spray transfer from globular transfer . the amount of metal vapor in the arc during spray transfer is large compared to that during globular transfer , and the properties of light emitted by the arc welding are affected by the amount of metal vapor blending into the arc . therefore , the cause of the rapid increase in the effective irradiance from 250 to 300 a is considered to be because of the change in the metal transfer mode ( globular transfer to spray transfer ) . the metal transfer for non - pulsed 80% ar+20% co2 welding the effective irradiance of the uvr emitted during 100% co2 welding was in the range of 0.69 - 7.4 mw / cm and increased with increasing welding current . takeuchi et al . reported that for 100% co2 welding , the transition from short - circuit transfer to globular transfer takes place at approximately 250 a. however , in the present study , no clear difference was seen in the effective irradiance vs. current trend between short - circuit transfer and globular transfer . the effective irradiance was approximately the same for non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less . however , a significant difference was observed at 300 a or higher . by observation of the arc during welding , the metal transfer mode of non - pulsed 80% ar + 20% co2 welding was spray transfer and that of 100% co2 welding was globular transfer . therefore , the effective irradiance is considered to depend on the metal transfer mode because of the differences in the shielding gas composition between them , and the effective irradiance increases with the transition to spray transfer . as shown above , the uvr emitted during the gmaw of mild steel was very hazardous during spray transfer . the welding operator must recognize that welding under spray transfer conditions is extremely dangerous , so it is necessary to take adequate protective measures . 3 , the effective irradiance of the uvr emitted during pulsed 80% ar + 20% co2 welding increased with increasing current and was 3.0 - 6.7 times larger compared with that emitted during non - pulsed 80% ar + 20% co2 welding at each welding current . the effective irradiance was observed in the vicinity of the arc and the metal transfer mode was spray transfer for the entire current range . 5 . the metal transfer for pulsed 80% ar+20% co2 welding ( spray transfer ) . ( the effective irradiance is 3.4 mw / cm . ) for non - pulsed 80% ar + 20% co2 welding , the short - circuit or globular transfer modes were observed at 250 a or less ( fig . the amount of metal vapor in the arc during spray transfer is large compared to that during short - circuit and globular transfer . in addition , the properties of the light emitted during the arc welding are affected by the amount of metal vapor blended into the arc . therefore , we believe that the effective irradiance of the uvr generated by spray transfer for a pulsed current is very large compared with that produced by the short - circuit and the globular transfer modes for a non - pulsed welding current . these results confirm that the effective irradiance is strongly influenced by the pulsed current , and the degree of the effect increases with decreasing pulsed current . in recent years , the demand for mild steel thin plate welding has increased to reduce the weight of equipment that needs to be transported . we used a low welding current below 100 a in the welding of thin plate . in addition , a digital inverter - type pulsed arc welding machine was used to increase the working efficiency . in this study , the effective irradiance of the uvr that occurred during pulsed 80% ar + 20% co2 welding at 100 a was 1.2 times greater than the effective irradiance during non - pulsed 80% ar + 20% co2 welding at 250 a. therefore , welding operators need to recognize the very high hazard of the uvr when they are welding with pulsed current , and supervisors must take adequate protection measures for the peripheral workers who are not welding . the effective irradiance measured in this study , regardless of the pulse current and the type of shielding gas , increased with increasing welding current ( figure 2 , 3 ) . this trend was also previously observed for the gmaw of mild steel using 100% co2 and 5% o2 + 95% ar as the shield gas . more recently , we also observed the trend for the gmaw and the gtaw of aluminum and magnesium alloys . thus , the welding current is an important factor influencing the degree of hazard of the uvr emitted during the welding process ; the uvr hazard can be understood to be a rapidly increasing function of the welding current . the effective irradiance of the uvr generated during non - pulsed 80% ar + 20% co2 welding was in the range of 0.51 - 12.9 mw / cm and increased with increasing welding current . in particular , a significant difference in the increase in effective irradiance for welding currents from 100 to 250 a and from 250 a upward was observed . to investigate the reason for this difference , we observed the arc during welding . 4 , short - circuit transfer was observed at 100 a. in short - circuit transfer , the tip of the welding wire is short - circuited with the base metal ( molten pool ) , and the molten metal is shifted to the base material . in addition , globular transfer was observed at 250 a. in globular transfer , the welding wire tip melted by the arc is transferred to the base metal in which the grain size grows larger than the wire diameter . as also shown in fig . 4 , it was not possible at 300 a to observe the droplets , which were blocked by the arc light . however , the wire tip was pointed , and because no change appeared in the shape of the wire tip with the passage of time , we assumed this was spray transfer . in spray transfer , the welding wire tip melted by the arc is transferred to the base material with a grain smaller than the wire diameter . these results were consistent with the research of takeuchi et al .. the effective irradiance of the uvr emitted by non - pulsed 80% ar + 20% co2 welding increased with increasing welding current from 100 to 250 a , and no change was seen in this increasing trend ( fig . , the effect of changing the metal transfer mode from short - circuiting to globular on the effective irradiance was considered to be small . however , the effective irradiance increased rapidly between 250 a and 300 a. the metal transfer mode changed to spray transfer from globular transfer . the amount of metal vapor in the arc during spray transfer is large compared to that during globular transfer , and the properties of light emitted by the arc welding are affected by the amount of metal vapor blending into the arc . therefore , the cause of the rapid increase in the effective irradiance from 250 to 300 a is considered to be because of the change in the metal transfer mode ( globular transfer to spray transfer ) . the metal transfer for non - pulsed 80% ar+20% co2 welding the effective irradiance of the uvr emitted during 100% co2 welding was in the range of 0.69 - 7.4 mw / cm and increased with increasing welding current . takeuchi et al . reported that for 100% co2 welding , the transition from short - circuit transfer to globular transfer takes place at approximately 250 a. however , in the present study , no clear difference was seen in the effective irradiance vs. current trend between short - circuit transfer and globular transfer . the effective irradiance was approximately the same for non - pulsed 80% ar + 20% co2 welding and 100% co2 welding at 250 a or less . however , a significant difference was observed at 300 a or higher . by observation of the arc during welding , the metal transfer mode of non - pulsed 80% ar + 20% co2 welding was spray transfer and that of 100% co2 welding was globular transfer . therefore , the effective irradiance is considered to depend on the metal transfer mode because of the differences in the shielding gas composition between them , and the effective irradiance increases with the transition to spray transfer . as shown above , the uvr emitted during the gmaw of mild steel was very hazardous during spray transfer . the welding operator must recognize that welding under spray transfer conditions is extremely dangerous , so it is necessary to take adequate protective measures . 3 , the effective irradiance of the uvr emitted during pulsed 80% ar + 20% co2 welding increased with increasing current and was 3.0 - 6.7 times larger compared with that emitted during non - pulsed 80% ar + 20% co2 welding at each welding current . the effective irradiance was observed in the vicinity of the arc and the metal transfer mode was spray transfer for the entire current range . 5 . the metal transfer for pulsed 80% ar+20% co2 welding ( spray transfer ) . ( the effective irradiance is 3.4 mw / cm . ) for non - pulsed 80% ar + 20% co2 welding , the short - circuit or globular transfer modes were observed at 250 a or less ( fig . the amount of metal vapor in the arc during spray transfer is large compared to that during short - circuit and globular transfer . in addition , the properties of the light emitted during the arc welding are affected by the amount of metal vapor blended into the arc . therefore , we believe that the effective irradiance of the uvr generated by spray transfer for a pulsed current is very large compared with that produced by the short - circuit and the globular transfer modes for a non - pulsed welding current . these results confirm that the effective irradiance is strongly influenced by the pulsed current , and the degree of the effect increases with decreasing pulsed current . in recent years , the demand for mild steel thin plate welding has increased to reduce the weight of equipment that needs to be transported . we used a low welding current below 100 a in the welding of thin plate . in addition , a digital inverter - type pulsed arc welding machine was used to increase the working efficiency . in this study , the effective irradiance of the uvr that occurred during pulsed 80% ar + 20% co2 welding at 100 a was 1.2 times greater than the effective irradiance during non - pulsed 80% ar + 20% co2 welding at 250 a. therefore , welding operators need to recognize the very high hazard of the uvr when they are welding with pulsed current , and supervisors must take adequate protection measures for the peripheral workers who are not welding . ( 1 ) it is more hazardous at higher welding currents than at lower welding currents . ( 2 ) at higher welding currents , it is more hazardous when 80% ar + 20% co2 is used as a shielding gas than when 100% co2 is used . ( 3 ) it is more hazardous for pulsed welding currents than for non - pulsed welding currents . ( 4 ) it appears to depend on the metal transfer ; the hazard of the uvr emitted during spray transfer is the highest .

objectives : ultraviolet radiation ( uvr ) emitted during arc welding frequently causes keratoconjunctivitis and erythema in the workplace . the degree of hazard from uvr exposure depends on the welding method and conditions . therefore , it is important to identify the uvr levels present under various conditions . methods : we experimentally evaluated the uvr levels emitted in gas metal arc welding ( gmaw ) of mild steel . we used both a pulsed welding current and a non - pulsed welding current . the shielding gases were 80% ar + 20% co2 and 100% co2 . the effective irradiance defined in the american conference of governmental industrial hygienists guidelines was used to quantify the uvr hazard . results : the effective irradiance measured in this study was in the range of 0.51 - 12.9 mw / cm2 at a distance of 500 mm from the arc . the maximum allowable exposure times at these levels are only 0.23 - 5.9 s / day . conclusions : the following conclusions were made regarding the degree of hazard from uvr exposure during the gmaw of mild steel : ( 1 ) it is more hazardous at higher welding currents than at lower welding currents . ( 2 ) at higher welding currents , it is more hazardous when 80% ar + 20% co2 is used as a shielding gas than when 100% co2 is used . ( 3 ) it is more hazardous for pulsed welding currents than for non - pulsed welding currents . ( 4 ) it appears to be very hazardous when metal transfer is the spray type . this study demonstrates that unprotected exposure to uvr emitted by the gmaw of mild steel is quite hazardous .

pubmed

a wide variety of taxonomically different groups of microorganisms are known to produce intracellular energy and carbon storage compounds , generally described as polyhydroxyalkanoates ( phas ) . it has been shown that bacterial cells with a higher content of phas can survive longer than those with a lower phas content , because they can utilize their reserve material longer and more efficiently . therefore , it was stated that the accumulation of phas might increase the survival capabilities of these bacteria in extreme environments or when nutrient availability is poor . phas are synthesized by many bacteria and archaea when in environment carbon is available , but other essential nutrients are not . phas are commonly divided into two groups based on the number of constituent carbon atoms in their monomer units : short - chain - length ( scl- ) phas and medium - chain - length ( mcl - phas ) . the former consists of monomers with 35 carbon atoms and the latter consists of monomers with 614 carbon atoms . p(3hb ) is highly crystalline , whereas mcl - phas are elastic with low melting temperature . these variations in the pha polymer family create the potential for a vast array of applications . the surface of pha granules is coated with phospholipids and proteins which play a major role in pha synthesis , degradation , and even in the process of pha synthesis regulation . the type of pha produced and accumulated in the granules depends on the metabolic pathway of the particular microorganism . however , independently of the type of synthesized phas , the enzyme called pha synthase always plays the main role . pha synthases were classified into four classes , according to their substrate specificities and subunits organization . classes i , iii , and iv prefer to synthesize scl - pha ( phac gene ) , whereas the ii class is responsible for mcl - pha synthesis . class i of pha synthase comprises enzyme consisting of single subunit , whereas classes iii and iv comprise enzymes that consist of two different types of subunits . the ii class of pha synthase is also built by single subunit but it is coded by two forms of gene that are called phac1 and phac2 . the nucleotide composition of phac1 and phac2 genes is very similar but their substrate specificity may differ . extremophiles inhabiting polar areas are a source of a novel enzymes , which have a great economic potential in many industrial processes , including agricultural , chemical , and pharmaceutical applications . especially , shallow antarctic reservoirs influenced by the forces of variable trophic conditions are underexploited sources of microorganisms with biotechnological potential . as a result of selection pressures in these reservoirs , there is a potential for the discovery of novel biochemical pathways leading to phas synthesis . psychrotrophic bacteria , which are adapted to variable temperatures and to other fluctuating conditions such as low nutrient availability and low water quality , may offer important advantages for industrial phas production . it is widely accepted that microorganisms from unusual environments provide not only valuable resources for exploiting novel biotechnological processes , but also serve as models for investigating how these biomolecules are stabilized when subjected to changing conditions . the main purpose of this study was to determine whether bacteria inhabiting freshwater reservoirs in glacier forefield areas have the potential to synthesize phas . samples of water and mats were collected in summer of 2011 year from five , shallow freshwater reservoirs and one stream . they were located in the foreland of ecology glacier near to the polish antarctic station arctowski 1map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . ( part of admiralty bay map modified from pudeko map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . ( part of admiralty bay map modified from pudeko selected reservoirs were different in age , location , and environmental parameters ( table 1 ) . located near to the front of ecology glacier , pond 7 was two years old . this reservoir was located on the ground moraine and was directly supplied with water from the glacier , with no signs of vegetation . pond 8 was located away from the front of the glacier , with abundant bloom of microalgae . reservoirs 10 and 11 , which have been noticed there since 1977 , were situated on the top of lateral moraine . both were situated close to each other and connected with small stream ( fig . 1 ) . samples were taken with a sampler on a bracket into sterile bottles and delivered to the laboratory within 1 hour . microbial mat samples were collected as cores ( 1 5 cm ) , and one gram of sample was aseptically weighed and homogenized in 9 ml of sterile cold.(4 c ) physiological saline ( 0.86 % nacl ) using a vortex . cultures were obtained by plating 100 l of a sample onto antarctic bacteriological medium ( abm ; ph 7.4 ) containing : peptone ( 0.5 % , w / v ) , yeast extract ( 0.2 % , w / v ) , and agar ( 2 % , w / v ) in three replicates . the well -separated colonies on medium were macroscopically described , photographed , than isolated under binocular , and transferred to slants of abm in the tubes . after transportation to poland the microscopic purity evaluation of gram - stained strains was done.table 1characteristics of sampling sitessampling sitevariables6781011gps position624826,4 s 0582853,9 w624825,2 s 0582850,1 w621002,3 s 0582740,9 w620923,2 s 0582820,2 w620937,0 s 0582823,7 welevation . ( m a.s.l.)879227225area ( m)266.91122.30117.60123.80nh4-n ( mg l)0.024 ( 0.004)0.035 ( 0.011)0.426 ( 0.071)0.161 ( 0.018)0.033 ( 0.002)po4-p ( mg l)0.148 ( 0.003)0.130 ( 0.00)0.120 ( 0.042)0.079 ( 0.02)0.154 ( 0.044)p - tot ( mg l)0.03 ( 0.00)0.035 ( 0.007)0.050 ( 0.028)0.020 ( 0.00)0.070 ( 0.00)n - tot ( mg l)0.650 ( 0.071)1.950 ( 1.768)4.100 ( 0.00)1.400 ( 0.00)1.700 ( 0.00)ph ( mg l)7.6 ( 0.5)7.7 ( 0.6)8.4 ( 0.7)6.5 ( 1.2)7.1 ( 0.8)temp ( c)2.8 ( 1.8)2.9 ( 1.6)5.0 ( 0.2)6.1 ( 2.1)5.8 ( 0.6)o2 ( mg l)11.85 ( 0.35)10.40 ( 0.42)10.86 ( 1.22)11.08 ( 0.60)10.92 ( 0.23)conductivity ( us / cm)79.4 ( 26.86)84.7 ( 41.37)166.1 ( 76.23)135.0 ( 32.85)160.3 ( 19.38)chlorophyll a ( g l)0.4000.4171.6660.9000.900pheophytin ( g l)2.8002.7912.1253.8001.800total chlorophyll ( g l)3.2003.2083.7914.7002.700doc ( mg l)0.5670.6531.1350.7870.826 characteristics of sampling sites in order to obtain dna , cells were scraped from the surface of slants and washed in sterile water . dna extraction was performed using commercial kit genomic mini ( a&a biotechnology ) according to the manufacturers instructions . the purified dna was suspended in 50 l of deionized , dnase free water and stored in 20 c . the partial 16s rrna gene was amplified using the primers 341 : 5-cctacgggaggcagcag -3 and 16sr : 5-taccttgttacgacttcacccca-3 described previously by rossau et al . . in order to detect microorganism producing phas , pcr was performed with two primer pairs . first one , recognizing both scl- and mcl - pha synthase genes was elaborated by romo et al . ( g - d : 5-gtgccgcc(gc)(ct ) ( ag)(gc)atcaacaagt-3 ; g1-r : 5 gttccag(at)acag(gc)a(gt)(ag ) tcg aa-3 ) . the second primers pair ( i-179l : 5-acagatcaacaagttctacatctt cgac-3 ; i-179r : 5-ggtgttgtcgttgttccagtagaggatgtc-3 ) was specific for both genes responsible for mcl - pha synthesis . the mixtures used for pcr amplification contained 50 ng of extracted dna , 0.5 m of each primer , 100 m of deoxynucleoside triphosphate ( promega , winsconsin , usa ) , 1 u of taq dna polymerase ( polgen , poland ) , and 5 l of reaction buffer ( 500 mm kcl , ph 8.5 ; triton x-100 ) . the temperature program for 16s rrna gene amplification was as follows : 94 c for 5 min ; 30 cycles of denaturation at 94 c for 30 s , annealing at 54 c for 30 s , extension at 72 c for 1 min , and single final elongation at 72 c for 5 min . the pcr amplicons of 16s rdna and pha synthase genes were resolved on 1.5 % agarose gels stained with ethidium bromide , and size of pcr products was estimated using molecular weight marker ( 100 bp , promega , winsconsin , usa ) . the sequencing of 16s rdna and scl - pha synthase ( phac ) pcr products was performed using the same pcr primers as for amplification . because pcr primers pair i-179l and i-179r amplifies both genes coding for mcl - pha synthase , we use primers orf2 ( 5-catgacagcggcctgttcacctgg-3 ) and i-179r using the same conditions as described in the work of ciesielski et al . . using this approach , it was possible to amplify and directly sequence a fragment of phac gene about 720 bp long . dna sequencing was performed using a perkin elmer abi 373 automated dna sequencer ( pe applied biosystems , foster city , ca , usa ) at the institute of biochemistry and biophysics in warsaw , poland . the nucleotide sequences were submitted to the genbank database under accession numbers from kf301568 to kf301599 . the sequences of genes coding for 16s rrna and pha synthases were compared with those from the genbank database using the ncbi blast program . the evolutionary distances were inferred using the neighbor - joining method with the mega5 program . to determine the degree of statistical support for branches in the phylogeny , 1,000 bootstrap replicates of data were analyzed . the gene sequences that were > 94.0 % identical to sequences of cultured species in the ncbi database were assigned genus names . a canonical correspondence analysis ( cca ) the presence of bacteria possessing specific form of pha synthase genes was analyzed in relation to the environmental background samples of water and mats were collected in summer of 2011 year from five , shallow freshwater reservoirs and one stream . they were located in the foreland of ecology glacier near to the polish antarctic station arctowski 1map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . ( part of admiralty bay map modified from pudeko map showing sampling location in the foreland of ecology glacier , king georg island , antarctica . ( part of admiralty bay map modified from pudeko selected reservoirs were different in age , location , and environmental parameters ( table 1 ) . located near to the front of ecology glacier , pond 7 was two years old . this reservoir was located on the ground moraine and was directly supplied with water from the glacier , with no signs of vegetation . pond 8 was located away from the front of the glacier , with abundant bloom of microalgae . reservoirs 10 and 11 , which have been noticed there since 1977 , were situated on the top of lateral moraine . both were situated close to each other and connected with small stream ( fig . 1 ) . samples were taken with a sampler on a bracket into sterile bottles and delivered to the laboratory within 1 hour . microbial mat samples were collected as cores ( 1 5 cm ) , and one gram of sample was aseptically weighed and homogenized in 9 ml of sterile cold.(4 c ) physiological saline ( 0.86 % nacl ) using a vortex . cultures were obtained by plating 100 l of a sample onto antarctic bacteriological medium ( abm ; ph 7.4 ) containing : peptone ( 0.5 % , w / v ) , yeast extract ( 0.2 % , w / v ) , and agar ( 2 % , w / v ) in three replicates . the well -separated colonies on medium were macroscopically described , photographed , than isolated under binocular , and transferred to slants of abm in the tubes . after transportation to poland the microscopic purity evaluation of gram - stained strains was done.table 1characteristics of sampling sitessampling sitevariables6781011gps position624826,4 s 0582853,9 w624825,2 s 0582850,1 w621002,3 s 0582740,9 w620923,2 s 0582820,2 w620937,0 s 0582823,7 welevation . ( m a.s.l.)879227225area ( m)266.91122.30117.60123.80nh4-n ( mg l)0.024 ( 0.004)0.035 ( 0.011)0.426 ( 0.071)0.161 ( 0.018)0.033 ( 0.002)po4-p ( mg l)0.148 ( 0.003)0.130 ( 0.00)0.120 ( 0.042)0.079 ( 0.02)0.154 ( 0.044)p - tot ( mg l)0.03 ( 0.00)0.035 ( 0.007)0.050 ( 0.028)0.020 ( 0.00)0.070 ( 0.00)n - tot ( mg l)0.650 ( 0.071)1.950 ( 1.768)4.100 ( 0.00)1.400 ( 0.00)1.700 ( 0.00)ph ( mg l)7.6 ( 0.5)7.7 ( 0.6)8.4 ( 0.7)6.5 ( 1.2)7.1 ( 0.8)temp ( c)2.8 ( 1.8)2.9 ( 1.6)5.0 ( 0.2)6.1 ( 2.1)5.8 ( 0.6)o2 ( mg l)11.85 ( 0.35)10.40 ( 0.42)10.86 ( 1.22)11.08 ( 0.60)10.92 ( 0.23)conductivity ( us / cm)79.4 ( 26.86)84.7 ( 41.37)166.1 ( 76.23)135.0 ( 32.85)160.3 ( 19.38)chlorophyll a ( g l)0.4000.4171.6660.9000.900pheophytin ( g l)2.8002.7912.1253.8001.800total chlorophyll ( g l)3.2003.2083.7914.7002.700doc ( mg l)0.5670.6531.1350.7870.826 characteristics of sampling sites in order to obtain dna , cells were scraped from the surface of slants and washed in sterile water . dna extraction was performed using commercial kit genomic mini ( a&a biotechnology ) according to the manufacturers instructions . the purified dna was suspended in 50 l of deionized , dnase free water and stored in 20 c . the partial 16s rrna gene was amplified using the primers 341 : 5-cctacgggaggcagcag -3 and 16sr : 5-taccttgttacgacttcacccca-3 described previously by rossau et al . . in order to detect microorganism producing phas , pcr was performed with two primer pairs . first one , recognizing both scl- and mcl - pha synthase genes was elaborated by romo et al . ( g - d : 5-gtgccgcc(gc)(ct ) ( ag)(gc)atcaacaagt-3 ; g1-r : 5 gttccag(at)acag(gc)a(gt)(ag ) tcg aa-3 ) . the second primers pair ( i-179l : 5-acagatcaacaagttctacatctt cgac-3 ; i-179r : 5-ggtgttgtcgttgttccagtagaggatgtc-3 ) was specific for both genes responsible for mcl - pha synthesis . the mixtures used for pcr amplification contained 50 ng of extracted dna , 0.5 m of each primer , 100 m of deoxynucleoside triphosphate ( promega , winsconsin , usa ) , 1 u of taq dna polymerase ( polgen , poland ) , and 5 l of reaction buffer ( 500 mm kcl , ph 8.5 ; triton x-100 ) . the temperature program for 16s rrna gene amplification was as follows : 94 c for 5 min ; 30 cycles of denaturation at 94 c for 30 s , annealing at 54 c for 30 s , extension at 72 c for 1 min , and single final elongation at 72 c for 5 min . the pcr amplicons of 16s rdna and pha synthase genes were resolved on 1.5 % agarose gels stained with ethidium bromide , and size of pcr products was estimated using molecular weight marker ( 100 bp , promega , winsconsin , usa ) . the sequencing of 16s rdna and scl - pha synthase ( phac ) pcr products was performed using the same pcr primers as for amplification . because pcr primers pair i-179l and i-179r amplifies both genes coding for mcl - pha synthase , we use primers orf2 ( 5-catgacagcggcctgttcacctgg-3 ) and i-179r using the same conditions as described in the work of ciesielski et al . . using this approach , it was possible to amplify and directly sequence a fragment of phac gene about 720 bp long . dna sequencing was performed using a perkin elmer abi 373 automated dna sequencer ( pe applied biosystems , foster city , ca , usa ) at the institute of biochemistry and biophysics in warsaw , poland . the nucleotide sequences were submitted to the genbank database under accession numbers from kf301568 to kf301599 . the sequences of genes coding for 16s rrna and pha synthases were compared with those from the genbank database using the ncbi blast program . the evolutionary distances were inferred using the neighbor - joining method with the mega5 program . to determine the degree of statistical support for branches in the phylogeny , 1,000 bootstrap replicates of data were analyzed . the gene sequences that were > 94.0 % identical to sequences of cultured species in the ncbi database were assigned genus names . a canonical correspondence analysis ( cca ) was conducted using a statistical package for windows v. canoco 4.5 . the presence of bacteria possessing specific form of pha synthase genes was analyzed in relation to the environmental background a total of 50 bacterial isolates from five ponds and one stream were characterized by sequencing of 1000 bp long fragment of 16s rrna gene . twenty , unique dna sequences were phylogenetically analyzed altogether with their closest relatives derived from genbank ( fig . 2 ) . isolated strains belonged mainly to the phylum of proteobacteria and represent classes betaproteobacteria ( 11 isolates ) , gammaproteobacteria ( 7 isolates ) , alphaproteobacteria ( 2 isolates ) and only two isolates belonged to bacteroidetes phylum ( fig . 2 ) . 2phylogenetic tree based on 16s rrna sequences , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . accession numbers of dna sequences are given in parentheses phylogenetic tree based on 16s rrna sequences , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . accession numbers of dna sequences are given in parentheses the most frequently recovered group of isolates was members of the pseudomonas genus ( 7 isolates ) . the second numerous group of isolates belonged to janthinobacterium genus ( 5 isolates ) . in the aim of detection of a microorganism possessing the potential ability of phas synthesis , the first one ( g - d and g-1r ) , is able to recognize both scl- and mcl - pha synthase genes . the second primers pair ( i-179l and i-179r ) was specific only for mcl - pha synthase genes . the amplification using primers g - d and g-1r gave a positive signal in 12 of the isolates corresponding to six pseudomonads strains , four janthinobacterium strains , and two undefined betaproteobacteria ( p21 and p25 ) . the amplification with pcr primers designed by solaiman and co - workers showed that six pseudomonas strains ( p1 , p4 , p19 , p27 , p32 , and p45 ) and two janthinobacterium strains ( p10 and p24 ) possessed genes responsible for mcl - pha synthesis . pcr products obtained by employing primer pairs g - d : g-1r and orf2 : i-179r were directionally sequenced . the results of dna sequencing of pcr products obtained using g - d and g-1r primers proved that studied dna of two janthinabacterium strains ( p38 and p39 ) , and two undefined betaproteobacteria strains ( p21 and p25 ) is responsible for scl - pha synthesis . nucleotide composition of pcr products amplified with orf2 : i-179r primers pair has provided evidence that all are typical for phac1 gene coding for synthase responsible for mcl - pha synthesis . for deeper analysis , all obtained dna sequences of phac and phac1 genes , after division into two group , were phylogenetically examined altogether with their closest relatives found in genbank . evolutionary tree presenting genetic distance between phac gene dna sequences is shown in the fig . very high similarity ( genetic distance 0.236 ) was revealed between janthinobacterium sp . p39 and antarctic proteobacterium p21 , inhabiting the same pond 7 . very close to them dna sequence of janthinobacterium sp . p38 was placed , and all three sequences obtained in this study were accompanied by dna sequence of zooglea ramigera . the fourth sequence , belonging to antarctic proteobacterium p25 was grouped with rhodoferax ferrireducens ( genetic distance 0.237 ) ( fig . the second group , collecting dna sequences of phac1 gene ( mcl - pha ) , was predominated by members of pseudomonas species . the highest similarity ( genetic distance 0.003 ) , was observed between pseudomonas sp . p19 ( genetic distance 0.009 ) and was collected from different , although neighboring ponds . surprisingly , these two sequences were grouped together with janthinobacterium sp . p24 sequence that came also from mat of pond 10 . 3phylogenetic tree based on phac gene coding for scl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . 4phylogenetic tree based on phac1 gene coding for mcl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . accession numbers of dna sequences are given in parenthesestable 2molecular identification of isolated strains , their nearest neighbors based on 16s rrna and phac / phac1 genes and their isolation locationisolateputative phaclosest relatives based on 16s rdna sequence ( accession no / identity % ) closest cultured based on phac / phac1 gene sequence ( accession no / identity % ) isolation source pseudomonas sp . p1mcl pseudomonas extremaustralis sy11 ( kc790323/99 ) pseudomonas fluorescens pf0 - 1 ( cp000094/91)mat ( pond 10 ) pseudomonas sp . p4mcl pseudomonas frederiksbergensis ( jf343187/100 ) pseudomonas fluorescens pf0 - 1 ( cp000094/90)mat ( pond 10 ) pseudomonas sp . ( fj472656/91)water ( pond 11 ) pseudomonas sp . p27mcl pseudomonas antarctica ( he586386/99 ) pseudomonas fluorescens sbw25 ( am181176/95)water ( pond 8) pseudomonas sp . p32mcl pseudomonas putida ( he586397/99 ) pseudomonas sp . p45mcl pseudomonas fragi ( ab685634/100 ) pseudomonas stutzeri 1317 ( ay278219/84)stream 6 janthinobacterium sp . p10mcl janthinobacterium lividum ( hq824865/100 ) pseudomonas extremaustralis ( fn435843/90)water ( pond 10 ) janthinobacterium sp . p24mcl janthinobacterium lividum ( hq824864/100 ) pseudomonas fluorescens bm07 ( fj472656/92)mat ( pond 10)antarctic betaproteobacterium p21scl burkholderia sp . era35(jq977167/99 ) zooglea ramigera ( u66242/83)water ( pond 7)antarctic betaproteobacterium p25scl rhodoferax ferrireducens ( hg003356/99 ) comamonas testosteroni cnb-2 ( cp001220/81)water ( pond 10 ) janthinobacterium sp . smn 33.6 ( jx624164/99 ) zooglea ramigera ( u66242/83)water ( pond 10 ) janthinobacterium sp . tmt4 - 26 - 6 ( jx949991/99 ) zooglea ramigera ( u66242/83)water ( pond 7 ) pseudomonas sp . p5- pseudomonas fragii ( ab685683/99)-water ( pond 8) iodobacter sp p11- iodobacter fluviatilis ( kc213858/100)-water ( pond 7 ) flavobacterium sp . p14- flavobacterium algicola ( ab455265/99)-water ( pond 10 ) yersinia sp . p16- yersinia intermedia ( nr_027545/99)-water ( pond 7 ) shewanella sp . sphingomonadaceae bacterium n ( dq497241/99)-water ( pond 7)antarctic betaproteobacterium p36- rhodoferax ferrireducens ( nr_074760/99)-water ( pond 8) * only cultured strains were considered during blast searching phylogenetic tree based on phac gene coding for scl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . accession numbers of dna sequences are given in parentheses phylogenetic tree based on phac1 gene coding for mcl - pha synthase , generated by the neighbor - joining method showing the genetic relationships among bacteria analyzed in this work . type strains are indicated with superscript t. the numbers on the branches refer to bootstrap values for 1,000 times . accession numbers of dna sequences are given in parentheses molecular identification of isolated strains , their nearest neighbors based on 16s rrna and phac / phac1 genes and their isolation location * only cultured strains were considered during blast searching the statistical analysis of principal components ( cca ) showed correlation between the presence of bacteria possessing particular pha synthase gene and environmental conditions . bacteria having genetic potential to synthesize scl - pha inhabited reservoirs with rather low trophy characterized by low levels of total phosphorus and high amounts of pheophytins ( fig . 5).fig . 5canonical correspondence analysis showing relation between the presence of bacteria possessing specific form of pha synthase genes and environmental data of sampling sites canonical correspondence analysis showing relation between the presence of bacteria possessing specific form of pha synthase genes and environmental data of sampling sites in the ponds that were studied , more than half of the isolated strains possessed genes responsible for phas synthesis . among them were mainly microbes belonging to the pseudomonas genus ( six strains ) and janthinobacterium genus ( four strains ) . they isolated from antarctic soil strains of bacteria having the ability to produce phas , among them twelve were belonging to pseudomonas spp . and three to janthinobacterium genus . showed that strains of this genus isolated from antarctica could be divided into two groups ; the first is jantinobacterium lividum group , whereas the second was called our analysis also indicates that isolated janthinobacterium strains were divided into two groups on the base of gene coding for 16s rrna ( fig . 2 ) . . are often isolated from cold environments and are known for their metabolic versatility and high genome plasticity . the possibility of phas accumulation and high tolerance to cold and oxidative stress is common features of many pseudomonas species . therefore , it is suggested that pha metabolism in antarctic strains could be an adaptation mechanism that is necessary to withstand hard conditions in polar regions . the high number of isolates possessing genes responsible for phas synthesis suggests that antarctic bacteria evolved , or obtained in another way , this metabolic ability to endure the extreme conditions typical for polar regions . accordingly to our results , isolated strains possessed genes responsible for scl- or mcl - pha synthesis . it is worth pointing out that bacteria having phac1 genes were isolated both from mats and water , and those having phac gene were only planktonic . it has previously been reported that survival in harsh conditions can be increased by belonging to a complex communities like microbial mats . recent studies have evaluated the potential of microbial mats as high - level pha production systems under natural conditions , and as a source of bacterial pha producers [ 3 , 13 ] . accordingly to these studies , microorganisms inhabiting microbial mats from marine coastal zones are able to synthesize and accumulate high quantities of phas . our results also show that microbial mats created in antarctic ponds could be a potential source of pha producing bacteria . our comparative analysis of dna sequences of 16s rdna and phac / phac1 genes suggested that some strains possess pha synthesis genes that might have not been obtained by gradual evolution . the best example are isolates belonging to janthinobacterium spp . among four members of this genus , possessing genes responsible for phas synthesis , two of them had phac genes , whereas two had phac1 genes . p38 and p39 was similar to a variant of this gene previously detected only in zooglea ramigera , whereas the phac1 genes of janthinobacterium sp . p10 and p24 were most similar to genes of pseudomonas fluorescences . the genes responsible for scl - pha synthesis differ so much from those responsible for mcl - pha synthesis that their presence in these closely related bacteria is most likely not the result of gradual evolution , but rather of horizontal gene transfer . it is interesting that isolates that had obtained these particular types of genes were divided into two subgroups on the basis of 16s rdna sequences ( fig it is possible that this evolutionary separation is related to some physiological differences that make a particular pha operon useful for these bacteria . have been reported , and ours is the first report of potential mcl - pha synthesis by members of this genus . 16s rdna gene analysis showed that the taxonomic position of each of the six pseudomonas isolates having the phac1 gene was different ( table 2 ) . whereas , variation between phac1 genes was lower , closest dna sequences of phac1 gene of examined strains obtained by blast searching belonged only to three genera . the dna sequences of p1 , p4 , and p19 were closest to the sequence of pseudomonas fluorescens , a well - known mcl - pha producer . the structure of the phac1 gene of pseudomonas sp . in the same study , the same pha operon was found in both cells of pseudomonas sp . p45 , inhabiting a glacial melt water stream , possessed a phac1 gene most similar to that of pseudomonas stutzeri , although the similarity was only 84 % . p45 16s rrna gene was most similar to p. fragi and identical to the dna sequence of the pseudomonas sp . p45 , in cells of strain p5 , genes responsible for pha synthesis were not detected . in general , comparative analysis of 16s rrna and phac / phac1 genes revealed that isolated bacteria gain the genetic ability to synthesize pha rather as a result of horizontal gene transfer than by gradual evolution . furthermore , it seems that some pha operons are favored during this unusual ecological event . bacteria possessing pha synthase coding genes were detected in each of the studied location suggesting that phas accumulation could increase the survival capabilities of microorganisms in this extreme environment . bacteria having genes responsible for both scl- and mcl - phas synthesis were found , but those able of scl - pha synthesis were discovered only in ponds 7 and 10 . the relation between the presence of scl - pha producing bacteria and environmental conditions is supported by cca ( fig . the highest number of different forms of phac / phac1 genes was founded in pond 10 , which was characterized by low ph ( 6.5 ) , highest level of pheophytin ( 3.800 g l ) , and total chlorophyll ( 4.700 g l ) , and with the highest temperature measured ( 6.1 c ) . p19 ) and what is interesting similar form of gene was found in cells of janthinobacterium sp . ponds 10 and 11 are connected with the stream and it is likely , that this form of phac1 was channeled from pond 11 to pond 10 . a very distant form of phac1 gene was found in the mat taken from the stream 6 , it might release from the fact that environmental conditions in the streams are different from those in ponds . the spatial distribution of microorganisms possessing particular form of genes in the area of glacier forefield results mainly from glacier melting . the flowing water carries not only nutrients and minerals , but also transfers microorganisms having accelerated ability to colonize new territories . this group can include bacteria having ability to utilize phas produced by themselves or other members of microbial community . it seems that bacteria having genetic ability to store and degrade phas can work as a pioneers during a succession process . thus , glacier forefields provide a unique opportunity as a natural laboratory to study the succession of microorganisms . to sum up , our study shows that many microorganisms inhabiting freshwater lakes charged with glacier meltwater possess the genes responsible for pha synthesis . all isolated pseudomonas species had the phac1 gene responsible for mcl - pha synthesis , whereas janthinobacterium isolates had phac and phac1 genes used for both scl- and mcl - pha synthesis . to our knowledge , this is the first evidence that janthinobacterium spp . another interesting observation is the lack of correspondence between the evolutionary history of the 16s rdna genes and those coding for pha synthases . , suggest that these genes could have been acquired by horizontal gene transfer . the obtained results allow us to speculate that the possession of these genes enabling pha synthesis may improve the fitness and survival of bacteria in harsh conditions additionally , the presented results show that microorganisms inhabiting extreme environments should be considered as potential producers of phas .

the diversity of polyhydroxyalkanoates - producing bacteria in freshwater reservoirs in the ecology glacier foreland , antarctica , was examined by a cultivation - dependent method . isolated strains were analyzed phylogenetically by 16s rrna gene sequencing , and classified as members of alpha- , beta- , or gammaproteobacteria classes . polymerase chain reaction was used to detect pha synthase genes . potential polyhydroxyalkanoates ( phas ) producers belonging mainly to pseudomonas sp . , and janthinobacterium sp . were isolated from all five sampling sites , suggesting that pha synthesis is a common bacterial feature at pioneer sites . all pseudomonas strains had the genetic potential to synthesize medium - chain - length phas , whereas some isolated janthinobacterium strains might produce short - chain - length phas or medium - chain - length phas . it is the first report revealing that janthinobacterium species could have the potential to produce medium - chain - length phas .

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the operations required to reclaim arid and semi - arid lands fall into several different categories : modifying topography ( slope , contours , and drainage ) , creating appropriate surface composition ( compaction , particle size , and origin of the final surface ) , and establishing vegetation . of these categories , establishing vegetation entails the most uncertainty . vegetation establishment is important for : reducing erosion from both wind and water ( guiterrez and hernandez 1996 ; de baets et al . 2008 ) , improving public perception by reducing the visual impact ( hands and brown 2002 ; svobodova et al . 2012 ) , allowing for productive reuse of the land for wildlife habitat or livestock grazing ( e.g. , steinke and majak 2010 ) , and otherwise reintegrating disturbed lands into the existing landscape . uncertainty in vegetation establishment comes primarily from reliance on unpredictable rainfall for germination and establishment . while reliance upon rainfall is not the only option , irrigation is expensive and generally not more successful ( roundy et al . 2001 ) possibly because a high intensity / long duration of water application is needed to establish plants , and even when plants are established with irrigation , they can lack water hardiness and die when the irrigation is eventually removed ( josa et al . irrigation can also benefit exotic weeds more than natives ( e.g. , banerjee et al . natural rainfall can allow the establishment of robust native or non - native vegetation albeit at densities similar to nearby undisturbed vegetation ( e.g. , fehmi and kong 2012 ) . while in the past non - natives were preferred for their ability to establish in years with limited rainfall , natives are now more recommended due to their known long - term stability , resource value for wildlife , and better public acceptance ( dantonio and meyerson 2002 ) . non - natives are currently less acceptable due to potential economic losses due to their invasions off - site , increases in the ability of reclaimed lands to carry wildfire ( mcdonald and mcpherson 2011 ) , and other negative impacts ( hobbs and cramer 2008 ) . to maximize the long - term positive impact of re - vegetation , a mixed plant community is needed that contains the same ratio of functional groups as a natural community in the same area . in addition to being self - sustaining , diversity increases the resiliency of the plant community to disturbance ( quijas et al . where they occur , perennial grasses are often the primary contributors to surface stability and erosion prevention ( guiterrez and hernandez 1996 ; de baets et al . 2009 ) through their life form which is extensive , low to the ground , and with spreading , fibrous roots near the soil surface . cox and jordan ( 1983 ) expected successful establishment of native perennial warm - season grasses only 10 % of the time ( 1 in 10 years ) in arid to semi - arid southeastern arizona , usa . this is similar to the estimates generated by peters ( 2000 ) for central new mexico . establishment success for a seedling depends on the seminal root staying ahead of the soil drying front . when native warm - season ( c4 ) grasses commonly planted in the southwestern usa germinate , they are supported by the seminal root ( often also called the primary root ) until the adventitious roots begin to grow . because these grasses establish the plant crown near the surface of the soil , adventitious root growth can be suppressed by dry conditions ( hyder et al . other than the predictions mentioned above and research studies that rarely consider more than a few years ( e.g. , woods et al . 2012 ) , little work has occurred to better assess the chance of seeding success for arid areas . other studies have shown that seedling recruitment is not dependent solely on high rainfall years but instead on the amount and distribution of rainfall events ( e.g. , peters 2000 ; zimmerman et al . 2012 ) . given the advances in both natural history information and in ease of computer modeling , cox and jordan s ( 1983 ) prediction about southern arizona should be revisited . modeling can resolve important management and ecological questions because it allows comparisons that are not possible in the field ( hardegree et al . the goal of this project is to estimate how frequently native perennial warm - season grasses may be established in southern arizona using 31 years of historical rainfall event data . it is important to research the likelihood of vegetation establishment to better develop mitigation strategies and to allow appropriate budgeting for them . to estimate the frequency of successful establishment , we simulated soil moisture for a 31-year period using a land surface model . the inputs included detailed long - term precipitation event data , data on movement of moisture through the soil , and data on evaporation from the santa rita experimental range ( described below ) . these data allowed analysis of moisture at the surface and at each soil layer as roots grew through them . using real - world data was important because having valid input parameters remains the most difficult aspect of modeling seed germination ( flerchinger and hardegree 2004 ) , and correctly representing rainfall variability is critical for modeling soil moisture ( laio et al . the specific practices selected for analysis included having seeded on 15 may ( 30 days ahead of the normal start of the monsoon season ) onto bare soil in southern arizona , usa . the seeds ( generic native warm - season grasses described below ) were assumed to be in the top 01 cm of the soil when the monsoon rains began . the soil type used in the simulations was a loamy sand because this soil texture was common in the region as well as being the primary soil type on a nearby mineland reclamation site . no existing vegetation was assumed at the time of the seeding ( bare soil ) which had the simplifying effect of having evaporation to be the primary evapotranspiration loss . germination and establishment were evaluated over the first 21 days , so competition among seedlings was assumed not to have an impact on plant growth or soil moisture in this period . rainfall data ( 19802010 , 31 years ) came from gages 5 ( 31.8152 n , 110.8516 w , 1,163 m asl , 398 mm average annual precipitation ) and 6 ( 31.8137 n , 110.8544 w , 1,215 m asl , 405 mm average annual precipitation ) from the santa rita experimental range ( srer ) which was roughly 45 km south of tucson , az , usa ( http://www.tucson.ars.ag.gov/dap/ ) . the gages are 311 m apart and record the date , time , amount , and duration of each rainfall event . the monsoon rainfall in this region was generally from convective cells which can result in different amounts of rainfall , even over short distances , so the data from each gage were evaluated separately . seven years ( 6 january 200431 december 2010 ) of eddy flux data were available , including the following measurements : temperature / relative humidity ; photosynthetically active radiation ; ground heat flux ; soil temperature ; precipitation ; volumetric soil water content at 5 , 10 , 20 , 30 , 50 , 70 , 100 , and 130 cm depths ; wind velocity vector ; sonic temperature and concentrations of water vapor ; and carbon dioxide measurements ( scott et al . 2009 ; hereafter , flux tower data ) . the measurements were from the santa rita mesquite savanna site which was also located on the srer ( 31.8214 n , 110.8661 w , 1,120 m asl ) . to model the expected soil moisture from the 31 years ( 19802010 ) of precipitation data , we ran a land surface model , which was driven by hourly atmospheric forcing data . we calibrated the model s hydraulic parameters to match the observed soil moisture and surface energy and water fluxes from the flux tower data and then applied the optimized parameters to the 31-year precipitation data set . the atmospheric forcing data to drive the model included : hourly downward shortwave and longwave radiation fluxes , precipitation , air temperature , humidity , pressure , and wind speed . these data were computed as distance - weighted averages of those at four surrounding points of the 0.125 degree , gridded north american land data assimilation system ( nldas ) surface atmospheric data ( mitchell et al . 2004 ) . to check the accuracy of the nldas data , sensitivity experiments during the calibration period were conducted by replacing an individual variable of the flux tower data with that of the nldas data . through this exercise , we found that nldas data overestimated wintertime precipitation and had the largest effect on the simulation of soil moisture , while other variables had less effect . we then replaced the nldas precipitation with the rainfall from gages 5 and 6 from the santa rita experimental range . thus , for the period from 19802003 , we ran the model using the optimized hydraulic parameters and the nldas atmospheric forcing with its rainfall data being replaced with data from gages 5 and 6 . the noah land surface model was used in this study with multiple options for parameterization schemes of various land surface processes ( niu et al . there were only four soil layers in the standard model that was coupled with the weather research and forecast model . in this study , to match the flux tower measurement depths , we divided the model soil into 16 soil layers ( breaking at 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 45 , 50 , 60 , 70 , 90 , 100 , 120 , 130 , and 200 cm ) . the model solved the one - dimensional richards equation to account for redistribution of soil moisture among these layers through diffusion and percolation processes . at the 2-m model bottom , we used a groundwater recharge rate , which was provided by a simple groundwater model , as the lower boundary condition to ensure a more precise simulation . the soil hydraulic parameters for loamy sand were set up through a look - up table using the clapp hornberger relationship ( clapp and hornberger 1978 ) . however , the amplitudes of the modeled soil moisture for most of the deep soil layers were too small to match observations , indicating the diffusion processes of soil water are relatively weak . the constant value 1.5 10 m / s was added to the diffusivity which resulted in comparable soil moisture for all measurement depths . the enhanced diffusivity accounted for extra diffusion of vaporized soil water under dry and hot conditions ( niu et al . our analysis assumed that the native , warm - season ( c4 ) grass seeds were in the soil from 01 cm deep as suggested by roundy et al . ( 1997 ) , which corresponds well to visual observations of seed depth after broadcast seeding on this soil type in this area . the seeds were assumed to remain available until they germinate rather than experiencing any other fate such as being eaten , buried , blown , or washed off site , etc . all seeds were assumed to germinate when exposed to suitable monsoon conditions ( per abbott and roundy 2003 ) . wetting events that did not result in germination were assumed to cause no injury to these warm - season perennial grass seeds ( roundy et al . germinating conditions that occurred before 15 may were not considered because this was before we assumed planting to occur . germinating conditions that occurred after 30 september were assumed to result in seeding failure due to the lack of suitable growing conditions ( cool weather ) in october . two scenarios were evaluated for each of the rain gages2 days of suitable conditions resulting in germination and 3 days of suitable conditions resulting in germination . plants were counted as germinated if the water volume in the 02.5 cm layer stayed above 0.09 cm / cm for 2 or 3 days ( analyzed separately ) an intermediate value for water volume ( roughly twice the wilting point , 0.046 cm / cm , laio et al . 2001 ) was chosen because the 02.5-cm layer dries quickly , but seeds can germinate if the relative humidity remains high at the soil surface ( wuest 2007 ) . ( 1997 ) that the surface soil becomes too dry within 1 day or less of the cessation of a storm . the 2-day scenario was a more cautious interpretation of the available data on seed germination . most warm - season perennial grasses germinate within 1.5 to 4 days of saturation of the surface soil at summer temperatures ( roundy and biedenbender 1996 ) . the 2-day scenario supported native warm - season grass species like bouteloua curtipendula ( sideoats grama ) which germinated and emerged within 2 days of suitable conditions50 % in 1 day ( simanton and jordan 1986 ) . another study observed emergence of b. curtipendula beginning in 18 h and ending after 3 days ( frasier et al . while different authors have tested germination in different ways , most typically , the reported times were for saturated soil and our standard was dryer than that . our 2-day germination scenario typically corresponded to one large or two medium - sized events . the 3-day scenario was a more lenient estimate of the time needed for germination and corresponds more closely to available data on native warm - season grass germination . ( 2000 ) found digitaria californica ( arizona cottontop ) required a minimum of 140 mm of water in a 3-day period for 50 % emergence . wilson and briske ( 1979 ) found bouteloua gracilis ( blue grama ) needed wet conditions for 24 days to germinate . after 2.3 days in wet field conditions , abbott and roundy ( 2003 ) found b. curtipendula had 90 % germination ; leptochloa dubia ( green sprangletop ) had 75 % ; d. californica had 70 % , and eragrostis intermedia ( plains lovegrass ) had 45 % . germinated plants die when seminal root growth does not keep up with the drying front in the soil which makes soil moisture the primary controlling factor for seedling mortality ( carren et al . 1987 ; zimmerman et al . 2008 ; perring and hovenden 2012 ; thapa et al . this process was important for 23 weeks after germination when 24 days of additional moisture was needed at the soil surface for development of the plant crown and adventitious roots ( carren et al . the drying front was defined as the wilting point ( 0.046 cm / cm ) for a loamy sand ( laio et al . the rate of 0.8 cm root elongation per day was chosen as a conservative estimate based on several studies of native warm - season perennial grasses . for b. gracilis , the seminal root grew 0.79 cm per day for the first 2 weeks ( carren et al . b. curtipendula averaged about 0.8 cm per day of seminal root elongation during the first 15 days ( roundy et al . simanton and jordan ( 1986 ) found an elongation rate of 0.97 cm per day for the first 7 days for b. curtipendula . using the modeled soil moisture , if sufficient water were available , plants may have initiated adventitious roots during our 19-day evaluation , but we evaluated soil moisture for the whole soil column and dryness below the wilting point would have affected adventitious roots as well as seminal roots . in our assessment , we checked the simulated water content of the soil column from the surface to the depth of the seminal root , and if the whole column was below the wilting point , the seeding was considered to have failed . rainfall data ( 19802010 , 31 years ) came from gages 5 ( 31.8152 n , 110.8516 w , 1,163 m asl , 398 mm average annual precipitation ) and 6 ( 31.8137 n , 110.8544 w , 1,215 m asl , 405 mm average annual precipitation ) from the santa rita experimental range ( srer ) which was roughly 45 km south of tucson , az , usa ( http://www.tucson.ars.ag.gov/dap/ ) . the gages are 311 m apart and record the date , time , amount , and duration of each rainfall event . the monsoon rainfall in this region was generally from convective cells which can result in different amounts of rainfall , even over short distances , so the data from each gage were evaluated separately . seven years ( 6 january 200431 december 2010 ) of eddy flux data were available , including the following measurements : temperature / relative humidity ; photosynthetically active radiation ; ground heat flux ; soil temperature ; precipitation ; volumetric soil water content at 5 , 10 , 20 , 30 , 50 , 70 , 100 , and 130 cm depths ; wind velocity vector ; sonic temperature and concentrations of water vapor ; and carbon dioxide measurements ( scott et al . the measurements were from the santa rita mesquite savanna site which was also located on the srer ( 31.8214 n , 110.8661 w , 1,120 m asl ) . the soils at the site are uniform sandy loams . to model the expected soil moisture from the 31 years ( 19802010 ) of precipitation data , we ran a land surface model , which was driven by hourly atmospheric forcing data . we calibrated the model s hydraulic parameters to match the observed soil moisture and surface energy and water fluxes from the flux tower data and then applied the optimized parameters to the 31-year precipitation data set . the atmospheric forcing data to drive the model included : hourly downward shortwave and longwave radiation fluxes , precipitation , air temperature , humidity , pressure , and wind speed . these data were computed as distance - weighted averages of those at four surrounding points of the 0.125 degree , gridded north american land data assimilation system ( nldas ) surface atmospheric data ( mitchell et al . 2004 ) . to check the accuracy of the nldas data , sensitivity experiments during the calibration period were conducted by replacing an individual variable of the flux tower data with that of the nldas data . through this exercise , we found that nldas data overestimated wintertime precipitation and had the largest effect on the simulation of soil moisture , while other variables had less effect . we then replaced the nldas precipitation with the rainfall from gages 5 and 6 from the santa rita experimental range . thus , for the period from 19802003 , we ran the model using the optimized hydraulic parameters and the nldas atmospheric forcing with its rainfall data being replaced with data from gages 5 and 6 . the noah land surface model was used in this study with multiple options for parameterization schemes of various land surface processes ( niu et al . there were only four soil layers in the standard model that was coupled with the weather research and forecast model . in this study , to match the flux tower measurement depths , we divided the model soil into 16 soil layers ( breaking at 2.5 , 5 , 10 , 15 , 20 , 25 , 30 , 45 , 50 , 60 , 70 , 90 , 100 , 120 , 130 , and 200 cm ) . the model solved the one - dimensional richards equation to account for redistribution of soil moisture among these layers through diffusion and percolation processes . at the 2-m model bottom , we used a groundwater recharge rate , which was provided by a simple groundwater model , as the lower boundary condition to ensure a more precise simulation . the soil hydraulic parameters for loamy sand were set up through a look - up table using the clapp . however , the amplitudes of the modeled soil moisture for most of the deep soil layers were too small to match observations , indicating the diffusion processes of soil water are relatively weak . the constant value 1.5 10 m / s was added to the diffusivity which resulted in comparable soil moisture for all measurement depths . the enhanced diffusivity accounted for extra diffusion of vaporized soil water under dry and hot conditions ( niu et al . our analysis assumed that the native , warm - season ( c4 ) grass seeds were in the soil from 01 cm deep as suggested by roundy et al . ( 1997 ) , which corresponds well to visual observations of seed depth after broadcast seeding on this soil type in this area . the seeds were assumed to remain available until they germinate rather than experiencing any other fate such as being eaten , buried , blown , or washed off site , etc . all seeds were assumed to germinate when exposed to suitable monsoon conditions ( per abbott and roundy 2003 ) . wetting events that did not result in germination were assumed to cause no injury to these warm - season perennial grass seeds ( roundy et al . germinating conditions that occurred before 15 may were not considered because this was before we assumed planting to occur . germinating conditions that occurred after 30 september were assumed to result in seeding failure due to the lack of suitable growing conditions ( cool weather ) in october . two scenarios were evaluated for each of the rain gages2 days of suitable conditions resulting in germination and 3 days of suitable conditions resulting in germination . plants were counted as germinated if the water volume in the 02.5 cm layer stayed above 0.09 cm / cm for 2 or 3 days ( analyzed separately ) . an intermediate value for water volume ( roughly twice the wilting point , 0.046 cm / cm , laio et al . 2001 ) was chosen because the 02.5-cm layer dries quickly , but seeds can germinate if the relative humidity remains high at the soil surface ( wuest 2007 ) . ( 1997 ) that the surface soil becomes too dry within 1 day or less of the cessation of a storm . the 2-day scenario was a more cautious interpretation of the available data on seed germination . most warm - season perennial grasses germinate within 1.5 to 4 days of saturation of the surface soil at summer temperatures ( roundy and biedenbender 1996 ) . the 2-day scenario supported native warm - season grass species like bouteloua curtipendula ( sideoats grama ) which germinated and emerged within 2 days of suitable conditions50 % in 1 day ( simanton and jordan 1986 ) . another study observed emergence of b. curtipendula beginning in 18 h and ending after 3 days ( frasier et al . while different authors have tested germination in different ways , most typically , the reported times were for saturated soil and our standard was dryer than that . our 2-day germination scenario typically corresponded to one large or two medium - sized events . the 3-day scenario was a more lenient estimate of the time needed for germination and corresponds more closely to available data on native warm - season grass germination . ( 2000 ) found digitaria californica ( arizona cottontop ) required a minimum of 140 mm of water in a 3-day period for 50 % emergence . wilson and briske ( 1979 ) found bouteloua gracilis ( blue grama ) needed wet conditions for 24 days to germinate . after 2.3 days in wet field conditions , abbott and roundy ( 2003 ) found b. curtipendula had 90 % germination ; leptochloa dubia ( green sprangletop ) had 75 % ; d. californica had 70 % , and eragrostis intermedia ( plains lovegrass ) had 45 % . die when seminal root growth does not keep up with the drying front in the soil which makes soil moisture the primary controlling factor for seedling mortality ( carren et al . 1987 ; zimmerman et al . 2008 ; perring and hovenden 2012 ; thapa et al . this process was important for 23 weeks after germination when 24 days of additional moisture was needed at the soil surface for development of the plant crown and adventitious roots ( carren et al . the drying front was defined as the wilting point ( 0.046 cm / cm ) for a loamy sand ( laio et al . the rate of 0.8 cm root elongation per day was chosen as a conservative estimate based on several studies of native warm - season perennial grasses . for b. gracilis , the seminal root grew 0.79 cm per day for the first 2 weeks ( carren et al . b. curtipendula averaged about 0.8 cm per day of seminal root elongation during the first 15 days ( roundy et al . simanton and jordan ( 1986 ) found an elongation rate of 0.97 cm per day for the first 7 days for b. curtipendula . using the modeled soil moisture , if sufficient water were available , plants may have initiated adventitious roots during our 19-day evaluation , but we evaluated soil moisture for the whole soil column and dryness below the wilting point would have affected adventitious roots as well as seminal roots . in our assessment , we checked the simulated water content of the soil column from the surface to the depth of the seminal root , and if the whole column was below the wilting point , the seeding was considered to have failed . the average germination date for the gage 5 2-day scenario was 20 july ( table 1 ) , and for the 3-day scenario , it was 1 august ( table 2 ) . the average germination dates for gage 6 2-day scenario was 19 july ( table 3 ) , and for the 3-day scenario , it was 27 july ( table 4 ) . in most years , there was little difference in germination date and success among the gages and the scenarios , but in a few years there were substantive differences among the gages and treatment germination dates ( fig . 1 ) . in 2001 , the expected germination in the 3-day evaluation of gage 5 was 111 days later than the 2-day evaluation of gage 6.table 1evaluation of plants taking 2 days to germinate at the gage 5 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5 cm 2.5 to 5.0 cm 5.0 to 10.0 cm 10.0 to 15.0 cm 198014 aug0.0510.0490.0530.055198111 jul0.1190.1180.0820.084failed 19828 jul0.048 0.045 0.0580.099198312 jul0.0670.0530.0550.049198430 jun0.0640.0810.0730.078198521 jul0.0730.0600.0750.083failed 198617 jul0.0600.057 0.041 0.043 failed 198722 may0.0580.052 0.039 0.037 failed 198811 jul 0.044 0.040 0.0520.107198928 jul0.0850.0760.0830.08319909 jul0.1290.0950.1330.136failed 199131 jul 0.035 0.033 0.041 0.067failed 19929 jul0.0880.065 0.044 0.05419934 aug0.1410.1130.0720.08219944 sep0.0600.0500.0890.076failed 199522 aug 0.046 0.049 0.039 0.040 19964 jul0.0720.0780.0940.070199719 aug0.0530.0640.0710.08019986 jul0.1290.0980.0720.085199927 jul0.0900.0670.0650.074200021 jun0.0590.0900.0910.090failed 20015 aug 0.034 0.0640.0680.068failed 200218 jul 0.045 0.0520.1030.087200327 jul0.0990.1110.0820.082failed 200415 jul 0.036 0.0470.0580.08920056 aug0.1160.0910.1010.082200628 jul0.0940.0940.0790.069200721 jul0.0640.0660.0760.089200813 jul0.0510.0470.0670.071failed 2009no germ201031 jul0.0750.0560.0540.054numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 20 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm no germinationtable 2evaluation of plants taking 3 days to germinate at the gage 5 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5 cm 2.5 to 5.0 cm 5.0 to 10.0 cm 10.0 to 15.0 cm failed 198015 aug0.048 0.043 0.0530.051198112 jul0.1040.1210.0750.084198223 jul0.0680.0630.0610.058failed 198313 jul0.064 0.044 0.055 0.046 198419 jul0.0910.1030.0980.096198522 jul0.0700.0600.1000.077failed 198618 jul0.0470.057 0.039 0.043 19876 aug0.0660.0850.0680.072failed 198812 jul 0.035 0.040 0.0850.108198929 jul0.0740.0760.0830.078199010 jul0.1010.0950.1330.130199110 aug0.0550.0710.0660.058failed 199213 jul 0.046 0.042 0.044 0.06419935 aug0.1410.0970.0720.08619945 sep0.0490.0500.0810.076199530 sep0.0640.0620.0530.051failed 199616 jul 0.042 0.043 0.0480.051199716 sep0.0540.0620.0620.05219987 jul0.1050.0980.0720.125199928 jul0.0850.0670.0650.074200025 jun0.0550.0710.0770.075failed 200110 oct0.0570.051 0.041 0.039 200225 jul0.1060.0780.0750.085200328 jul0.0960.1230.0780.082failed 200417 aug0.049 0.044 0.044 0.039 20057 aug0.1030.0910.0920.082200629 jul0.0960.0830.0740.067200722 jul0.0560.0630.0810.083200822 jul0.0990.0710.0490.062failed 2009no germ20101 aug0.0570.0510.0490.054numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 22 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm germination too late in season no germinationtable 3evaluation of plants taking 2 days to germinate at the gage 6 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5 cm 2.5 to 5.0 cm 5.0 to 10.0 cm 10.0 to 15.0 cm 198014 aug0.0550.0510.0550.057198111 jul0.1170.1220.0950.094failed 19828 jul0.048 0.044 0.0570.102198312 jul0.0650.0510.0530.048198430 jun0.0690.0840.0750.095198516 jul0.0630.1180.0810.102failed 198617 jul0.0570.054 0.042 0.043 failed 198722 may0.0530.048 0.037 0.036 failed 198811 jul0.047 0.042 0.0550.107198928 jul0.0830.0760.0830.067199010 jul0.1320.0980.1350.13719919 aug0.0710.0710.0630.063failed 199210 jul0.0900.064 0.044 0.05319934 aug0.1370.1130.0720.08419944 sep0.0570.0510.0890.079failed 199522 aug 0.041 0.044 0.036 0.040 19964 jul0.0600.0720.0920.067199719 aug0.0510.0620.0710.08019986 jul0.1250.0960.0720.082199927 jul0.0950.0670.0620.068200024 jun0.0660.0710.0840.081failed 200122 jun 0.031 0.035 0.030 0.034 failed 200218 jul 0.044 0.0510.1000.087200327 jul0.0950.1070.0800.077200430 jul0.0650.0700.0480.05620056 aug0.0920.0910.1010.083200628 jul0.0940.0940.0780.069200721 jul0.0640.0670.0760.089200813 jul0.0510.0470.0670.070failed 2009 no germ 201031 jul0.0750.0560.0540.054numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 22 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm no germination also failed 5 may which is outside the planting windowtable 4evaluation of plants taking 3 days to germinate at the gage 6 siteminimum soil moisture cm / cm during root elongation through zonesuccess / failureyeargermination datesurface to 2.5 cm 2.5 to 5.0 cm 5.0 to 10.0 cm 10.0 to 15.0 cm failed 198015 aug0.051 0.045 0.0550.054198112 jul0.1100.1220.0860.094198223 jul0.0770.0690.0670.064failed 198313 jul0.060 0.043 0.053 0.046 19841 jul0.0660.0770.0750.112198520 jul0.0990.0780.0810.085failed 198618 jul 0.045 0.054 0.039 0.043 19876 aug0.0600.0780.0660.069failed 198812 jul 0.037 0.042 0.0920.115198929 jul0.0750.0760.0830.063199010 jul0.1050.0980.1350.128199110 aug0.0550.0710.0610.060failed 199210 jul0.0750.054 0.044 0.08019935 aug0.1370.0970.0720.08319945 sep0.0480.0510.0820.079failed 199530 sep0.0650.0630.0560.057failed 199616 jul 0.038 0.040 0.046 0.051failed 199716 sep0.0520.0610.0620.05219987 jul0.1020.0960.0710.123199928 jul0.0850.0640.0620.068200025 jun0.0540.0710.0770.077200117 jul0.0660.0620.0610.051200225 jul0.0980.0740.0730.084200328 jul0.0910.1190.0770.077failed 200417 aug 0.044 0.041 0.042 0.038 20057 aug0.1030.0910.0920.083200629 jul0.0960.0830.0740.067200722 jul0.0560.0630.0810.083failed 200822 jul0.0990.0700.0490.062failed 2009 no germ 20101 aug0.0570.0510.0490.054numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 23 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm germinated too late in season for successful establishment no germinationfig . julian date 150 is 29 may , 200 is 18 july , and 250 is 6 sept evaluation of plants taking 2 days to germinate at the gage 5 site numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 20 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm evaluation of plants taking 3 days to germinate at the gage 5 site numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 22 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm germination too late in season evaluation of plants taking 2 days to germinate at the gage 6 site numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 22 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm also failed 5 may which is outside the planting window evaluation of plants taking 3 days to germinate at the gage 6 site numbers at or below the wilting point ( 0.046 cm / cm ) for this soil type are in bold . of the 31 years evaluated , 23 years had successful establishment in the first 19 days after germination too little rain , hits wilting point at 2.5 to 5.0 cm too little rain , hits wilting point at 5.0 to 10.0 cm too little rain , hits wilting point at surface to 2.5 cm germinated too late in season for successful establishment graph of the germination dates by year from 1980 to 2010 . julian date 150 is 29 may , 200 is 18 july , and 250 is 6 sept for the gage 5 two - day scenario ( table 1 ) , 35 % of the years failed to have successful establishment , and 29 % failed for the 3-day scenario ( table 2 ) . for the gage 6 two - day scenario ( table 3 ) , 29 % of the years failed , and 35 % failed for the 3-day scenario ( table 4 ) . for gage 5 , there were 6 years where the seeding failed for both scenarios ( 19 % failure rate ) , and for gage 6 , there were only 4 years ( 13 % failure rate ) where it failed for both scenarios ( table 5 ) . these same 4 years also failed for gage 5 , giving a failure rate common across both gages and both scenarios of 13 % .table 5the overlap ( consistency ) among establishment failure across the gages and germination scenariosgage 5 sitegage 6 site2-day3-day2-day3-dayfailures119911overlapgage 5 site2-day6963-day658gage 6 site2-day9543-day684average germination date20 jul1 aug19 jul27 jul the overlap ( consistency ) among establishment failure across the gages and germination scenarios the worst 10-year span was gage 5 for the 2-day scenario where between 1986 and 1995 , six of ten plantings would have failed ( table 1 ) . the best 10-year span was gage 6 for the 3-day scenario where , between 1998 and 2007 , only one of ten was projected to fail ( table 4 ) . at most , 3 years in a row failed , and 6 years in a row were successful . our modeling results showed six times more potential for seeding success ( 68 % ) than was expected from the 10 % estimate by cox and jordan ( 1983 ) for southern arizona . while this may show substantial potential for reclamation in most years , there are numerous factors , such as germination response , root growth , and other sources of mortality , that bear further consideration . the germination periods of 2 or 3 days which we assumed for analysis do not necessarily reflect the full scope of plant responses in real - world situations . for instance , non - germinating rains ( not long enough in duration ) that cause more than 1 day of adequate soil moisture can reduce seed viability ( emmerich and hardegree 1996 ) . but less than 1 day of soil moisture before the eventual germinating rain can prime seeds and allow them to germinate faster than unexposed seeds once germinating rains occur ( emmerich and hardegree 1996 ) . in addition , a small amount of the native grass seed ( 12 % ) will not germinate until the second or third germinating rain ( abbott and roundy 2003 ) which is also not included in the model . these factors were not included because not enough information was available to determine the expected average outcome , and the magnitude of the effect on reclamation success would likely be small . the period after germination is similarly more complex than our model represents , although we have incorporated plant death due to lack of rain after germination . these deaths are similar to those observed by abbott and roundy ( 2003 ) who found two sowing dates in two separate years where the drying front exceeded seminal root depth for native perennial grasses . the complexity comes in because their field data did not show 100 % mortality but instead had 0.6 % and 7.9 % survival . while this may not result in a stand of vegetation that allows one to claim reclamation success , even with sufficient water , survival rates of native perennial grasses are not near 100 % in arid and semi - arid areas . with 32 days of available water ( the drying front did not exceed the seminal root depth ) , there was 28 % survival of b. curtipendula , 27 % survival of l. dubia , 10 % survival of d. californica , and 11 % survival of e. intermedia ( abbott and roundy 2003 ) . nor are low survival rates unique to arizona . in a study in wyoming , chambers ( 2000 ) found an overall survival rate of 1 % for seeded plants . pyke ( 1990 ) had less than 1 % on a study in utah , and james et al . given the relatively low survival rates of these common native perennial grasses , small differences may be more important than they appear , although poor survival possibly can be managed in the field by increasing the seeding rate or changing seed sources to more drought - tolerant collections . the bottleneck for revegetation appears to be primarily in the establishment phase which occurs in the weeks following germination . this appears applicable across many different systems , including minnesota ( fay and shultz 2009 ) , utah ( pyke 1990 ) , and new mexico ( peters 2000 ) , although other studies from wyoming ( chambers 2000 ) and oregon ( james et al . 2011 ) found out that mortality primarily occurred between germination and emergence . in the period after germination , elongation rate of the seminal root and adventitious root initiation are the important factors for survival . b. curtipendula averaged about 0.8 cm per day of seminal root elongation , but the lengths varied 0.51.1 cm per day , depending on the amount and frequency of water with faster elongation associated with more water ( roundy et al . 1993 ) . for b. curtipendula , adventitious roots initiated on day 9 when watered every 3 days compared with day 13 when only watered on days 1 and 7 ( roundy et al . this would trigger the need for 24 days of available surface moisture to foster adventitious root elongation . b. gracilis is similar in that it needs wet conditions 28 weeks after germination to allow adventitious root establishment ( frasier et al . the variability of root elongation rates , the lack of studies on the non - genetic causes of rate differences , and inherent real - world variability in soil conditions make it difficult to know how they would change the outcome of our analysis . ( 2008 ) found that the primary driver of seedling survivorship in a climate change scenario was soil moisture and that warming and co2 increases only affected survivorship indirectly through changing soil moisture . ( 2012 ) predicted that southern arizona will have warmer and dryer weather in the future but that much of this will be decreased winter precipitation which may have a limited impact on the dominant warm - season ( c4 ) perennial grasses such as those modeled in this study . consistent with perring and hovenden ( 2012 ) , the total annual rainfall appears only slightly correlated with our predicted seeding success or failure . a logistic regression analysis showed that total seasonal precipitation was a significant predictor of germination success ( p = 0.027 ) , but the analysis accounted for little of the variability ( nagelkerke r = 0.059 ) . the 6 years with the lowest rainfall ( below 300 mm compared with a 400 mm average ) had a 50 % failure rate which is more than the average rate of 32 % . the six years with the highest observed average rainfall ( above 465 mm ) had a 33 % failure rate . the distribution of rainfall events is clearly a variable that benefits seeds or seedlings , but larger event sizes during the growing season might not increase establishment . once a storm event saturates the soil , a higher intensity event may not benefit seedling establishment because the seedlings are unable access moisture which has infiltrated deeper in the soil column and the excess water is lost as runoff . plant establishment is sensitive to small changes in rainfall timing which is not well specified in predictions of future rainfall . our observation of the common presence of events conducive to seeding success across the range of annual rainfall values makes it appear that warm - season perennial grasses may not be at risk from the kinds of changes predicted by notaro et al . the overall management implication for this study is that funds for reseeding must be available for at least a second year . this will allow reseeding the following year if there is a reclamation failure the year before . further funds / reseeding effort may be needed because seeding failure 3 years in a row appears possible . a variety of species in a diverse mix , while often recommended for creation of a stable and self - sustaining plant community , may also help reduce the risk of seeding failure in years with marginally poor rainfall distribution because different species have different water and time requirements for germination . field experiments would verify the germination and establishment modeling results and allow better predictions of seeding success .

of the operations required for reclamation in arid and semi - arid regions , establishing vegetation entails the most uncertainty due to reliance on unpredictable rainfall for seed germination and seedling establishment . the frequency of successful vegetation establishment was estimated based on a land surface model driven by hourly atmospheric forcing data , 7 years of eddy - flux data , and 31 years of rainfall data at two adjacent sites in southern arizona , usa . two scenarios differing in the required imbibition time for successful germination were evaluated2 or 3 days availability of sufficient surface moisture . establishment success was assumed to occur if plants could germinate and if the drying front in the soil did not overtake the growth of seminal roots . based on our results , vegetation establishment could be expected to fail in 32 % of years . in the worst 10-year span , six of ten plantings would have failed . in the best 10-year span , only one of ten was projected to fail . across all assessments , at most 3 years in a row failed and 6 years in a row were successful . funding for reclamation seeding must be available to allow reseeding the following year if sufficient amount and timing of rainfall does not occur .

pubmed

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 .

pubmed

small rna molecules are important in the regulation of various molecular and biological activities in the cell , and it is now well known that short rna sequences play a critical role in regulating the expression levels of specific genes in a targeted manner . rna interference ( rnai ) has become widely recognized to be an important gene regulatory mechanism that causes sequence - specific downregulation of mrnas . acting through this mechanism , double - stranded short interfering rnas ( sirnas ) can knock down expression levels via the rna - induced silencing complex , which mediates degradation or translational inhibition of the targeted mrnas . moreover , in addition to rna - induced silencing complex - mediated regulation at the post - transcriptional level , rnai can also modulate gene transcription itself . in fission yeast , homologs of the rna - induced silencing complex can regulate chromatin through recruitment of histone - modifying proteins to loci transcribing small noncoding rna , a mechanism also seen in plants , ciliates , nematodes , and flies . small promoter - targeted rnas have also been shown to repress transcription and induce epigenetic changes in eukaryotic cells through a mechanism called transcriptional gene silencing . in human cells , it has recently been reported that short rnas targeted to the promoter regions of certain genes can activate expression at the transcriptional level . this phenomenon has been called rna activation ( rnaa ) , and has been shown to be conserved in other mammalian species , including mouse , rat , and nonhuman primates . promoter - targeted small hairpin rnas have also been shown to efficiently upregulate genes in vivo . while the mechanism is not completely understood , it appears that naturally occurring antisense transcripts arising from or near the same genetic locus are able to direct recruitment of argonaute proteins and histone methyltransferases . short - activating rnas ( sarna ) may regulate transcription by targeting these antisense transcripts for degradation , resulting in a reversal of this epigenetic silencing and upregulation of sense mrna . based on this approach , we have developed a method to design sarnas for upregulation of specific cellular genes . in the present study , we focused on the feasibility and genetic consequences of upregulating important target genes involved in stem cell regulation and reprogramming . combined expression of the transcription factors kruppel - like factor 4 ( klf4 ) , pou5f1 ( also called oct3/4 ) , sox2 , and c - myc has been shown to reprogram mouse and human fibroblasts into induced pluripotent stem ( ips ) cells . in particular , klf4 is important for maintenance of embryonic stem cells , and has been reported to be a master regulator in embryonic stem cells that controls the expression of other pluripotency factors including pou5f1 , sox2 , c - myc , and nanog . it has previously been reported that direct reprogramming can be achieved in murine fibroblasts with only klf4 , oct4 , and sox2 . however , it has recently been shown that c - myc is critical for efficient induction of pluripotency in the early phases of reprogramming , by altering the metabolic state of the cell . accordingly , in the present study , we employed a genomic bioinformatic approach to design sarnas that specifically target two of these key reprogramming genes , klf4 and myc . these sarnas were tested for their ability to upregulate the targeted reprogramming factors , as well as their respective downstream genes , in human mesenchymal stem cells ( mscs ) , adult bone marrow - derived tissue - specific stem cells that already have multilineage differentiation potential . the effects of sarna transfection on endogenous gene expression profiles were compared with those resulting from lentiviral vector - mediated overexpression of the exogenous klf4 and c - myc transgenes . to date , there have been no studies comprehensively examining the cellular gene expression profiles after sarna - mediated gene activation of endogenous genes , particularly pluripotency - related genes , and few studies comparing how different reprogramming methods might differentially affect various cellular pathways . these results indicate that the use of sarna shows significant potential , both as a tool for studying stem cell biology , as well as a safe method to manipulate stem cell gene expression without altering the genome . to design sarna candidates for activation of stem cell factors , we developed a novel bioinformatic approach . the klf4 gene , located on chromosome 9 ( 9q31.2 ) , and c - myc gene , located on chromosome 8 ( 8q24.21 ) , were our initial targets for activation ( figure 1a , b ) . to identify potential antisense transcripts from the klf4 and c - myc loci , we searched the genomic region surrounding each locus for spliced expressed sequence tags ( ests ) which mapped to the positive strand . although it is usually difficult to determine the transcriptional orientation of ests , orientation can be determined by using splice site signatures of spliced ests . we found no spliced ests that overlapped klf4 , but the scan identified one antisense est ( db461753 ) ~15 kb upstream of klf4 's annotated transcription start site ( tss ) . we were also unable to find a spliced ests that overlapped c - myc , but identified one antisense est ( bc042052 ) ~2 kb upstream of c - myc 's tss . recent deep sequencing experiments have revealed that antisense rnas often are found in the region surrounding tsss . therefore , this region was chosen to design sarna sequences that targeted potential antisense transcripts from the promoter region . we used the antisense sequence 500 nts upstream and downstream from the tss ( abbreviated klf4_as_tss+/500 and myc_as_tss+/500 ) as a second target candidate . our goal was to design short sense rnas that could potentially bind and degrade antisense rnas generated from the two candidate sequences ( ests and the regions surrounding tsss ) with the hypothesis that this binding and degradation of the antisense rnas would activate gene expression . candidate sarnas targeting the 500 nt upstream of the tss were designated pr1 , whereas those targeting the 500 nt downstream of the tss were designated pr2 . to give effective antisense targeting and degradation and to minimize off - target effects , we used the gpboost sirna design algorithm to identify potential short rnas for downregulating the two candidate sequences . from the lists of predicted sirna candidates , we selected the two most promising non - overlapping sirna target sites on the antisense est db461753 and bc042052 , and the most promising sirna target site on each side of the klf4 and c - myc tss within the antisense promoter sequence ( klf4_as_tss+/500 and myc_as_tss+/500 ) . reasoning that activation of pluripotency factor gene expression might be more readily achieved in adult tissue - derived stem cells that retain restricted multilineage potential , we then tested whether these sarna candidates could upregulate klf4 or c - myc expression , respectively , in primary human mscs derived from adult bone marrow . target gene expression levels were examined by quantitative pcr of reverse - transcribed mrna from msc cultures after transfection of each individual sarna candidate oligonucleotide , as compared with transfection with an alexa fluor 555- or fam - labeled negative control oligo . a transfection efficiency of > 95% was determined by flow cytometry and by knockdown with control sirnas using the same transfection conditions ( see supplementary materials and methods and supplementary figures s1 and s2 ) . initially , none of the sarna oligos appeared to show any effect on target gene expression after 48 hours . however , upon continued transfection with klf4-pr1 sarna every other day , significant upregulation of klf4 mrna ( figure 2a ) was observed , on the order of 2.5-fold over controls by day 4 , and reaching approximately fourfold by day 6 of treatment ( p < 0.01 ) . target gene mrna levels after treatment with klf4-pr1 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50 nmol / l , as compared with 5 nmol / l ( figure 2b ) . increased klf4 protein was confirmed in mscs treated with klf4-pr1 sarna ( figure 2c , left panel ) by western blot analysis , and densitometric quantitation of these blots showed over threefold upregulation of klf4 protein relative to -actin internal control ( figure 2c , right panel ) , correlating closely with the level of mrna upregulation . this time - dependent upregulation of klf4 gene expression by the klf4 promoter - targeted pr1 sarna was observed consistently over multiple experiments . none of the other three klf4-targeted sarna candidate sequences was able to upregulate target gene expression , although conversely , klf4-db1 appeared to reduce klf4 mrna levels in mscs to about 60% that of scrambled oligo - treated controls by day 6 . similarly , we found that among the four sarna candidate sequences targeted to the c - myc promoter and antisense ests , both myc - pr1 and myc - pr2 were able to induce consistent upregulation of myc mrna ( figure 2d ) by up to 1.8-fold ( p < 0.01 ) and 1.6-fold ( p < 0.01 ) , respectively , during the 6-day interval of treatment , as compared with scrambled sequence controls . while myc - bc1 and myc - bc2 also appeared to affect myc mrna levels to some extent , these effects were more modest , and were not consistent between days 4 and 6 . again , target gene mrna levels after treatment with myc - pr1 and myc - pr2 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50 nmol / l , as compared with 5 nmol / l ( figure 2e ) . image densitometry analysis of western blots from mscs treated with myc - pr1 or myc - pr2 again confirmed upregulation of c - myc protein by over 2.5-fold relative to -actin internal control ( figure 2f ) . all western blots were performed in triplicate with replicates shown in supplementary figure s3 . to determine whether this upregulation of klf4 and myc is due to true transcriptional activation , expression levels of nascent rna were assessed . mscs were pulsed with ethynyl uridine ( eu ) during sarna treatment and total rna was isolated . newly transcribed eu - rna was separated from total rna by biotinylation of eu in a copper - catalyzed click reaction , followed by purification on streptavidin magnetic beads . quantitative pcr of reverse - transcribed nascent rna showed that the level of nascent klf4 mrna was significantly upregulated with klf4-pr1 treatment across a 6-day timecourse ( figure 3a ) . the level of nascent myc mrna was significantly upregulated with myc - pr2 treatment , whereas no significant change in nascent myc mrna expression was seen with myc - pr1 treatment ( figure 3b ) . to verify that these sarnas do not act through interferon response pathways , no significant upregulation of interferon response genes was detected 24 hours after sarna treatment ( figure 3c ) . in contrast , overexpression of klf4 and c - myc by lentiviral transduction showed dose - dependent induction of interferon response genes ( supplementary figure s4 ) . to further elucidate the mechanism of activation by sarna , the presence of promoter - associated antisense rnas was investigated using 5 rapid amplification of cdna ends ( race ) . since any antisense rnas involved in regulating gene expression may not be polyadenylated , random hexamers were used to prime cdna synthesis from total msc rna . antisense strand - specific primers matching the klf4-pr1 , myc - pr1 , and myc - pr2 sarna sequences were used for 5 race to amplify potential antisense rnas that are targeted by each sarna and may be involved in regulation of expression . race reactions were run on an agarose gel , followed by purification , cloning , and sequencing of any products ( see supplementary materials and methods ) . despite the presence of bands from each sarna primer , each was identified to be a known mrna with homology to the 3 end of the sarna sequence , likely a result of mispriming at permissive annealing temperatures ( supplementary figure s5 ) . repeats of this assay with more restrictive annealing temperatures yielded no products ( data not shown ) . to further rule out involvement of possible antisense rnas arising downstream of each sarna target sequence , cdna was synthesized from total msc rna using antisense strand - specific primers for klf4-pr1 , myc - pr1 , and myc - pr2 . this cdna was used as template for pcrs using primers downstream of the sarna target sequence , which would be in the direction of the 5 end of any putative antisense transcript . no antisense transcripts were amplified up to 525 bp from the klf4-pr1 target site ( supplementary figure s6a ) . of the priming sites up to 667 bp from myc - pr1 , including and surpassing the myc - pr2 target site , two products were amplified , cloned , and sequenced corresponding to the 63 and 136 bp downstream of myc - pr1 ( supplementary figure s6b ) . the expression level of this antisense transcript was significantly upregulated with myc - pr2 treatment , whereas its upregulation with myc - pr1 was not statistically significant ( p = 0.0682 ) ( supplementary figure s6c ) . to assess the biological significance of sarna activation of klf4 and myc in mscs , cells were monitored for morphological changes during oligo treatment with klf4-pr1 and myc - pr2 , which had been confirmed to be associated with increased nascent mrna transcribed from their respective target genes . after 6 days of treatment , klf4-pr1treated mscs showed marked differences in cell morphology compared with control , whereas myc - pr2 had modest changes ( figure 4 ) . in contrast to scrambled sequence oligo - treated controls , which appeared as progressively more densely packed fibroblastic cells over time , klf4-pr1treated mscs were less confluent and predominantly arranged in clusters with epitheloid cell - like morphology . myc - pr2treated cells were less confluent than controls but appeared more heterogeneous with numerous epitheloid cells containing enlarged nuclei . thus , target gene mrna levels after treatment with klf - pr1 and myc - pr2 , were significantly higher when mscs were exposed to these sarna oligos at concentrations of 25 or 50 nmol / l over a 6-day treatment period . hence , these promoter - targeted sarna oligos consistently induced increases in both mrna and protein expression of klf4 and c - myc , respectively , in a time- and dose - dependent manner . neither of these sarnas activated the interferon response , and both appear to act through specific activation of transcription . we therefore focused on these two positive sarnas , using the 6-day treatment protocol , in further experiments examining how sarna - mediated upregulation might affect downstream targets . microarray analysis was performed to determine the global gene expression profile and to investigate possible off - target effects with sarna treatment . differential gene expression profiles after upregulation of endogenous klf4 and c - myc by treatment with their respective sarnas versus control oligo were examined , and compared with that after overexpression of exogenous klf4 and c - myc delivered by viral gene transfer using commercially available second - generation lentivirus vectors . mscs were transduced with 0.1 pg p24 protein per cell as determined by p24 elisa , a viral dose that does not result in significant induction of interferon response genes ( supplementary figure s4 ) . only those cellular genes showing upward or downward changes in their expression levels at a significance level of at least p < 0.1 , as compared with their levels in scrambled sequence oligo - treated controls , were included in these analyses . interestingly , analysis of the overall gene expression profile in human mscs after treatment with klf4 pr-1 ( figure 5a ) and myc - pr2 ( figure 5b ) showed that the majority of cellular genes exhibited concordant changes in expression , but with some notable differences , between upregulation by sarna and overexpression by lentiviral transduction . for mscs treated with klf4-pr1 sarna , 68% of the cellular genes showing significant changes in their expression levels ( 971 out of 1,429 genes ) showed the same pattern of regulation as klf4 lentivirus - transduced mscs . for mscs treated with myc - pr2 , 64% ( 273 out of 429 genes ) exhibited the same pattern of regulation . however , this indicates that roughly a one - third of the cellular genes with significant changes in expression after sarna transfection or lentiviral gene transfer showed discordant regulation between these two groups . to determine whether these differences were due to off - target effects or resulted from the different methods used to activate expression , we used metacore pathway analysis from genego ( carlsbad , ca ) to determine what pathways were significantly enriched in those genes that were differentially regulated between sarna and virus samples . notably , the pathways that were most significantly differentially regulated between klf4-pr1 sarna - treated mscs and klf4 virus - treated mscs were those involved in cytoskeletal remodeling , macropinocytosis , and regulation of epithelial - to - mesenchymal transition ( emt ) , including transforming growth factor ( tgf)- induction of emt ( table 1 ) . the pathways that were most significantly differentially regulated between myc - pr2 sarna - treated mscs and the c - myc virus - treated mscs included cell survival and proliferation pathways such as granzyme a signaling , tgf- regulation , and telomere length , as well as macropinocytosis and emt pathways ( table 2 ) . that is , for klf4-targeted sarna versus klf4-virus , as well as myc - targeted sarna versus myc - virus , in both cases genes involved in the same pathways were found to be discordantly regulated in the same manner . the high degree of similarity in differentially regulated pathways observed with two different sets of sarnas versus two different lentivirus vectors suggests that the discordant gene expression patterns primarily arise due to characteristic cellular changes in response to oligo transfection versus viral transduction , rather than off - target effects that are shared by each set of sarnas . to determine whether narrowing the focus of the differential expression analysis to those types of genes we expect to be regulated by klf4 and c - myc would yield greater similarity , we generated lists of genes involved in stem cell maintenance , development , and proliferation , as well as cell cycle - related genes from the amigo gene ontology database . lists of genes used in this analysis are included in supplementary tables s2 and s3 . heatmaps visualizing the differential expression profiles of klf4 sarna - treated and klf4 virus - treated msc compared with scrambled oligo - treated control msc for stem cell - related genes ( figure 5c ) and cell cycle - related genes ( figure 5d ) showed much greater similarity , with 74% ( 26 out of 35 ) exhibiting concordant regulation for stem cell - related genes , and 80% ( 132 out of 165 ) exhibiting concordant regulation for cell cycle - related genes . heatmaps focused on cell cycle - related genes in myc - pr2 ( figure 5e ) sarna - treated msc compared to myc virus - treated msc also showed a higher degree of similarity , with 67% of cell cycle - related genes ( 8 out of 12 ) for myc - pr2 exhibiting concordant regulation . further david gene ontology ( go ) analysis of all genes revealed that the most significantly enriched go terms in the klf4-pr1 sarna - treated samples were the same as those in the klf4 virus - treated samples ( table 3 ) . similarly , the majority of the most significantly enriched go terms in the myc - pr2 sarna - treated samples were also significantly enriched in the c - myc virus - treated samples ( table 4 ) . to further validate the gene expression profile , we chose several well - known transcriptional gene targets of klf4 and c - myc to verify the results seen in the microarray data by real - time pcr . we found that klf4-pr1 sarna transfection resulted in significantly increased expression of the klf4 target genes cyclin d1 , ornithine decarboxylase ( odc1 ) , p21 , and p53 ( figure 6a ) ; klf4 virus - transduced cells also showed significantly increased expression of cyclin d1 and p21 to a similar degree , although not of odc1 or p53 . similarly , myc - pr2 sarna transfection resulted in significantly increased expression of c - myc target genes odc1 , p21 , and p53 , as did transduction with c - myc virus ( figure 6b ) . finally , to assess the ability of the klf4-pr1 sarna to activate the expression of other reprogramming factors , we analyzed mrna expression of oct4 , sox2 , and myc , as well as the stem cell marker nanog , by real - time pcr . we found that klf4 activation by sarna was also able to activate expression of oct4 , sox2 , myc , and nanog ( figure 6c ) . further analysis of oct4 mrna isoforms showed that oct4a was being significantly upregulated , whereas oct4b showed no difference ( figure 6d ) . conversely , the ability of myc - pr2 sarna to activate endogenous klf4 gene expression was also analyzed . in response to myc - pr2 transfection , there was an approximately twofold increase in klf4 mrna levels ( figure 6e ) . notably , this effect of myc - pr2 sarna was corroborated by the microarray analysis ( supplementary table s1 ) . to design sarna candidates for activation of stem cell factors , we developed a novel bioinformatic approach . the klf4 gene , located on chromosome 9 ( 9q31.2 ) , and c - myc gene , located on chromosome 8 ( 8q24.21 ) , were our initial targets for activation ( figure 1a , b ) . to identify potential antisense transcripts from the klf4 and c - myc loci , we searched the genomic region surrounding each locus for spliced expressed sequence tags ( ests ) which mapped to the positive strand . although it is usually difficult to determine the transcriptional orientation of ests , orientation can be determined by using splice site signatures of spliced ests . we found no spliced ests that overlapped klf4 , but the scan identified one antisense est ( db461753 ) ~15 kb upstream of klf4 's annotated transcription start site ( tss ) . we were also unable to find a spliced ests that overlapped c - myc , but identified one antisense est ( bc042052 ) ~2 kb upstream of c - myc 's tss . recent deep sequencing experiments have revealed that antisense rnas often are found in the region surrounding tsss . therefore , this region was chosen to design sarna sequences that targeted potential antisense transcripts from the promoter region . we used the antisense sequence 500 nts upstream and downstream from the tss ( abbreviated klf4_as_tss+/500 and myc_as_tss+/500 ) as a second target candidate . our goal was to design short sense rnas that could potentially bind and degrade antisense rnas generated from the two candidate sequences ( ests and the regions surrounding tsss ) with the hypothesis that this binding and degradation of the antisense rnas would activate gene expression . candidate sarnas targeting the 500 nt upstream of the tss were designated pr1 , whereas those targeting the 500 nt downstream of the tss were designated pr2 . to give effective antisense targeting and degradation and to minimize off - target effects , we used the gpboost sirna design algorithm to identify potential short rnas for downregulating the two candidate sequences . from the lists of predicted sirna candidates , we selected the two most promising non - overlapping sirna target sites on the antisense est db461753 and bc042052 , and the most promising sirna target site on each side of the klf4 and c - myc tss within the antisense promoter sequence ( klf4_as_tss+/500 and myc_as_tss+/500 ) . reasoning that activation of pluripotency factor gene expression might be more readily achieved in adult tissue - derived stem cells that retain restricted multilineage potential , we then tested whether these sarna candidates could upregulate klf4 or c - myc expression , respectively , in primary human mscs derived from adult bone marrow . target gene expression levels were examined by quantitative pcr of reverse - transcribed mrna from msc cultures after transfection of each individual sarna candidate oligonucleotide , as compared with transfection with an alexa fluor 555- or fam - labeled negative control oligo . a transfection efficiency of > 95% was determined by flow cytometry and by knockdown with control sirnas using the same transfection conditions ( see supplementary materials and methods and supplementary figures s1 and s2 ) . initially , none of the sarna oligos appeared to show any effect on target gene expression after 48 hours . however , upon continued transfection with klf4-pr1 sarna every other day , significant upregulation of klf4 mrna ( figure 2a ) was observed , on the order of 2.5-fold over controls by day 4 , and reaching approximately fourfold by day 6 of treatment ( p < 0.01 ) . target gene mrna levels after treatment with klf4-pr1 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50 nmol / l , as compared with 5 nmol / l ( figure 2b ) . increased klf4 protein was confirmed in mscs treated with klf4-pr1 sarna ( figure 2c , left panel ) by western blot analysis , and densitometric quantitation of these blots showed over threefold upregulation of klf4 protein relative to -actin internal control ( figure 2c , right panel ) , correlating closely with the level of mrna upregulation . this time - dependent upregulation of klf4 gene expression by the klf4 promoter - targeted pr1 sarna was observed consistently over multiple experiments . none of the other three klf4-targeted sarna candidate sequences was able to upregulate target gene expression , although conversely , klf4-db1 appeared to reduce klf4 mrna levels in mscs to about 60% that of scrambled oligo - treated controls by day 6 . similarly , we found that among the four sarna candidate sequences targeted to the c - myc promoter and antisense ests , both myc - pr1 and myc - pr2 were able to induce consistent upregulation of myc mrna ( figure 2d ) by up to 1.8-fold ( p < 0.01 ) and 1.6-fold ( p < 0.01 ) , respectively , during the 6-day interval of treatment , as compared with scrambled sequence controls . while myc - bc1 and myc - bc2 also appeared to affect myc mrna levels to some extent , these effects were more modest , and were not consistent between days 4 and 6 . again , target gene mrna levels after treatment with myc - pr1 and myc - pr2 were significantly higher when mscs were exposed to sarna at concentrations of 25 or 50 nmol / l , as compared with 5 nmol / l ( figure 2e ) . image densitometry analysis of western blots from mscs treated with myc - pr1 or myc - pr2 again confirmed upregulation of c - myc protein by over 2.5-fold relative to -actin internal control ( figure 2f ) . all western blots were performed in triplicate with replicates shown in supplementary figure s3 . to determine whether this upregulation of klf4 and myc is due to true transcriptional activation , expression levels of nascent rna were assessed . mscs were pulsed with ethynyl uridine ( eu ) during sarna treatment and total rna was isolated . newly transcribed eu - rna was separated from total rna by biotinylation of eu in a copper - catalyzed click reaction , followed by purification on streptavidin magnetic beads . quantitative pcr of reverse - transcribed nascent rna showed that the level of nascent klf4 mrna was significantly upregulated with klf4-pr1 treatment across a 6-day timecourse ( figure 3a ) . the level of nascent myc mrna was significantly upregulated with myc - pr2 treatment , whereas no significant change in nascent myc mrna expression was seen with myc - pr1 treatment ( figure 3b ) . to verify that these sarnas do not act through interferon response pathways , no significant upregulation of interferon response genes was detected 24 hours after sarna treatment ( figure 3c ) . in contrast , overexpression of klf4 and c - myc by lentiviral transduction showed dose - dependent induction of interferon response genes ( supplementary figure s4 ) . to further elucidate the mechanism of activation by sarna , the presence of promoter - associated antisense rnas was investigated using 5 rapid amplification of cdna ends ( race ) . since any antisense rnas involved in regulating gene expression may not be polyadenylated , random hexamers were used to prime cdna synthesis from total msc rna . antisense strand - specific primers matching the klf4-pr1 , myc - pr1 , and myc - pr2 sarna sequences were used for 5 race to amplify potential antisense rnas that are targeted by each sarna and may be involved in regulation of expression . race reactions were run on an agarose gel , followed by purification , cloning , and sequencing of any products ( see supplementary materials and methods ) . despite the presence of bands from each sarna primer , each was identified to be a known mrna with homology to the 3 end of the sarna sequence , likely a result of mispriming at permissive annealing temperatures ( supplementary figure s5 ) . repeats of this assay with more restrictive annealing temperatures yielded no products ( data not shown ) . to further rule out involvement of possible antisense rnas arising downstream of each sarna target sequence , cdna was synthesized from total msc rna using antisense strand - specific primers for klf4-pr1 , myc - pr1 , and myc - pr2 . this cdna was used as template for pcrs using primers downstream of the sarna target sequence , which would be in the direction of the 5 end of any putative antisense transcript . no antisense transcripts were amplified up to 525 bp from the klf4-pr1 target site ( supplementary figure s6a ) . of the priming sites up to 667 bp from myc - pr1 , including and surpassing the myc - pr2 target site , two products were amplified , cloned , and sequenced corresponding to the 63 and 136 bp downstream of myc - pr1 ( supplementary figure s6b ) . the expression level of this antisense transcript was significantly upregulated with myc - pr2 treatment , whereas its upregulation with myc - pr1 was not statistically significant ( p = 0.0682 ) ( supplementary figure s6c ) . to assess the biological significance of sarna activation of klf4 and myc in mscs , cells were monitored for morphological changes during oligo treatment with klf4-pr1 and myc - pr2 , which had been confirmed to be associated with increased nascent mrna transcribed from their respective target genes . after 6 days of treatment , klf4-pr1treated mscs showed marked differences in cell morphology compared with control , whereas myc - pr2 had modest changes ( figure 4 ) . in contrast to scrambled sequence oligo - treated controls , which appeared as progressively more densely packed fibroblastic cells over time , klf4-pr1treated mscs were less confluent and predominantly arranged in clusters with epitheloid cell - like morphology . myc - pr2treated cells were less confluent than controls but appeared more heterogeneous with numerous epitheloid cells containing enlarged nuclei . thus , target gene mrna levels after treatment with klf - pr1 and myc - pr2 , were significantly higher when mscs were exposed to these sarna oligos at concentrations of 25 or 50 nmol / l over a 6-day treatment period . hence , these promoter - targeted sarna oligos consistently induced increases in both mrna and protein expression of klf4 and c - myc , respectively , in a time- and dose - dependent manner . neither of these sarnas activated the interferon response , and both appear to act through specific activation of transcription . we therefore focused on these two positive sarnas , using the 6-day treatment protocol , in further experiments examining how sarna - mediated upregulation might affect downstream targets . microarray analysis was performed to determine the global gene expression profile and to investigate possible off - target effects with sarna treatment . differential gene expression profiles after upregulation of endogenous klf4 and c - myc by treatment with their respective sarnas versus control oligo were examined , and compared with that after overexpression of exogenous klf4 and c - myc delivered by viral gene transfer using commercially available second - generation lentivirus vectors . mscs were transduced with 0.1 pg p24 protein per cell as determined by p24 elisa , a viral dose that does not result in significant induction of interferon response genes ( supplementary figure s4 ) . only those cellular genes showing upward or downward changes in their expression levels at a significance level of at least p < 0.1 , as compared with their levels in scrambled sequence oligo - treated controls , were included in these analyses . interestingly , analysis of the overall gene expression profile in human mscs after treatment with klf4 pr-1 ( figure 5a ) and myc - pr2 ( figure 5b ) showed that the majority of cellular genes exhibited concordant changes in expression , but with some notable differences , between upregulation by sarna and overexpression by lentiviral transduction . for mscs treated with klf4-pr1 sarna , 68% of the cellular genes showing significant changes in their expression levels ( 971 out of 1,429 genes ) showed the same pattern of regulation as klf4 lentivirus - transduced mscs . for mscs treated with myc - pr2 , 64% ( 273 out of 429 genes ) exhibited the same pattern of regulation . however , this indicates that roughly a one - third of the cellular genes with significant changes in expression after sarna transfection or lentiviral gene transfer showed discordant regulation between these two groups . to determine whether these differences were due to off - target effects or resulted from the different methods used to activate expression , we used metacore pathway analysis from genego ( carlsbad , ca ) to determine what pathways were significantly enriched in those genes that were differentially regulated between sarna and virus samples . notably , the pathways that were most significantly differentially regulated between klf4-pr1 sarna - treated mscs and klf4 virus - treated mscs were those involved in cytoskeletal remodeling , macropinocytosis , and regulation of epithelial - to - mesenchymal transition ( emt ) , including transforming growth factor ( tgf)- induction of emt ( table 1 ) . the pathways that were most significantly differentially regulated between myc - pr2 sarna - treated mscs and the c - myc virus - treated mscs included cell survival and proliferation pathways such as granzyme a signaling , tgf- regulation , and telomere length , as well as macropinocytosis and emt pathways ( table 2 ) . that is , for klf4-targeted sarna versus klf4-virus , as well as myc - targeted sarna versus myc - virus , in both cases genes involved in the same pathways were found to be discordantly regulated in the same manner . the high degree of similarity in differentially regulated pathways observed with two different sets of sarnas versus two different lentivirus vectors suggests that the discordant gene expression patterns primarily arise due to characteristic cellular changes in response to oligo transfection versus viral transduction , rather than off - target effects that are shared by each set of sarnas . to determine whether narrowing the focus of the differential expression analysis to those types of genes we expect to be regulated by klf4 and c - myc would yield greater similarity , we generated lists of genes involved in stem cell maintenance , development , and proliferation , as well as cell cycle - related genes from the amigo gene ontology database . lists of genes used in this analysis are included in supplementary tables s2 and s3 . heatmaps visualizing the differential expression profiles of klf4 sarna - treated and klf4 virus - treated msc compared with scrambled oligo - treated control msc for stem cell - related genes ( figure 5c ) and cell cycle - related genes ( figure 5d ) showed much greater similarity , with 74% ( 26 out of 35 ) exhibiting concordant regulation for stem cell - related genes , and 80% ( 132 out of 165 ) exhibiting concordant regulation for cell cycle - related genes . heatmaps focused on cell cycle - related genes in myc - pr2 ( figure 5e ) sarna - treated msc compared to myc virus - treated msc also showed a higher degree of similarity , with 67% of cell cycle - related genes ( 8 out of 12 ) for myc - pr2 exhibiting concordant regulation . further david gene ontology ( go ) analysis of all genes revealed that the most significantly enriched go terms in the klf4-pr1 sarna - treated samples were the same as those in the klf4 virus - treated samples ( table 3 ) . similarly , the majority of the most significantly enriched go terms in the myc - pr2 sarna - treated samples were also significantly enriched in the c - myc virus - treated samples ( table 4 ) . to further validate the gene expression profile , we chose several well - known transcriptional gene targets of klf4 and c - myc to verify the results seen in the microarray data by real - time pcr . we found that klf4-pr1 sarna transfection resulted in significantly increased expression of the klf4 target genes cyclin d1 , ornithine decarboxylase ( odc1 ) , p21 , and p53 ( figure 6a ) ; klf4 virus - transduced cells also showed significantly increased expression of cyclin d1 and p21 to a similar degree , although not of odc1 or p53 . similarly , myc - pr2 sarna transfection resulted in significantly increased expression of c - myc target genes odc1 , p21 , and p53 , as did transduction with c - myc virus ( figure 6b ) . finally , to assess the ability of the klf4-pr1 sarna to activate the expression of other reprogramming factors , we analyzed mrna expression of oct4 , sox2 , and myc , as well as the stem cell marker nanog , by real - time pcr . we found that klf4 activation by sarna was also able to activate expression of oct4 , sox2 , myc , and nanog ( figure 6c ) . further analysis of oct4 mrna isoforms showed that oct4a was being significantly upregulated , whereas oct4b showed no difference ( figure 6d ) . conversely , the ability of myc - pr2 sarna to activate endogenous klf4 gene expression was also analyzed . in response to myc - pr2 transfection , there was an approximately twofold increase in klf4 mrna levels ( figure 6e ) . notably , this effect of myc - pr2 sarna was corroborated by the microarray analysis ( supplementary table s1 ) . we have been able to identify and characterize two sarnas , klf4-pr1 and myc - pr2 , that specifically activate transcription of endogenous klf4 and myc genes , respectively , in primary human mscs . interestingly , in both cases , it was a promoter - targeted sarna that gave the desired effect . given that studies implicate antisense rnas that overlap the gene of interest as the targets for degradation in rnaa , it is conceivable that these promoter - targeted sarnas are targeting an antisense rna that has not yet been discovered . however , we were unable to identify any antisense transcripts arising from the region of the klf4-pr1 target site . antisense transcripts have been reported to arise from the vicinity of the c - myc promoter in human cells such as prostate cancer cell lines , and we were able to identify one such antisense rna in primary human mscs . however , this antisense rna does not overlap the myc - pr2 target site and is therefore unlikely to be the rna target of myc - pr2 . interestingly , this antisense rna does overlap the myc - pr1 target site , but cells transfected with myc - pr1 sarna did not show increased transcription of nascent myc mrna . hence it is difficult to ascertain whether myc - pr1 does target this antisense rna , perhaps resulting in increased accumulation of myc sense mrna at a post - transcriptional level , or if the observed upregulation of myc mrna levels by myc - pr1 is an off - target effect . due to the relatively small activation of myc , it would be difficult to completely rule out off - target effects even with the use of more sophisticated techniques such as chromatin immunoprecipitation analysis . furthermore , specific sarna - targeted effects on myc expression by transcriptional or post - transcriptional mechanisms , and coexisting off - target effects , are not necessarily mutually exclusive . although it may not be possible for every gene to be activated by rnaa , our results suggest that this method could be used to generate sarna candidates for activation of other endogenous genes for which a promoter - associated antisense rna has not yet been defined . focusing on stem cell- and cell cycle - related genes , which were expected to be altered by klf4 and c - myc expression , we found the similarities between sarna - transfected and virus - transduced samples to be quite striking . this was confirmed by go analysis , as the most significantly enriched terms were nearly identical in klf4-pr1 oligo and klf4 virus samples , and most of the top myc - pr2 terms were also significantly enriched in the c - myc virus samples . in addition , several well - known klf4 and c - myc target genes were validated by real - time pcr , and showed a similar pattern of expression in the sarna - treated samples as in the virus - transduced samples . furthermore , we have shown that klf4 sarna is able to activate expression of endogenous pluripotency factors including oct4 , sox2 , nanog , and myc . activation of oct4 was specific to oct4a , which has been reported to be essential for stemness in human embryonic stem cells . the highly specific nature of oct4a upregulation in response to transfection of sarna targeting klf4 makes this likely to be a true downstream event caused by specific activation of klf4 . furthermore , the sarna - treated mscs showed marked differences in cell morphology , supporting our conclusion that this activation is biologically relevant . interestingly , for some downstream gene targets , our results were different from what was expected , as klf4 is expected to downregulate expression of cyclin d1 , ornithine decarboxylase , and p53 , whereas c - myc is expected to downregulate p21 . however , these previously observed results are often cell- and tissue type - dependent , as evidenced by more recent reports showing activation of cyclin d1 and p53 by klf4 , and activation of p21 through p53 by c - myc in different cell types . in addition , our data are consistent across both sarna- and virus - treated samples in independent microarray and real - time pcr experiments . as with rnai , a primary concern for using rnaa to study biological processes is the minimization of potential off - target effects . to evaluate possible off - target effects , we compared the gene expression profile of sarna activation of klf4 and myc to lentiviral - mediated expression . some significant differences were observed in the total gene expression profile , but this is perhaps not surprising , as one method employed transfection of sarna and the other lentiviral transduction . indeed , the use of any transduction method involving introduction of long stretches of exogenous nucleic acids , including both viral vectors as well as plasmids , will likely activate a variety of innate signaling mechanisms . this may result in unwanted upregulation of interferons and related genes that may not only cause protein synthesis shutdown and effects on cell proliferation , but may also impair normal stem cell function , as has been described in hematopoietic stem cells . notably , our interferon response gene expression analysis as well as our microarray analysis of gene functional annotation and differentially regulated pathways showed no evidence of interferon response upregulation after sarna oligo transfection . in contrast , we observed significant induction of interferon responses upon viral transduction at a dose of 1 pg p24 per cell . this corresponds roughly to an multiplicity of infection of 10 , which is within the range of multiplicity of infections typically used in reprogramming protocols . hence , it may be highly advantageous that rna duplexes < 23 bp in length will not cause induction of interferon , while effecting transcriptional gene activation of endogenous pluripotency factors . in fact , when we focused on the genes that were differentially expressed between the sarna and virus samples , the most significantly enriched pathways implicated the use of lentiviral vector - mediated gene transfer as the cause of many of the expression profile changes that were discrepant . notably , cytoskeletal remodeling , including tgf - mediated remodeling , were upregulated in both klf4 virus- and c - myc virus - treated samples . it is well known that retroviruses manipulate the host cytoskeleton to facilitate virus entry and integration . furthermore , the presence of tgf signaling pathways is also not surprising , considering that carryover of hiv tat can occur after packaging of second - generation lentivirus vectors , and that tat protein has been associated with the induction of tgf- , which likely serves multiple functions for the virus , including immunosuppression and facilitation of cytoskeletal remodeling . the differential regulation of macropinocytosis in both klf4 virus- and c - myc virus - treated samples is also likely due to viral entry , as both native hiv- and vesicular stomatitis virus - g ( vsv - g)pseudotyped hiv vectors have been reported to use macropinocytosis for entry . notably , several emt regulation pathways were observed to be differentially regulated between sarna- and virus - treated samples , irrespective of whether it was klf4 or c - myc being targeted or transduced . this was quite striking , as it has been reported that suppression of emt signals is required for reprogramming mouse fibroblasts . klf4 serves to activate the mesenchymal - to - epithelial transition ( met ) that is required for reprogramming , whereas oct4 , sox2 , and c - myc suppress tgf-induced emt . the differential regulation of these pathways in our experiments fits well into this model , as in both cases klf4 and c - myc are acting in competition with the tgf- induced by lentiviral infection . this suggests that using lentiviral vectors to activate reprogramming factors may actually hinder the reprogramming process , as these vectors have been shown to activate tgf- signaling . this underscores the need for alternative methods of gene activation in reprogramming . in recent years , invaluable information has been obtained from the use of rnai to study stem cell biology by inhibiting expression of specific genes involved in the regulation of pluripotency within embryonic as well as somatic stem cells . here , we have shown the potential of using sarnas that , conversely , upregulate expression of endogenous genes in stem cells . as each gene can be selectively targeted for activation , the use of sarnas may also provide a highly useful tool in studying the contribution of individual factors in ips cell reprogramming . several studies have used inducible systems to study the reprogramming process , but these have been limited by the inability to activate or repress the activity of each factor individually . since rnaa is a transient process , it may be possible to develop an optimized protocol for ips cell production wherein each factor can be activated when needed by the transfection of its specific sarna , and similarly removed when it is no longer necessary . in this context , current methods for upregulating klf4 and c - myc require transfection or viral transduction of klf4 or c - myc expression vectors into cells . as noted above , this study suggests that the tgf- induced by lentiviral vectors may actually be detrimental to reprogramming . further , oncogenic reactivation of stably integrated c - myc transgenes poses a serious safety issue to the use of ips cells . in addition , evidence that latent viral expression of reprogramming factors impairs normal differentiation of ips cells , and intolerance to genomic damage caused by exogenous dna or transposon integration further emphasizes the need for a method of ips cell generation that uses endogenous cellular processes and requires no foreign dna . in this regard , while reprogramming to full pluripotency has not , to date , been demonstrated with this method , other groups have recently shown sarna - mediated upregulation of endogenous oct4 in a breast cancer cell line , and endogenous klf4 in prostate cancer cell lines . notably , downstream gene expression and phenotypic changes induced by sarna - mediated upregulation of klf4 in prostate cancer cell lines were reported to be comparable to those obtained by retroviral vector transduction . this is consistent with our results obtained in primary human mscs , and suggests that the use of synthetic sarna oligos may prove highly advantageous as a safe and efficient alternative for upregulation of endogenous reprogramming genes . four parameters were used : ( i ) download target gene annotations ; ( ii ) identify antisense rna target sequences ; ( iii ) select promoter antisense sequences ; and ( iv ) identify candidate sarnas . first , the method downloads information about the target 's genomic location , orientation , and transcriptional structure from available databases such as the refseq database at ucsc ( university of california , santa cruz ) . second , given a database of rna transcripts with known read direction , such as the ucsc spliced est track , our method searches the database for transcripts that are antisense to and in the vicinity of the target gene . more specifically , the method identifies antisense transcripts that ( i ) overlap the target 's promoter and the target mrna 's 5 end ; ( ii ) overlap the target mrna ; ( iii ) are at most 20100 kb upstream of the target 's tss ; or ( iv ) are at most 20100 kb downstream of the target 's polyadenylation site . the method uses these four criteria as hierarchical filters such that if it finds antisense transcripts that for example satisfy criterion ( i ) , the method does not consider the three other criteria . third , based on the target 's tss , the method downloads the antisense genomic sequence from a fixed size region upstream and downstream of the tss . the typical region size used by the method is 500 nts upstream and downstream of tss , but larger or smaller sizes can also be used . fourth , the method designs sirnas that give effective and specific downregulation of the antisense target sequence . the method ( i ) uses a sirna design algorithm , such as gpboost , to identify candidate effective sirnas ; ( ii ) removes all candidate sirnas with aaaa , cccc , gggg , or uuuu motifs and gc content < 20% or > 55% ; ( iii ) removes all candidates that have hamming distance < 2 to all potential off - target transcripts ; and ( iv ) returns a given number of remaining non - overlapping sirnas sorted by their predicted sirna knockdown efficacy . bone marrow - derived adult human mesenchymal stem cells ( lonza , basel , switzerland ) were cultured in the manufacturer 's media as instructed . the klf4 , myc , and control duplex rna oligonucleotides were transfected into mscs using lipofectamine rnaimax reagent ( invitrogen , carlsbad , ca ) following the manufacturer 's protocol with 30 pmol oligo to 1 l reagent in a 24-well plate to a final oligo concentration of 50 nmol / l . the block - it alexa fluor red fluorescent control ( invitrogen ) and silencer fam labeled negative control # 1 sirna ( applied biosystems , carlsbad , ca ) , which have no homology to any known gene , were used as negative controls and to assess transfection efficiency by fluorescence microscopy and flow cytometry . images were taken at 100 magnification on a nikon ts100 microscope ( nikon instruments , melville , ny ) . the plasmids psin - ef2-klf4-pur and psin - ef2-c - myc - pur were generated by cloning human klf4 and c - myc transgenes from plasmids pmxs - hklf4 or pmxs - hcmyc , respectively , into the psin - ef2-pur lentiviral vector backbone . vsv - g pseudotyped second - generation lentivirus preparations were produced using standard protocols ; briefly , packaging plasmids pmd2.g , pspax2 , and transfer vector were cotransfected into 293 t cells with jetprime reagent ( polyplus - transfection , new york , ny ) , and 48 hours later virus - containing supernatant was collected , filtered , and concentrated by ultracentrifugation . vector titers were determined by p24 elisa , performed by the ucla virology core . quantitative reverse transcription - pcr . total rna was isolated from mscs using the rneasy micro plus kit to remove gdna ( qiagen , valencia , ca ) . rna was reverse - transcribed to cdna using the high capacity cdna kit ( applied biosystems ) . for nascent rna analysis , experiments were performed using the click - it nascent rna capture kit ( invitrogen ) according to the manufacturer 's protocol with a 1 hour eu pulse before sample collection on each day of the experiment . quantitative real - time pcr was performed using taqman gene expression master mix ( applied biosystems ) on a myiq2 thermal cycler ( bio - rad , hercules , ca ) according to the manufacturer 's standard protocols . the taqman primer sets used were as follows : klf4 , hs00358836_m1 ; pou5f1 ( oct4a and oct4b isoform ) , hs00999632_g1 ; pou5f1 ( oct4a isoform ) , hs01895061_u1 ; pou5f1 ( oct4b isoform ) , hs00742896_s1 ; sox2 , hs00602736_s1 ; nanog , hs02387400_g1 ; myc , hs00153408_m1 ; ccnd1 , hs00277039_m1 ; cdkn1a , hs00355782_m1 ; odc1 , hs00159739_m1 ; tp53 , hs99999147_m1 ; actb , hs00357333_g1 ( applied biosystems ) . for interferon response gene expression , primers from the interferon response detection kit ( system biosciences , mountain view , ca ) were used for sybr green real - time pcr with ssofast evagreen supermix ( bio - rad ) . as suggested by the manufacturer 's protocol , samples were collected for expression analysis 24 hours after sarna transfection or viral transduction . expression of -actin mrna was used as an internal control and samples were normalized to the scrambled sequence control oligonucleotide or untreated samples . statistical significance was determined by student 's t - test , with p values < 0.05 considered significant . cells were lysed and protein concentration was determined using coomassie plus assay reagent ( thermo scientific , waltham , ma ) . each sample was loaded onto a nupage bis - tris gel ( invitrogen ) at 30 g / well and electrophoresed and transferred according to the manufacturer 's specifications . primary antibodies used were gklf ( sc-20691 ; santa cruz biotechnology , santa cruz , ca ) and c - myc ( sc-764 ; santa cruz biotechnology ) . -actin primary antibody ( ab8227 ; abcam , cambridge , ma ) was used as an internal control and for quantitation . protein was detected using anti - rabbit hrp conjugated secondary antibody ( haf008 ; r&d systems , minneapolis , mn ) , developed using immun - star westernc reagent ( bio - rad ) , and visualized on a chemidoc xrs+ ( bio - rad ) . statistical significance was determined by student 's t - test , with p values < 0.05 considered significant . rna was processed and hybridized to a genechip human gene 1.0 st array ( affymetrix , santa clara , ca ) in triplicate by the city of hope microarray core facility ( duarte , ca ) . samples were normalized using the exonrma16 summarization algorithm and filtered on expression percentile in the raw data ( 20100% ) . differential expression analysis compared to control samples was performed using an unpaired t - test with asymptotic p value computation and benjamini - hochberg multiple testing correction . heatmaps were generated using hierarchical clustering using centroid linkage and euclidean similarity measure . for pathway analysis , lists of genes differentially regulated between sarna and virus samples were used to generate significantly enriched pathways in metacore ( version 6.3 build 25177 by genego ) . for go analysis , lists of differentially expressed genes with the indicated adjusted p values and absolute fold changes were generated for the sarna- and virus - treated samples versus control . these lists were used to generate functional annotation charts using david bioinformatic analysis with the goterm_bp_fat category on a hugene-1_0-st - v1 background . transfection efficiency of positive control sirnas as evaluated by rt - qpcr for expression levels of targeted genes . rt - qpcr of klf4 and c - myc virus transduction in mscs for interferon response genes at the indicated p24 amount per cell . list of genes with stem cell - related gene ontology , used to generate figure 3c . table s3 . list of genes with cell cycle - related gene ontology , used to generate figure 3d f . transfection efficiency of positive control sirnas as evaluated by rt - qpcr for expression levels of targeted genes . rt - qpcr of klf4 and c - myc virus transduction in mscs for interferon response genes at the indicated p24 amount per cell . list of genes with stem cell - related gene ontology , used to generate figure 3c . list of genes with cell cycle - related gene ontology , used to generate figure 3d f .

it is now recognized that small noncoding rna sequences have the ability to mediate transcriptional activation of specific target genes in human cells . using bioinformatics analysis and functional screening , we screened short - activating rna ( sarna ) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors , kruppel - like factor 4 ( klf4 ) and c - myc . we identified klf4 and c - myc promoter - targeted sarna sequences that consistently induced increases in their respective levels of nascent mrna and protein expression in a time- and dose - dependent manner , as compared with scrambled sequence control oligonucleotides . the functional consequences of sarna - induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis , which revealed significant increases in mrna levels of their respective downstream pathway genes . notably , the microarray profile after sarna - mediated induction of endogenous klf4 and c - myc showed similar gene expression patterns for stem cell- and cell cycle - related genes as compared with lentiviral vector - mediated overexpression of exogenous klf4 and c - myc transgenes , while divergent gene expression patterns common to viral vector - mediated transgene delivery were also noted . the use of promoter - targeted sarnas for the activation of pluripotency reprogramming factors could have broad implications for stem cell research .

pubmed

chronic kidney diseases , which lead to end - stage kidney failure , are associated with changes in kidney structure and fibrosis regardless of the underlying cause . urinary tract obstruction is characterized by tubular atrophy or dilation , tubular cell death by apoptosis and necrosis , interstitial leukocyte infiltration , and increased interstitial matrix deposition . . such an obstruction might be observed after benign prostatic hyperplasia ; renal , ureteral , or bladder calculi ; urethral stricture ; and neoplasm of the bladder , prostate , or urethra . the hydrostatic pressure , which is the result of the blockage , initiates renal injuries . the injuries are characterized by tubular dilatation or atrophy , inflammatory infiltration of leucocytes , fibroblast activation , proliferation , increase in matrix proteins , and progressive interstitial fibrosis with the loss of renal parenchyma . unilateral ureteral obstruction ( uuo ) is an experimental rat model of renal injury that imitates the process of obstructive nephropathy in an accelerated manner . evidence supports a key role of the inflammatory process in the renal structural changes that occur after uropathy . taurine ( 2-aminoethanesulfonic acid ) is an organic acid and a major component of the bile . taurine has been shown to have beneficial effects on the renal fibrosis that occurs after cisplatin - induced injuries , diabetic nephropathy , and age - related renal changes . it also plays an important role in some biochemical and physiological processes in the kidney . in a review , chesney et al . described the renal - taurine interactions related to ion reabsorption , renal blood flow , renal vascular endothelial function , and antioxidant properties ( especially in the glomerulus ) . in addition , it has been reported that taurine plays a major role in the renal cell cycle as well as in apoptosis and also functions as an osmolyte during the stress response . taurine has been shown to play an important role in four different forms of kidney diseases : glomerulonephritis , diabetic nephropathy , chronic renal failure , and acute kidney injury . paclitaxel stabilizes microtubules and as a result interferes with the normal breakdown of the microtubules during cell division . sun et al . and zhang et al . have reported that paclitaxel ameliorates fibrosis in the uuo rat kidney model . in addition , sommardahl et al . showed the efficacy of taxol in the mouse model of polycystic kidney disease . in addition , zhou et al . reported the ameliorative effects of paclitaxel on liver fibrosis . in this study , we tried to determine whether taurine and taxol can attenuate the tubulointerstitial fibrosis and preserve the normal renal histology in a rat model of uuo . moreover , stereological survey was applied to investigate structural changes in the uropathic kidney , including the volume of the cortex , medulla , glomeruli , proximal convoluted tubules ( pct ) , distal convoluted tubules ( dct ) , henle tubules , collecting ducts , vessels , and fibrous tissue . the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences . the rats were randomly divided into four groups each including six animals , which is considered acceptable for stereological studies . in group one ( sham - operated animals ) , laparotomy only was done without any procedures and the rats did not receive any treatments . on the other hand , groups the third group received taxol ( 0.3 mg / kg twice per week intraperitoneally ) for 28 days . finally , the fourth group was treated with taurine ( 7.5 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 28 days . after the rats were anesthetized with ketamine / xylosine ( 25 mg / kg i.p . and 2.5 mg / kg i.p . , respectively ) , the abdominal cavity was opened by an incision to reach the left ureter . a 3 - 0 silk ligature was tied around the ureter , after which the incision was closed . after 28 days , the rats ' kidneys were removed . the primary volume of the kidney , vprimary , was measured by using sherle 's immersion method . then , the tissue shrinkage produced by fixation , processing , and staining was estimated . estimation of the shrinkage and the length of the renal tubules as well as the vessels requires isotropic , uniform random sections [ 15 - 17 ] . 2 . the estimated shrinkage was also used to estimate the final volume of the kidney to avoid the consecutive sectioning that is required for the cavalieri method . the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields then , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . the volume density of each structure ( the fraction of the unit volume of the kidney that is occupied by the structure ) was estimated by using the point - counting method . the volume fraction of the cortex and the medulla were estimated at a final magnification of 375 and other parameters at 1,500 . the length density ( the length of each tubular structure in the unit volume of the kidney ) of the proximal and the dct , collecting ducts , henle 's loop , and vessels was estimated by randomly overlaying an unbiased counting frame with an area of 2,700 m on the live monitor images . finally , the total volume of the parameters and the total length of the tubules as well as the vessels were estimated by multiplying the fractional volume or the length density by the final volume of the kidney to prevent the " reference trap " [ 15 - 17 ] . statistical comparisons between the group means were done by kruskall - wallis and mann - whitney u - test . moreover , p0.01 was considered as statistically significant . the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences . the rats were randomly divided into four groups each including six animals , which is considered acceptable for stereological studies . in group one ( sham - operated animals ) , laparotomy only was done without any procedures and the rats did not receive any treatments . on the other hand , groups the third group received taxol ( 0.3 mg / kg twice per week intraperitoneally ) for 28 days . finally , the fourth group was treated with taurine ( 7.5 mg / kg / d ) dissolved in a distilled water vehicle given by gavage for 28 days . after the rats were anesthetized with ketamine / xylosine ( 25 mg / kg i.p . and 2.5 mg / kg i.p . , respectively ) , the abdominal cavity was opened by an incision to reach the left ureter . a 3 - 0 silk ligature was tied around the ureter , after which the incision was closed . after 28 days , the rats ' kidneys were removed . the primary volume of the kidney , vprimary , was measured by using sherle 's immersion method . then , the tissue shrinkage produced by fixation , processing , and staining was estimated . estimation of the shrinkage and the length of the renal tubules as well as the vessels requires isotropic , uniform random sections [ 15 - 17 ] . 2 . the estimated shrinkage was also used to estimate the final volume of the kidney to avoid the consecutive sectioning that is required for the cavalieri method . 3 . after estimating the volume shrinkage [ 15 - 17 ] , the final volume of the kidney ( the reference space ) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage ) . each sampled section was analyzed by using a video microscopy system that was made up of a microscope ( e-200 , nikon , tokyo , japan ) linked to a video camera , a computer , and a monitor to determine the parameters . between 8 and 12 microscopic fields were selected in a systematic random manner . then , the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center , shiraz university of medical sciences , shiraz , iran . the volume density of each structure ( the fraction of the unit volume of the kidney that is occupied by the structure ) was estimated by using the point - counting method . the volume fraction of the cortex and the medulla were estimated at a final magnification of 375 and other parameters at 1,500 . the length density ( the length of each tubular structure in the unit volume of the kidney ) of the proximal and the dct , collecting ducts , henle 's loop , and vessels was estimated by randomly overlaying an unbiased counting frame with an area of 2,700 m on the live monitor images . finally , the total volume of the parameters and the total length of the tubules as well as the vessels were estimated by multiplying the fractional volume or the length density by the final volume of the kidney to prevent the " reference trap " [ 15 - 17 ] . statistical comparisons between the group means were done by kruskall - wallis and mann - whitney u - test . moreover , p0.01 was considered as statistically significant . the histological changes of the kidney after the uuo procedure as well as after the uuo procedure plus taxol or taurine treatment can be observed in fig . renal disruption , tubular atrophy , degeneration , necrosis , and fibrosis were observed in the uuo animals . after treatment with taxol or taurine , the mean and the standard deviation of the kidney volume , the volume of the cortex , and the volume of the medulla are presented in table 1 . the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , degenerative tubules , and necrotic and fibrotic tissue in the different groups is shown in table 2 . the volume of the glomeruli , pct , dct , henle 's loop , and collecting ducts was reduced in the rats after uuo . less than -8% of these structures remained intact on average . after treatment of the uuo rats with taxol , -87% , -32% , -51% , -72% , and -88% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . after treatment of the uuo rats with taurine , -25% , -16% , -16% , -40% , and -46% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . table 2 depicts the increase in degenerative tubules and necrotic and fibrotic tissues in the uuo groups . in addition , after treating the uuo animals with taxol , the volume of the degenerative tubules and necrotic and fibrotic tissues was decreased -42% and -53% , respectively , compared with that in the uuo rats . after treating the uuo rats with taurine , the volume of the degenerative tubules increased 1.8-fold , whereas necrotic and fibrotic tissues decreased -63% compared with that in the uuo rats without any treatments ( p<0.01 ) . on average , less than -3% of the lengths of these structures remained intact in the uuo rats . after treatment of the uuo rats with taxol , -65% , 70% , 56% , and 61% of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . on the other hand , after treatment of the uuo rats with taurine , -49% , -53% , -43% , and -45% , of the length of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) ( table 3 ) . the histological changes of the kidney after the uuo procedure as well as after the uuo procedure plus taxol or taurine treatment can be observed in fig . renal disruption , tubular atrophy , degeneration , necrosis , and fibrosis were observed in the uuo animals . after treatment with taxol or taurine , the mean and the standard deviation of the kidney volume , the volume of the cortex , and the volume of the medulla are presented in table 1 . the total volume of the glomeruli , pct , dct , henle 's loop , collecting duct , degenerative tubules , and necrotic and fibrotic tissue in the different groups is shown in table 2 . the volume of the glomeruli , pct , dct , henle 's loop , and collecting ducts was reduced in the rats after uuo . less than -8% of these structures remained intact on average . after treatment of the uuo rats with taxol , -87% , -32% , -51% , -72% , and -88% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . after treatment of the uuo rats with taurine , -25% , -16% , -16% , -40% , and -46% of the glomeruli , pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . table 2 depicts the increase in degenerative tubules and necrotic and fibrotic tissues in the uuo groups . in addition , after treating the uuo animals with taxol , the volume of the degenerative tubules and necrotic and fibrotic tissues was decreased -42% and -53% , respectively , compared with that in the uuo rats . after treating the uuo rats with taurine , the volume of the degenerative tubules increased 1.8-fold , whereas necrotic and fibrotic tissues decreased -63% compared with that in the uuo rats without any treatments ( p<0.01 ) . on average , less than -3% of the lengths of these structures remained intact in the uuo rats . after treatment of the uuo rats with taxol , -65% , 70% , 56% , and 61% of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) . on the other hand , after treatment of the uuo rats with taurine , -49% , -53% , -43% , and -45% , of the length of the pct , dct , henle 's loop , and collecting ducts remained intact , respectively ( p<0.01 ) ( table 3 ) . the present study confirmed through a quantitative survey the protective role of taxol and taurine on renal tissue damage after the induction of uuo in rats . furthermore , the findings of the present study revealed that taxol was more protective than taurine . paclitaxel is an alkaloid plant that is derived from the bark of the pacific yew tree . paclitaxel works by disrupting the microtubular network essential for cell division and other normal cellular functions , which eventually leads to cell death . the positive effects of taxol have been reported in cisplatin - induced and ischemia - reperfusion injuries . moreover , a study demonstrated that taxol can attenuate oxidative / nitrosative stress , glutathione depletion , enhanced urinary hydrogen peroxide excretion , and the decrease in antioxidant enzymes ( catalase , glutathione peroxidase , and glutathione - s - transferase ) . in addition , yoon et al . reported that taxol can induce cytoprotective enzymes and thereby improve the ischemia - reperfusion - induced changes in lipid hydroperoxides , glutathione , creatinine clearance , kidney weight , and histological abnormalities . the protective effect of taurine on nephrotoxicity after cyclosporine or cisplatin treatment has been reported . it is believed that taurine , owing to its antioxidant properties , avoids cyclosporine - induced reactive oxygen species and , consequently , nephrotoxicity . in a recent article by yousef et al . , it was shown that taurine induces a protective property after paracetamol - induced oxidative injury of the kidney . articles have discussed various mechanisms by which taxol and taurine may protect renal structure changes after uuo . for example , the results of a study by sun et al . revealed that a low dose of paclitaxel ameliorates renal fibrosis via the modulation of microrna-192 pathobiology and transforming growth factor- signaling . transforming growth factor- has been shown to play a central role in the development of the tubulointerstitial fibrosis . guerrero - beltran et al . reported that taxol can prevent cell death and inflammation in nephropathy . the findings of the study by zhou et al . indicate that 200 nmol / l paclitaxel ameliorates hepatic fibrosis by modulating transforming growth factor - beta signaling . in addition , koz et al . showed that paclitaxel provided antifibrotic effects during conjunctival wound healing . these major roles include regulating the blood flow in the vasculature of the kidney , scavenging the reactive oxygen species in glomerulus , na transport in the proximal tubules , and osmoregulation as well as cell volume regulation in the medulla . in addition to the preventive roles of taurine in renal fibrosis , other tissues can also be protected . reported that taurine reduces iron - potentiated alcoholic liver fibrosis by reducing oxidative stress , inflammatory and fibrogenic mediators , and stellate cell activation . in addition , the results of the study by devi and anuradha showed that the restorative effect of taurine in an experimental model of liver fibrosis involved amelioration of protein and lipid damage by decreasing oxidative and nitrosative stresses . therefore , a variety of mechanisms can be considered responsible for the protective effects of taxol and taurine in the present study . it can be concluded that taxol and taurine are both effective in preventing some structural renal damage in the direct obstruction model in rats . the volume of the necrotic and fibrotic tissue was found to be less in the taxol- and taurine - treated groups .

purposeblockage of the urinary tract induces changes in renal structure including tubular dilatation or atrophy , tubular cell death , inflammatory processes , and progressive interstitial fibrosis with the loss of renal parenchyma . the present study was conducted to survey the protective effects of taxol and taurine on the renal structure after unilateral ureteral obstruction ( uuo).materials and methodsuuo was induced in three groups of rats ( n=6 ) who then received distilled water , taxol ( 0.3 mg / kg / d ) , or taurine ( 7.5 mg / kg / d ) . stereological methods were used to gather quantitative as well as comparative data.resultsless than -8% of the volume of the glomeruli , proximal convoluted tubules ( pct ) , distal convoluted tubules ( dct ) , henle 's loop , and collecting ducts were preserved after uuo . after treatment of the uuo rats with taxol , between -32% and 88% of the parameters mentioned above remained intact , and after treatment of the uuo rats with taurine , between -16% and 46% of the parameters remained intact ( p<0.01 ) . compared with the untreated uuo animals , the volume of necrotic and fibrotic tissues decreased -53% and -63% in the uuo rats treated with taxol and taurine , respectively ( p<0.01 ) . less than -3% of the lengths of the renal tubules ( pct , dct , henle 's loop , and collecting ) were preserved in the uuo rats . after treatment with taxol and taurine , -61% to 70% and -43% to 53% of the length of the renal tubules were preserved , respectively ( p<0.01).conclusionstaurine and taxol are effective in preventing some structural renal damage in a direct ureteral obstruction model . taxol was more effective in renal protection .

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pathogen recognition receptors ( prrs ) trigger innate immune responses after sensing microbial components such as lipopolysaccharide ( lps ) , lipoprotein , flagellin , and nucleic acids and are necessary for the protection of a host suffering from microbial infection ( beutler , 2009 ; kawai and akira , 2009 ) . after detecting microbial components , prrs drive the coordinated activation of transcription factors , leading to the expression of inflammatory cytokines , chemokines , type i interferons ( ifns ) , and antimicrobial genes . although innate immunity is essential for host defense , aberrant activation of innate immune responses results in the development of inflammatory diseases ; e.g. , autoimmune disease and septic shock . thus , the signaling pathways of the innate immune system are tightly regulated to avoid either deficient or excessive responses . protein degradation is critically involved in the control of innate immune responses ( beutler , 2009 ; kawai and akira , 2009 ) . although the regulation of immune responses by the ubiquitin proteasome ( a selective protein degradation system ) has long been studied ( liu et al . , 2005 ; bhoj and chen , 2009 ) , recent research has shown the involvement of another clearance system , autophagy , in the innate immune response . autophagy is a bulk degradation system that delivers cytoplasmic constituents into lysosomes ( fig . 1 ; this process enables the reuse of intracellular constituents and supplies an amino acid pool during periods of starvation . indeed , mice deficient in atg3 , atg5 , or atg7 die within 1 d of delivery , indicating that autophagy is essential for survival during neonatal starvation ( kuma et al . , 2004 ; komatsu et al . autophagy is also involved in the clearance of old / damaged organelles , long - lived proteins , insoluble protein aggregates , and lipid droplets , thus regulating cellular homeostasis , cell death / survival , and lipid metabolism ( yoshimori and noda , 2008 ; mizushima , 2009 ; czaja , 2010 ) . several essential components of the autophagic machinery have been identified by yeast genetic screening , the so - called autophagy - related proteins ( atgs ; he and klionsky , 2009 ; nakatogawa et al . , 2009 ) . recent studies have identified mammalian counterparts of yeast atgs , such as ulk1 ( atg1 ) , atg35 , beclin ( atg6 ) , atg7 , lc3 ( atg8 ) , atg9a , atg10 , atg12 , atg13l , atg14l , atg16l1 , fip200 ( atg17 ) , and wipi-1 ( atg18 ) , indicating that atgs are phylogenetically highly conserved between yeast and mammals ( mizushima et al . , 2003 ; proikas - cezanne et al . , 2004 ; yamada et al . , 2005 ; young et al . , 2006 ; hara et al . , 2008 ; itakura et al . , ; chang et al . , 2009 ; ganley et al . , 2009 ; hosokawa et al . , 2009 ; jung et al . , 2009 ; matsunaga et al . , 2009 ; the coordinated action of atgs mediates the membrane trafficking required for autophagosome formation ( fig . autophagy , a bulk degradation system , is induced after sensing various types of stress , such as nutrient starvation , the accumulation of protein aggregates , and invasion by microbes . this system enables the reuse of cellular components and the clearance of unfavorable substances , thus contributing to the maintenance of cellular homeostasis and the prevention of disease . atgs are recruited to the isolation membrane , a source membrane of autophagosomes , and play a vital role in driving the membrane trafficking necessary for the generation of autophagosomes . autophagosomes then fuse with lysosomes to become autolysosomes , leading to the degradation of the constituents trapped inside . autophagy plays a critical role in host defense responses by promoting the elimination of pathogens and the induction of acquired immunity ( deretic , 2005 ; deretic and levine , 2009 ; virgin and levine , 2009 ) . the rab7-dependent formation of bacteria - containing autophagosomes is critical for the killing of invading bacteria , such as streptococcus pyogenes and coxiella burnetii , in nonphagocytic cells ( bern et al . , 2002 ; , 2004 ; romano et al . , 2007 ; yamaguchi et al . , 2009 ) . bacteria , such as shigella flexneri , escape from the autophagic machinery via expression of the virg protein , which is capable of inhibiting atg5 function in nonphagocytic cells ( ogawa et al . , 2005 ) . immunity - related gtpase family m , an ifn-inducible gtpase , promotes autophagy , and the autophagic machinery effectively eliminates mycobacteria in macrophages ( gutierrez et al . , 2004 ; singh et al . , 2006 ) . sequestosome 1 ( sqstm1)/p62 , which interacts with both lc3 and ubiquitinated proteins , is recruited to bacteria - containing ubiquitinated vacuoles upon infection and promotes the killing of invading pathogens by autophagy ( bjrky et al . , 2005 ; komatsu et al . , 2007 ; yoshikawa et al . , 2009 ; ponpuak et al . , in addition to the formation of autophagosomes , atgs are responsible for the transport of irga6 , an immunity - related gtpase , from the er golgi to the vacuoles containing invading microbes , which is a function required for the elimination of toxoplasma gondii by macrophages ( zhao et al . , 2008 ) . these findings clearly demonstrate that atgs are critical for the direct elimination of infectious agents . autophagy also plays an indispensable role in antigen presentation to antigen - specific t cells ( a process essential for the induction of acquired immunity ; gannag and mnz , 2009 ) . major histocompatibility complex ( mhc ) class ii molecules are localized on autophagosomes , and the autophagic machinery promotes the presentation of viral and self - antigens by mhc class ii molecules to antigen - specific cd4 t cells ( nimmerjahn et al . , 2003 ; dengjel et al . , 2005 ; paludan et al . , 2005 ; zhou et al . autophagy controls the mhc class i dependent presentation of viral antigens to cd8 t cells ( english et al . , 2009 ) . in addition to these functions , recent studies reveal that atgs are important for innate immune responses provoked by the engagement of prrs with microbial components . in this review recent genome - wide association studies have identified atg16l1 as a candidate gene responsible for susceptibility to the inflammatory bowel disease , crohn s disease ( hampe et al . atg16l1 forms an 800-kd protein complex with atg12-atg5 conjugates , and the self - multimerization of atg16l1 via its coiled - coil domain is required for the recruitment of this conjugate into an isolation membrane ( a source membrane of autophagosomes ; fig . 1 ; mizushima et al . , 2003 ; fujita et al . , . the atg12-atg5atg16l1 complex recruits an atg3-lc3 intermediate to the isolation membrane , and in doing so , defines the site at which lc3 is conjugated to phosphatidylethanolamine ( fujita et al . , 2008 ) consistently , cells lacking atg16l1 are deficient in the conjugation of lc3 to phosphatidylethanolamine , an essential process for the elongation of autophagosomes ( saitoh et al . , 2008 ) . this phenotype is similar to that observed in other atg - deficient mice ( kuma et al . , 2004 ; komatsu et al . , 2005 ) thus , atg16l1 is an essential component of autophagy under both nutrient - rich and nutrient - starved conditions . using atg16l1-deficient mice , we have shown that the loss of atg16l1 enhances endotoxin - induced inflammatory immune responses ( fig . commensal bacteria are thought to be one of the major causative agents of bowel disease . when intestinal epithelial cells are damaged , commensal bacteria are able to pass through the epithelial layer and stimulate prrs , leading to the induction of intestinal inflammation . toll - like receptors ( tlrs ) , one family of prrs , detect microbial components and induce the production of inflammatory cytokines such as tnf- , il-1 , il-6 , il-12 , and il-18 by macrophages / dendritic cells ( dcs ) , resulting in the infiltration of activated lymphocytes into the intestine ( beutler , 2009 ; kawai and akira , 2009 ) . macrophages lacking atg16l1 produce high amounts of il-1 and il-18 in response to lps , a ligand for tlr4 . macrophages from atg7-deficient mice also show enhanced production of il-1 , indicating the importance of atg in the regulation of the inflammatory response . stimulation of the other tlr family members ( except for tlr3 ) fails to induce this enhanced production of il-1 by atg16l1-deficient macrophages . trif ( toll / il-1 receptor domain containing adaptor inducing ifn- ) , the adaptor protein involved in tlr3/4 signaling pathways , also mediates il-1 production in atg16l1-deficient macrophages . it was shown that atp , uric acid crystals , silicas , and asbestos induce the generation of reactive oxygen species ( ros ) , leading to the activation of caspase-1 , an essential protease for il-1 production ( dostert et al . , 2008 ; stutz et al . , 2009 ; zhou et al . , 2010 ) . in atg16l1-deficient macrophages , the enhanced il-1 production is induced by trif - dependent generation of ros and is blocked by ros scavengers . mice with hematopoietic cells lacking atg16l1 are highly susceptible to dextran sulfate sodium induced colitis , which is relieved by the injection of anti these findings indicate that the loss of atg16l1 , an essential component of the autophagic machinery , results in the production of inflammatory cytokines in response to endotoxin . however , the source of the ros has not yet been identified . other possible sources of ros include mitochondria because autophagy is required for the clearance of old / damaged mitochondria . recent studies have shown that loss of the atg results in the accumulation of ros in immune - competent cells , such as t cells , as a result of the disruption of mitochondrial turnover ( pua et al . further studies are needed to elucidate the source of ros in macrophages deficient in atgs . the ikk-ikk-nemo complex mediates the activation of the transcription factor nf-b , which in turn induces the transcription of proinflammatory cytokines and pro il-1. the tbk1ikk - i complex mediates the activation of the transcription factor irf3 , which then induces the transcription of type i ifns and ifn - inducible genes . in autophagy - deficient cells , high levels of ros are generated , which mediate trif - dependent caspase-1 activation , resulting in the processing of il-1. however , in wild - type macrophages , limited amounts of il-1 are induced by lps as the result of a lack of ros generation . after detection of the fungal cell wall component zymosan , tlr2 induces the maturation of phagosomes , leading to the elimination of the fungus . atgs such as atg5 , atg7 , and pi3k are involved in the fusion of phagosomes with lysosomes . ( c ) ligands for tlr7 such as ssrna and imiquimod induce the formation of autophagosomes via myd88 , an essential adaptor molecule , and promote the elimination of bacillus calmette - guerin . lps , a ligand for tlr4 , induces the formation of autophagosomes via the trif - p38 signaling axis , leading to the elimination of mycobacterial bacilli . atgs such as atg5 , beclin , and pi3k are required for the formation of autophagosomes by tlr stimulation . recent studies have demonstrated that tlr signaling induces the maturation of phagosomes after exposure to bacteria and promotes mhc class ii ( 2007 ) assessed the involvement of atgs in the maturation of phagosomes after exposure to microbial components and found that zymosan particles ( cell wall components of fungi ) stimulate tlr2 , thus promoting the fusion of gfp - lc3positive phagosomes with lysosomes in macrophages ( fig . surprisingly , myd88 ( myeloid differentiation primary response gene 88 ) , an essential adaptor molecule for cytokine production via tlr2 stimulation , is not necessary for the recruitment of gfp - lc3 to phagosomes by zymosan , suggesting that tlr2 triggers an as yet unidentified pathway to induce the maturation of phagosomes . atgs such as atg5 , atg7 , and pi3k are required for the efficient maturation of phagosomes induced by tlr2 . ( 2007 ) also mentioned that pam3csk4 , a soluble tlr2 ligand , fails to increase the number of gfp - lc3positive phagosomes , but pam3csk4 fused with latex beads induces the maturation of phagosomes in primary macrophages . thus , tlr2 signaling is necessary but not sufficient for the induction of phagosome maturation . although atgs are important for the maturation of phagosomes after tlr2 stimulation , subsequent characterization by electron microscopy shows that the gfp - lc3positive phagosomes do not have the morphological characteristics of autophagosomes , indicating that tlr2 stimulation induces the atg - dependent , but autophagy - independent , exclusion of fungi in phagolysosomes . it would be of interest to examine how zymosan activates the function of atgs via a tlr2-dependent but myd88-independent pathway and how atgs promote the fusion of phagosomes with lysosomes in macrophages . because the receptor dectin-1 , which recognizes the -glucan structure in zymosan , can activate the tyrosine protein kinase syk - dependent signaling pathway and promote uptake of zymosan ( brown and gordon , 2001 ; brown et al . , 2003 ; gantner et al . , 2003 ; rogers et al . , 2005 ) , it will be important to assess the involvement of this receptor in the atg - dependent maturation of phagosomes after infection by fungal pathogens . the tlr - dependent induction of autophagy in macrophages / monocytes has been reported by other groups . delgado et al . ( 2008 ) reported that the engagement of tlr7 induces autophagy and promotes the elimination of bacillus calmette - guerin in autolysosomes ( fig . 2 c ) . both atg5 and beclin are required for the induction of autophagy in macrophages after tlr7 stimulation . although myd88 is not necessary for the formation of gfp - lc3 dots after stimulation by zymosan , it is involved in the formation of gfp - lc3 dots after stimulation of tlr7 ( sanjuan et al . it would be of interest to examine the reasons why myd88 is differentially involved in the formation of gfp - lc3 dots after the engagement of tlrs and how myd88 signals atgs to induce the formation of autophagosomes . however , xu et al . ( 2007 ) reported that tlr4 stimulation results in the pi3k - dependent formation of gfp - lc3 dots and enhances the elimination of mycobacteria in macrophage cell lines ( fig . lps stimulation increases the number of autophagosomes in primary human monocytes , although it fails to induce autophagy in primary mouse macrophages ( xu et al . the signal transducers , trif , ripk1 ( receptor - interacting protein kinase 1 ) , and p38 , are required for the tlr4-induced formation of gfp - lc3 dots in macrophage cell lines . further studies are required to explain the cell type or species - specific induction of autophagy by lps and to identify the target of p38 , whose phosphorylation is responsible for the activation of atgs . nod1 ( nucleotide - binding oligomerization domain 1 ) and nod2 are intracellular sensors that recognize the unique bacterial polypeptides ie - dap ( -d - glutamyl - meso - diaminopimelic acid ) and muramyl dipeptide ( mdp ) , respectively ( franchi et al . , 2009 ) . thus , nod1 and nod2 detect invading bacteria and play a central role in the production of cytokines and antimicrobial peptides . importantly , a strong relationship has been found between nod2 mutation and the development of crohn s disease ( cho and weaver , 2007 ) . it has also been shown that nod1 is a candidate gene responsible for susceptibility to crohn s disease ( mcgovern et al . , 2005 ) . indeed , immune responses to bacterial polypeptides and microbial infection are impaired in cells isolated from patients harboring nod2 mutations ( franchi et al . , 2009 ) . in mouse models , nod1 or nod2 deficiency results in enhanced intestinal inflammation upon bacterial infection as a result of the disruption of innate immune responses . furthermore , nod2 mutant mice , whose nod2 locus harbors the homologue of the most common crohn s disease susceptibility allele 3020insc , are highly sensitive to chemical - induced colitis because of the enhanced production of proinflammatory cytokines such as il-1 and il-1 ( maeda et al . , 2005 ) . because a single nucleotide polymorphism in the atg16l1 gene is also associated with the development of crohn s disease ( hampe et al . , 2007 ; rioux et al . , 2007 ) , the involvement of nod1 and nod2 in the induction of autophagy was assessed . ( 2010 ) found that stimulation of nod2 by mdp induces the formation of autophagosomes , thus promoting antigen presentation on mhc class ii molecules by human dcs ( fig . atgs such as atg5 , atg7 , and atg16l1 are required for the induction of autophagy in human dcs and the subsequent promotion of antigen presentation by mdp ( cooney et al . , 2010 ) . ripk2 , one of the downstream regulators of the nod2 signaling pathway , is also involved in mdp - induced formation of autophagosomes . importantly , dcs expressing crohn s disease associated nod2 or atg16l1 variants fail to induce autophagosome formation and antigen presentation in response to mdp . ( 2010 ) also found that stimulation with both nod1 and nod2 triggers the induction of autophagy in mouse embryonic fibroblasts ( mefs ) , mouse macrophages , human lymphoblasts , and human cell lines ( fig . however , nod1 and nod2 are not necessary for the formation of autophagosomes induced by nutrient starvation and rapamycin treatment , indicating that nod1 and nod2 are specifically involved in the formation of bacteria autophagosomes . in mefs , nod1 , but not the downstream signaling molecules ripk2 and ib kinase-/nemo , is required for the generation of s. flexneri containing autophagosomes and promotes the elimination of the invading bacteria . interestingly , both nod1 and nod2 associate with atg16l1 and recruit it to the bacterial entry sites after bacterial infection . the most common nod2 mutation associated with crohn s disease fails to induce the movement of atg16l1 and the formation of autophagosomes . these studies indicate a role for both nod1 and nod2 in the formation of bacteria autophagosomes and in the regulation of antimicrobial responses . however , the signaling pathways downstream of nod1 and nod2 that are responsible for the induction of bacteria autophagosomes remain unclear . nod1 and nod2 are composed of n - terminal cards , a centrally located nucleotide - binding oligomerization domain ( nod ) , and multiple c - terminal leucine - rich repeats ( lrrs ) . the leucine - rich repeats of nod1 and nod2 detect ie - dap and mdp , respectively . after sensing these ligands , nod1 and nod2 induce the formation of autophagosomes , leading to the promotion of antigen presentation and the enhancement of bactericidal responses . atgs such as atg5 , atg7 , and atg16l1 are involved in nod1- and nod2-mediated formation of autophagosomes . for host defense against rna viruses , type i ifns ( ifn- and ifn- ) induce the expression of a series of antiviral factors and play crucial roles in the establishment of an antiviral state ( beutler , 2009 ; kawai and akira , 2009 ) . plasmacytoid dcs ( pdcs ) detect the single - stranded rna ( ssrna ) of rna viruses via tlr7 , which is expressed on endolysosomes . tlr7 stimulates irf7 ( ifn regulatory factor 7 ) , a transcription factor that induces ifn stimulation - responsive element - dependent transcription via the adaptor molecule myd88 and mediates the production of type i ifns by pdcs . in cell types retinoic acid inducible gene i ( rig - i ) and mda-5 , caspase recruitment domain ( card)containing rna helicases called rig - like receptors ( rlrs ) sense the cytoplasmic double - stranded rna ( dsrna ) of rna viruses and mediate signals to ips-1 ( ifn- promoter stimulator-1 ; also called mavs / visa / cardif ) . ips-1 , a card - containing mitochondrial protein , activates the transcription factors irf3 and nf-b via the ib kinase family , leading to the production of type i ifn and the expression of ifn - inducible genes . recent studies have shown an important role for atgs in both tlr - dependent and rlr - dependent antiviral innate immune responses ( jounai et al . , 2007 ; lee et al . two models have been proposed for the involvement of atgs in rlr - mediated innate immune responses . ( 2007 ) found a possible role for atg12-atg5 conjugates in the disruption of the rlr 4 a ) . the atg12-atg5 conjugate associates with the card domains of rlr and ips-1 , and ectopic expression of atg12 or atg5 prevents rlr- or ips-1mediated activation of the type i ifn promoter . consistently , loss of atg5 enhances the production of type i ifn by vesicular stomatitis virus ( vsv ) infection and dsrna treatment and limits the replication of vsv in mefs . loss of atg7 , an essential requirement for the conjugation of atg12 with atg5 , also results in enhanced type i ifn production by dsrna ( jounai et al . , 2007 ) 2009 ) showed that the disruption of cellular homeostasis by a deficiency in autophagy affects rlr ips-1 signaling pathways ( fig . damaged mitochondria accumulate because of the loss of autophagy , resulting in the elevated expression of the ips-1 protein . rlr stimulation triggers an elevation in ros production by the accumulated mitochondria , leading to the activation of ips-1dependent innate immune responses in atg5-deficient cells . further analysis will be required to reveal the mechanisms responsible for the ros - dependent activation of rlr signaling . ( a ) rig - i and mda-5 , card - containing rna helicases , sense viral dsrna and signal to ips-1 , a card - containing adaptor molecule , to induce the expression of type i ifn / ifn - inducible genes and proinflammatory cytokines . atg12-atg5 conjugates are recruited to the card domains of rig - i , mda-5 , and ips-1 and suppress the dsrna - induced innate immune response . ( b ) tlr7 detects viral ssrna and induces innate immune responses . targeted disruption of atg5 or pharmacological inhibition of pi3k results in the impairment of tlr7-mediated expression of type i ifn / ifn - inducible genes and proinflammatory cytokines . ( c ) sting , a multispanning membrane protein , and tbk1 , an irf3 kinase , mediate the induction of innate immune responses by dsdna . after sensing dsdna , sting moves from the er to the golgi apparatus and finally reaches the cytoplasmic punctate structures to assemble with tbk1 , resulting in the irf3-dependent expression of type i ifns / ifn - inducible genes . sting colocalizes with atg9a in the golgi apparatus after dsdna stimulation . the loss of atg9a greatly enhances the dsdna - induced assembly of sting and tbk1 , leading to aberrant activation of the innate immune response . ( 2007 ) reported that autophagy is necessary for the tlr7-dependent production of type i ifns and cytokines in pdcs after rna virus infection ( fig . loss of atg5 , or pharmacological inhibition of pi3k , severely impairs the production of both type i ifn and cytokine il-12 p40 by pdc infected with vsv and sendai virus . the number of autophagosomes in pdcs does not increase after tlr7 stimulation , indicating that basal autophagy , which constitutively occurs under nutrient - rich conditions , is responsible for the tlr7-dependent induction of innate immune responses in pdcs . these findings suggested that the viral ssrna from replicating vsv or sendai virus is generated in autophagosomes , or autolysosomes , in pdcs , resulting in the tlr7-dependent production of type i ifns and proinflammatory cytokines . ( 2007 ) also reported that the production of type i ifn but not il-12 p40 by a / d - type unmethylated cpg oligonucleotides ( a ligand for tlr9 ) is severely impaired in atg5-deficient pdcs . because tlr7 and tlr9 share the signaling pathway that induces type i ifn , autophagy or atgs might be involved in signaling events required for tlr7/tlr9-dependent type i ifn production in pdcs . it is known that hyperactivation of b cells by nucleic acid containing antigens results in the development of autoimmune diseases such as systemic lupus erythematosus ( marshak - rothstein , 2006 ) . although b cell receptors ( bcrs ) on the cell surface and tlr7/tlr9 in the endolysosomes are involved in hyperimmune responses to nucleic acid containing antigens , it is unclear how nucleic acid containing antigens induce the aberrant activation of b cells via these receptors . ( 2008 ) found that stimulation of bcr by dna - containing antigens results in the translocation of both the bcr and tlr9 to autophagosome - like compartments where the dna - containing antigens are accumulated ( fig . inhibition of pld , but not diacylglycerol kinase , blocks the translocation of tlr9 after it engages the bcr , suggesting that bcr stimulation induces the pld - dependent generation of phosphatidic acid to promote the recruitment of tlr9 to autophagosome - like compartments . the movement of tlr9 from the endosome into the autophagosome - like compartments on bcr stimulation depends on a functional microtubular network . treatment with nocodazole , an inducer of tubulin depolymerization , inhibits the translocation of tlr9 and the subsequent hyperactivation of mapk by dna - containing antigens . thus , the movement of bcr and tlr9 induced by dna - containing antigens and the colocalization of the receptors and dna - containing antigens in autophagosome - like compartments facilitates the efficient engagement of the receptors , resulting in the synergistic activation of downstream signaling cascades . study of primary b cells lacking atgs is needed to elucidate the requirement for autophagosomes in the bcr- and tlr - dependent activation of b cells by dna - containing antigens . dna - containing antigens stimulate bcrs , leading to the translocation of tlr9 from the endosomes to autophagosome - like compartments . dna - containing antigens also promote the movement of bcrs from the cell surface to autophagosome - like compartments . in these autophagosome - like compartments , dna - containing antigens trigger both bcr and tlr9 signaling pathways , resulting in the synergistic activation of mapk . functional pld and microtubules are required for the translocation of these receptors and play a critical role in the hyperresponse to dna - containing antigens . microbial dna induces the expression of type i ifns and proinflammatory cytokines , leading to the potent induction of innate immunity ( stetson and medzhitov , 2006a ; kawai and akira , 2009 ) . furthermore , synthesized dna stimulates the innate immune system and acts as a good adjuvant to induce the efficient induction of acquired immune responses ( ishii et al . , 2008a ) . indeed , tlr9 , the receptor for single - stranded dnas containing unmethylated cpg motifs , is involved in the protection of hosts suffering dna virus infection , and the ligands for tlr9 efficiently induce acquired immune responses upon vaccination . however , dsdna derived from bacteria and dna viruses , as well as host genomic dna from dying cells , could induce the expression of both type i ifns and ifn - inducible genes via a tlr - independent pathway ( okabe et al . , 2005 ; ishii et al . , 2006 ; stetson and medzhitov , 2006b ; stetson et al . , 2008 ) . although the specific sensors involved in dsdna - induced innate immune responses are still unclear , recent studies have revealed that tbk1 ( tank - binding kinase 1 ) and stimulator of ifn genes ( sting ; also called mpys / mita / eris ) , function as mediators in dsdna - induced production of type i ifn ( ishii et al . , 2006 ; stetson and medzhitov , 2006b ; ishikawa and barber , 2008 ; jin et al . , 2008 are also required for the efficient induction of acquired immune responses by dna - based vaccines and play a key role in host defense against the dna virus herpes simplex virus 1 ( ishii et al . , 2008b ; ishikawa et al . , 2009 ) . upon stimulation by dsdna , tbk1 phosphorylates the transcription factor irf3 , leading to the activation of ifn - stimulated response element dependent transcription of type i ifn / ifn - inducible genes . sting is a multispanning membrane protein that associates with tbk1 to mediate dsdna - induced signaling pathways . recently , we and other groups reported that the translocation and assembly of these essential signal transducers ( sting and tbk1 ) are required for dsdna - triggered innate immune responses ( fig . after stimulation with dsdna , sting moves from the er to the golgi apparatus , finally reaching the cytoplasmic punctate structures to assemble with tbk1 . exoc2/sec5 , a component of the exocyst complex responsible for targeting exocytic vesicles to specific docking sites on the plasma membrane , colocalizes with sting and regulates innate immune responses to dsdna . thus , it is clear that a membrane trafficking system mediates the dynamic movement of sting , leading to the efficient induction of innate immune responses to dsdna . we assessed involvement of atgs in the translocation of sting and found that , after dsdna stimulation , sting colocalizes with atg9a in the golgi apparatus and with lc3 in cytoplasmic punctate structures but not with other atgs ( fig . characterization by electron microscopy revealed that the sting - positive puncta induced by dsdna stimulation did not have the morphological characteristics of autophagosomes but were unidentified membrane - bound compartments , suggesting a unique function of atg9a or lc3 in the regulation of innate immune responses . atg9a is the only multispanning membrane protein identified as an atg in mammals and localizes to the golgi apparatus and late endosomes ( yamada et al . , 2005 ; webber and tooze , 2010 ) . atg9a does not reside at one site , but rather , it dynamically cycles between these organelles under starvation conditions , mediating membrane transport to generate autophagosomes . mice lacking atg9a can not survive neonatal starvation periods , and cells lacking atg9a are deficient in the generation of autophagosomes under both nutrient - rich and nutrient - starved conditions , indicating that atg9a is an essential component of autophagy ( saitoh et al . interestingly , the loss of atg9a greatly enhances the assembly of sting and tbk1 triggered by dsdna , leading to aberrant activation of the innate immune response ( fig . 4 c ) . however , both the localization of sting and cytokine production are almost normal in dsdna - stimulated mefs lacking either atg7 or atg16l1 . thus , deficiency of atg9a , but not of autophagy , affects the movement of sting and tbk1 after dsdna stimulation . further studies are needed to reveal the unique function of atg9a involved in the regulation of the membrane trafficking responsible for the assembly of sting and to assess importance of atg9a in host defense against dna viruses and pathogenic bacteria . recent advances in the study of the involvement of atgs in innate immunity have clearly demonstrated the importance of membrane trafficking systems to host defense . however , in some cases , it is still unclear whether autophagy or atgs are involved in the control of prr - mediated innate immune responses . furthermore , it is difficult to judge whether the observed phenotypes represented by a deficiency of atgs are caused by the inhibition of transiently induced autophagy because autophagy is constitutively induced , even under normal conditions , and maintains cellular homeostasis , the disruption of which often affects the regulation of innate immune responses . for example , sqstm1/p62 , accumulated through a deficiency in autophagy , might indirectly affect innate immune responses by inhibiting the function of the ubiquitin ligase keap1 , leading to the activation of nf - e2related factor 2 , a transcription factor involved in the regulation of prr - mediated innate immune responses ( bjrky et al . , 2005 ; thimmulappa et al . , 2006 ; komatsu et al . , 2007 , 2010 in future studies , it will be important to identify the precise molecular mechanisms underlying the disruption of prr - mediated innate immune responses because of the loss of atgs . although the role of atgs in host defense responses is extensively investigated in vitro , it is important that future studies assess its role in vivo . this is because certain cell types are difficult to maintain in vitro and because atgs might be required for the development of unique but important cell types in vivo . indeed , cadwell et al . ( 2009 ) reported an essential role for atgs in the development of paneth cells , intestinal epithelial cells that produce the antimicrobial peptides defensin via nod2 ( kobayashi et al . interestingly , t300a mutation of the atg16l1 protein disrupts the development of paneth cells in patients suffering from crohn s disease , suggesting that atg16l1 is important for the development of this inflammatory disorder ( cadwell et al . , 2008 ) . furthermore , it is possible that the loss of atgs by a particular cell type in vivo indirectly affects innate immune responses . for example , hepatocyte - specific deletion of atgs results in compromised lipid metabolism , which controls the level of triglycerides and cholesterol ( shibata et al . , 2009 ; singh et al . , 2009 ) . altered lipid storage in the liver might indirectly affect the innate immune response because oxidized lipids and cholesterol crystals are potent inducers of inflammation ( febbraio et al . , 2000 ; ricci et al . , 2004 ; duewell et al . , 2010 ; stewart et al . , 2010 ) . specific knockout mice are required for the detailed analysis of atg - dependent regulation of the innate immune response in vivo . recent advances in the study of atgs provide strong evidence that atgs influence both the host defense response and the regulation of inflammation . consistently , it has been proposed that the compromised function of autophagy regulators results in the development of immune - related disease such as crohn s disease ( hampe et al . . therefore , enhancement of autophagic activity is an attractive approach to protect the host against infectious diseases and inflammatory disorders . it would be of interest to assess the therapeutic effects of strategies to increase autophagic function , such as a hypocalorific diet and autophagy - inducible drugs , in the treatment of these diseases .

pattern recognition receptors detect microbial components and induce innate immune responses , the first line of host defense against infectious agents . however , aberrant activation of immune responses often causes massive inflammation , leading to the development of autoimmune diseases . therefore , both activation and inactivation of innate immune responses must be strictly controlled . recent studies have shown that the cellular machinery associated with protein degradation , such as autophagy , is important for the regulation of innate immunity . these studies reveal that autophagy - related proteins are involved in the innate immune response and may contribute to the development of inflammatory disorders .

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the association of this habit with oral cancer was noted over a century ago ; yet the chewing of betel quid in india was considered to be a comparatively innocuous practice with complete socio - cultural acceptance . in recent years it has emerged as a significant public health problem in india . the normal cultural practice of betel nut ended up to a health hazard when tobacco was introduced by the european settlers four centuries ago . in addition to its use for smoking , as the europeans demonstrated , tobacco somehow got mixed with betel quid and prepared to be chewed . in india tobacco was culturally accepted as it was used with a culturally accepted betel quid substance . during the second half of the 20 th century , the causal association between chewing of betel quid with tobacco and oral cancer was well established , so it could no longer be regarded as a completely innocuous practice . the major culprit was clearly tobacco in the quid ; however , probably because of its ' exotic ' appeal , areca nut was erroneously thought of as playing a more important role . betel nut was reported safe in many studies where only tobacco in the quid was held responsible for oral cancers . later it was clear that betel nut chewing was responsible for oral submucous fibrosis ( osmf ) , an established precancerous condition with trismus . osmf was a rare disease in the early 80 s , occurring generally among older age groups who chew betel quid frequently . a later incidence of osmf increased amongst the indian young population as a result of increasing gutkha chewing . the frequency of oral cancer has increased among young adults as a result of increasing in number of osmf ( a precancerous condition ) . the increasing incidence of osmf and oral cancers during the last two decades was due to industrial manufacturing and marketing of these chewable mixtures gutkha . the incidence of oral cancer is 12.5/100,000 and the largest numbers of oral cancer patients are present in india . in india , this epidemic proportion of oral cancer in india is due to the rampant use of betel nut , betel quid , and gutkha and pan masala chewing which are proven to be carcinogens . oral cancer is uncommon disease in the west but continue to be the major cause of cancer related death in indian men . over last few decades , large funds have been spent to educate public , implement laws effectively , rehabilitate tobacco growers , build cessation facility , create health care infrastructure , etc to reduce the betel nut , betel quid , gutkha and other forms of chewable tobacco usage . this is a retrospective study of 63 cases of oral cancer patients who were managed in kvg medical college sullia . sixty three patients , 38 males and 25 females , were included in our study . the youngest patient was 47 years old and the oldest was 73 . a detailed history of the patients was taken plus oral and oropharyngeal examination . chest x - ray along with ap neck x - ray , lateral view and panoramic view were taken ( figure 1 ) . the biopsy of the ulceroproliferative mass was taken ; histopathology of all specimens showed squamous cell carcinoma . tnm staging ( tumor size , lymph nodes , metastasis ) showed most of the cases were in advanced stages . all of 63 patients were managed according to the oral cancer management protocol and all signed written informed consent . the mean age in males was 61.20 years and female median age was 59.92 years . the mean duration of betel chewing in males was 18.56 years and in females was 20 years ( figure 2 ) . all patients in our study changed to chewing packaged panmasala and gutkha which were easily available . the mean duration of panmasala and gutkha chewing in males was 8.66 years and in females was 7.36 years ( figure 3 ) . thirteen males ( mean 11.2 years ) in our study were tobacco smokers and 12 males ( mean 9.4 years ) were occasional alcohol drinkers . twelve males ( mean 10.3 years ) and 14 females ( mean 8.4 years ) were workers in tobacco processing industry . ischemic heart disease and hypertension were seen in 22 patients ( 16 males and 6 females ) . oral submucous fibrosis and leukoplakia were excluded from the study after histopathological examination ( figure 4 ) . out of 63 cases , 14 had lip carcinoma , 41 with carcinoma of the buccal mucosa and 7 had carcinoma of tongue and there was 1 case of carcinoma of upper alveolus . out of 14 cases of carcinoma of lip , 2 cases were in stage1 , 3 cases in stage 2 , 7 cases in stage 3 , and 2 cases in stage 4 . out of 41 cases of buccal mucosa carcinoma , 4 were in stage 1 , 11 in stage 2 , 18 in stage 3 , and 8 cases in stage 4 ( figure 5 ) . out of 7 cases of tongue carcinoma , 2 were in stage 1 , no cases in stage 2 , 2 cases in stage 3 , and 3 cases in stage 4 ( figure 6 ) . the case of carcinoma of upper alveolus refused treatment and was lost in follow up ( figure 7 ) . all stage 1 oral cancer patients in our study were treated by wide excision only and followed up for 6 months to 2 years . three cases of lip carcinoma with stage 2 were treated by wide excision and postoperative radiotherapy ( figure 8) . two cases of buccal mucosa carcinoma with stage 2 were widely excised and were sent for postoperative radiotherapy , 8 patients were sent for radiotherapy as they refused surgery and 1 refused radiotherapy ( table 1 ) . out of 7 cases of lip carcinoma with stage 3 , 3 patients refused treatment and 4 had wide excision with neck dissection and postoperative radiotherapy . out of 18 cases of carcinoma of buccal mucosa stage 3 , 7 patients underwent wide excision with neck dissection plus radiotherapy , 11 refused surgery so they were referred to radiotherapy . one case of carcinoma stage 3 had wide excision with radiotherapy and the other refused surgery but took radiotherapy . two cases of lip carcinoma with stage 4 refused surgery so they were given radiotherapy . all buccal mucosa carcinoma patients with stage 4 refused surgery but only 2 of them received radiotherapy . a direct relation between oral cancer and duration of betel quid , gutkha and panmasala chewing was seen in our study . betel nut may be consumed alone or as a betel quid in combination with betel leaf and slaked lime and may contain other substances like tobacco , catechu , spices or sweeteners [ 9 , 10 ] . betel leaf that is commonly consumed with this nut contains a known carcinogen named safrole [ 9 , 10 ] . the lime in the betel quid enhances the extraction of the betel nut alkaloids by salivary degradation and promotes carcinogenesis [ 9 , 10 ] . the alkaloids in betel nut which are responsible for the biological actions in humans are arecoline , arecaidine , guvacine , and guvacoline [ 9 , 10 ] . asians and indians even after immigrating to western countries still continue the habit of betel nut chewing which show an addiction among these populations [ 9 , 10 ] . the betel nut derived alkaloids are cholinergic agonists and they affect nervous system via acetylcholine . prolonged use of betel nut causes peculiar facial and systemic features , staining of teeth , attrition of teeth , and creation of wear facets with higher incidence of periodontitis . betel nut for consumption is obtained from areca catechu which is the fourth most common addictive substance after tobacco , alcohol , and caffeine . it is possibly the second most consumed carcinogen after tobacco in the indian subcontinent . in india , betel nut is chewed for variety of reasons such as stress reliever , mouth freshener , improving concentration , and for digestion after food . addiction withdrawal symptoms are mood swings , anxiety , and irritability , loss of concentration , sleep disturbance and craving which were seen in most cases in our study . betel nut psychosis has been reported in heavy users following sudden cessation of the habit . india is the greatest producer of betel nut something like 330,000 million tonnes per year ( nearly half of global betel nut production ) . the total area under betel nut cultivation in india is estimated to be around 372,000 hectares and total production is 340,000 tonnes . it is estimated that nearly 10 million people depend on betel nut industry for their livelihood in india . most of the betel nut production is consumed within india because it has limited export potential . betel nut products ( gutkha , panmasala ) are being advertised as mouth fresheners which are misleading indian youth . gutkha and panmasala are more dangerous than betel nut alone because they are kept in mouth for a longer time . the other promoting factors in this industry are lack of statutory warning , low cost , easy availability , attractive packaging and aggressive marketing . panmasala is basically a preparation of betel nut , catechu , cardamom , lime and a number of natural , artificial perfuming and flavoring materials . gutkha is a variant of panmasala , in which in addition to these ingredients flavored chewing tobacco is added . both products are often sweetened to enhance the taste .betel leaf is perishable and preparation of betel quid is somewhat complex or requires visits to shops selling pan / bq . with the emergence of commercial pan masala and gutkha about three decades ago , not only did the indian market witness massive growth in the sales of smokeless tobacco and betel nut products , but also a huge worldwide export market developed . all our patients earlier chewed betel quid but turned to gutkha , pan masala chewing over the last two decades . just like tobacco , the cancer causing properties of betel nut have been well - reported in animal , human and epidemiologic studies . cancers caused by betel nut chewing also shows a strong dose - response relationship for frequency and duration of chewing . n - nitroso compounds converted from betel nut alkaloids are responsible for oral and pharyngeal cancer . also betel nut chewing ( with or without tobacco ) is associated with hepatocellular carcinoma , cholangiocarcinoma , cancers of the larynx , stomach , lung and cervix in humans . betel nut is a synergistic carcinogen along with tobacco with which it is commonly consumed in india . all patients in our study had betel quid and gutkha panmasala chewing as the etiology with smoking , alcohol consumption and working in tobacco processing factories as other etiologies . the who and international agency for research on cancer classified betel nut as a group 1 human carcinogens with sufficient evidence of increased risk of osmf ( precancerous oral lesion ) and cancers of the oral cavity , pharynx and esophagus [ 17 - 20 ] . cancers caused by betel nut chewing also shows a strong dose - response relationship for frequency and duration of chewing . it is postulated that betel nut - specific n - nitroso compounds converted from alkaloids are responsible for oral and pharyngeal cancer . there are several other reports that have linked betel nut chewing ( with or without tobacco ) with hepatocellular carcinoma , cholangio - carcinoma , laryngeal cancers , stomach , lung and cervix in humans . studies in mice have shown that , betel quid extract given by gavages produced carcinoma of lung , stomach , liver and salivary gland . fibrosarcomas at the injection site were observed by subcutaneous injections of betelnut extracts in mice . animal research has shown that panmasala in the diet led to tumors in various organs , most frequently adenocarcinoma of lung . in another experiment , arecoline was given as gavage and it produced carcinoma of lung , stomach and hemangiomas of liver . esophageal cancers increased 195 times , pharyngeal cancers 96.9 times and laryngeal cancers 40.3 times when betel quid chewers had habits of smoking and drinking . betel quid chewing resulted in a statistically significant increase in the risk of overall mortality and cerebrovascular deaths in the elderly population [ 22 , 23 ] . betelnut addiction has also been found to be associated with heart attacks , arrhythmia , metabolic syndrome and diabetes [ 24 - 27 ] . chronic betel nut chewers women give birth to low weight babies similar to smoker women [ 29 , 30 ] . betel nut and betel quid chewing has been a popular oral habit in india and many other southeast asian countries for a long time [ 31 , 16 ] . betel nut , betel quid and gutkha chewing habit has become the top five oral habits ( including smoking , alcohol , tea , coffee , and betel chewing ) in the world . there are about 200 - 600 million betel nut , betel quid and gutkha chewers in the world [ 28 , 29 ] . they are proven carcinogens resulting in oral cancer , oral precancerous lesions , hepatocarcinoma , diabetes mellitus ( dm ) , cardiovascular diseases and other systemic diseases [ 31 , 16 ] . india accounts for 1/3 of an estimated 3 million betel and tobacco related deaths in the world per year . in 2001 , the sale of cigarettes was banned to people under 18 . in view of high usages of betel nut , betel quid and other tobacco containing products such as gutkha and their proven role as a carcinogen , it is necessary to look into the hazardous effects of these addictions and their impact on health and society . so a ban on betel nut , betel quid and tobacco in any form is immediately necessary to eradicate the menace of the morbidity . panmasala and gutkha have flooded the indian market as cheap and convenient betel quid substitutes and become popular across all age groups wherever betel nut is abundantly used . there is sufficient evidence that chewing of betel nut with or without tobacco and lime are carcinogenic in humans . some restrictions on the manufacture and sale of these products are being implemented but because of seriousness of the situation , a permanent ban on gutkha and panmasala is necessary as they are well - established oral cancer causing agents . an emphasis on health education aiming to reduce and later eliminating betel nut and tobacco products should be placed . panmasala and gutkha have flooded the indian market as cheap and convenient betel quid substitutes and become popular across all age groups wherever betel nut is abundantly used . there is sufficient evidence that chewing of betel nut with or without tobacco and lime are carcinogenic in humans . some restrictions on the manufacture and sale of these products are being implemented but because of seriousness of the situation , a permanent ban on gutkha and panmasala is necessary as they are well - established oral cancer causing agents . an emphasis on health education aiming to reduce and later eliminating betel nut and tobacco products should be placed .

backgroundbetel nut and betel quid chewing are from major etiological factor for oral cancer . they also increase the risk of systemic diseases such as asthma , diabetes mellitus , metabolic syndrome , myocardial infarction , hypertension , and other cardiovascular diseases . methodssixty three patients of oral cancer in our institution during jan . 2007 to may 2011 were included in our study . this study included 14 cases of lip carcinoma , 41 cases of buccal mucosa carcinoma , 7 cases of tongue carcinoma , and 1 case of carcinoma of upper alveolus . duration of use of betel quid , pan masala , and gutkha were studied as well as management.resultsall patients in our study have been chewing betel - quid for 6 - 31 years ( mean19.42 years ) . all of them quit betel quid and used easily available panmasala and gutkha for 4 - 13 years ( mean 8.28 years ) . nine cases of lip carcinoma , 13 cases of buccal mucosa carcinoma and 3 cases of tongue carcinoma were treated with surgery . seven cases of lip carcinoma , 30 cases of buccal mucosa carcinoma and 5 cases of tongue carcinoma were treated with post operative or palliative radiotherapy.conclusionbetel nut chewing with or without tobacco and lime are proven to be carcinogens in human . direct relationship between oral cancer and betel quid , gutkha , and panmasala use has been shown in our study . as betel quid , panmasala and gutkha chewing were proven to be carcinogens , a permanent ban on manufacturing and sale of these products should be implemented .

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the concept of patient blood management is gaining increased attention . during the last decade numerous reports have emphasised the need for reductions in transfusions of blood and blood products as allogeneic red blood cell ( rbc ) transfusions are associated with increased morbidity and mortality [ 110 ] , increased risk of severe postoperative infections , adverse effects or risk of transferring pathogens [ 11 , 12 ] , relatively high costs , and shortage of blood bank products [ 6 , 1214 ] . postoperative severe bleeding is relatively common following cardiac surgery compared to other surgical specialties and is considered a serious complication associated with increased morbidity and mortality [ 1421 ] . within the cardiac surgery population , patients with advanced age and long cardiopulmonary bypass ( cpb ) are especially at risk of postoperative bleeding [ 18 , 19 ] . moreover , it is well known that excessive bleeding may be caused by surgical factors and impaired haemostasis due to enhanced fibrinolysis , platelet dysfunction , haemodilution , acidosis , hypothermia , and consumption of coagulation factors as well as the surgical trauma alone [ 22 , 23 ] . during recent years several cardiac surgery studies have reported on the long term mortality after transfusion with blood and blood products [ 610 ] . although not all , the majority of studies report a higher long term mortality after blood transfusion [ 6 , 9 , 10 , 24 ] ( figure 1 ) . however , the reports are mainly from designated types of surgery [ 79 ] or single centre studies [ 6 , 8 , 9 ] . in some studies the kaplan - meyer plots tend to run parallel after the first month when including the immediate postoperative mortality , indicating less impact on the long term survival [ 6 , 9 , 10 ] . one of the last published evidence based guidelines for the transfusion of rbc concluded that red blood cell transfusions should not be dictated by a single haemoglobin ( hb ) transfusion trigger , but instead should be based on the patient 's risk of developing complications of inadequate oxygenation . furthermore , that rbc transfusion would generally be indicated , but not mandatory , at hb levels lower than 6.0 g / dl ( 3.7 mmol / l ) and rarely indicated in patients with haemoglobin higher than 10.0 g / dl ( 6.1 mmol / l ) . in many types of major surgery these recommendations have been followed and the use of transfusions reduced . however , in cardiac and vascular surgery the use of blood and blood products is still common clinical practice . from a survey of cardiac procedures covering monitoring , anaesthesia and transfusions carried out in 2005 , it was found that almost 40% of european institutions used blood or blood products in more than half of their cardiac surgery patients . when calculating the number of surgical procedures in each institution and number of patients not receiving blood or blood products , 55.7% of european cardiac surgery patients received blood or blood products in the perioperative period . however , the difference in transfusions from less than 10% of patients to 100% that do receive blood or blood product transfusion ( figure 2 ) is interesting and indicates that transfusions are guided by local policies and not evidence based practice . patients referred for coronary artery bypass graft ( cabg ) are more commonly treated with antiplatelet agents especially aspirin and oral adenosine diphosphate ( adp ) receptor antagonists together with newer alternative low molecular heparin drugs , like fondaparinux . long term aspirin therapy is the standard of care in patients with coronary artery disease , while concomitant treatment with oral adp receptor antagonists is recommended for patients with recent acute coronary syndrome ( acs ) or percutaneous coronary intervention ( pci ) [ 2729 ] . studies have found both reduced mortality and no difference in bleeding or no difference in bleeding complications in patients treated with antiplatelet drugs [ 31 , 32 ] . this is in contrast to other studies reporting higher transfusion requirements without effect on mortality or increased bleeding and transfusion as well as increased risk of myocardial infarction . in the crusade study following established guidelines a significant increase in transfusion requirements in patients continuing on clopidogrel was observed compared to patients where the drug was discontinued more than five days before surgery . similarly , large cohort studies found significant increases in major bleeding and reoperation rates in clopidogrel - exposed cabg patients [ 33 , 36 ] . the use of antiplatelet agents at the time of cabg carries both benefits and risks . the drugs are effective in reducing ischemic events in high - risk patients awaiting surgery . after surgery drugs may prevent graft occlusion and recurrence of ischemic episodes . however , antiplatelet drugs also aggravate perioperative bleeding followed by increased perioperative blood loss , increased transfusion requirements , and hemodynamic instability [ 35 , 37 , 38 ] . as the indications for oral antiplatelet drugs expand , cardiac surgeons frequently have to make decision about the timing of surgery in patients exposed to these drugs . current international guidelines recommend discontinuing aspirin two to ten days before elective cardiac surgery , while the adp receptor antagonist clopidogrel should be withdrawn at least five days before elective cabg [ 39 , 40 ] . the basic role of blood is to transport and deliver oxygen to the organs to maintain organ function . it is evident that clinical and some hemodynamic measurements are imperative to gain the required information for decision making . even with the most extensive invasive haemodynamic monitoring the obtained information is not fully adequate , as individual organ monitoring is not possible or available in clinical practice . we do have some intervention possibilities for blood pressure , heart rate , haemoglobin level , and volume status and it is possible to operate influence factors such as oxygen saturation , vascular resistance , preload , and cardiac output , while organ blood flow and organ oxygenation are difficult to influence directly . the correlation between blood flow , oxygen delivery , and consumption together with haemoglobin can easily be visualised ( figure 4 ) . the figure demonstrates that oxygen delivery in principle is determined by only three variables , cardiac output ( co ) , haemoglobin ( hb ) level , and oxygenation ( sao2 ) . . if oxygen delivery is out of balance with oxygen consumption , the compensation mechanisms are relatively simple , as a decrease in haemoglobin must result in a compensatory increase in either cardiac index or oxygen extraction or a combination . furthermore , the relation is independent of the fact that co changes with age and that the ratio of co to total blood volume changes from roughly 3 : 1 in small children to 2 : 3 or lower in the elderly cardiac patient . a valuable parameter in evaluating need for inotropic support or rbc transfusion is whether oxygen consumption is adequately covered by oxygen delivery . in general , when oxygen consumption is high ( i.e. , during exercise ) the increased oxygen requirement is usually provided by an increased cardiac output . however , low cardiac output , low haemoglobin concentration ( anaemia ) , or low haemoglobin o2 saturation will result in an inadequate delivery of oxygen , unless a compensatory change occurs in one of the other factors . alternatively , if oxygen delivery falls relative to oxygen consumption , the tissues extract more oxygen from the haemoglobin and the saturation of mixed venous blood ( svo2 ) falls below 70% ( a - b in figure 5 ) . a reduction below point c in figure 4 can not be compensated by an increased oxygen extraction and results in anaerobic metabolism and lactic acidosis , and oxygen consumption becomes totally dependent on oxygen supply . oxygen is carried in the blood in two forms : primarily by haemoglobin but a very small amount is dissolved in the plasma . when fully oxygen saturated ( po2 > 13.3 kpa ) only 3 ml of oxygen will be dissolved in every litre of plasma . if the po2 of oxygen in arterial blood ( pao2 ) is increased significantly ( breathing 100% oxygen ) , a small amount of extra oxygen will dissolve in the plasma at a rate of 0.023 ml o2/100 ml blood / kpa po2 . normally , there will be no significant increase in the amount carried by haemoglobin if oxygen saturation is already higher than 95% . in the literature there is some indications that patients with coexisting cardiac diseases may have a particular risk of developing impaired oxygenation at lower haemoglobin levels , but the documentation for this is not that convincing . in patients undergoing cardiac surgery studies of cardiac surgery patients have shown increased mortality after rbc transfusions [ 42 , 43 ] . further , others have found that lowering of the hb threshold for transfusion in patients undergoing coronary artery bypass surgery ( cabg ) from 9 to 8 g / dl ( 5.5 to 5.0 mmol / l ) was neither followed by a higher mortality nor followed by an increase in adverse effects by accepting the lower postoperative haemoglobin . this indicates that patients with coexisting cardiac diseases , including coronary artery disease ( cad ) , tolerate moderate anaemia . another study showed that patients may even benefit from a restrictive transfusion regimen followed by a lower morbidity and mortality . the mean haemoglobin at transfusion in this study was 8.4 g / dl ( 5.1 mmol / l ) and both icu and overall mortality rate were higher in transfused patients . the findings were further supported in an icu study where patients with known cardiovascular disease had equal level of mortality and less organ dysfunction if in a transfusion restrictive group . in contrast , one study indicated that rbc transfusions may decrease mortality in elderly patients with acute myocardial infarction and an admission haematocrit below 30% . another study shows increased in - hospital mortality in patients with lower preoperative haemoglobin levels . however , the first study is not without methodological challenges and needs careful analysis as patients with normal haemoglobin levels seemed to be treated more intensely . previously , we have demonstrated a correlation between preoperative haematocrit ( hct ) and 30-day mortality . however , when compensated for euroscore [ 4850 ] the 30-day mortality was independent of haemoglobin level and only correlated with the euroscore . the data showed a strong relationship between actual rbc transfusion and residual euroscore ( euroscore minus age factor ) and interestingly also the lack of correlation between age higher than 74 years and rbc transfusion ( table 1 ) . the first priority of perioperative fluid therapy in surgical patients is to achieve optimal filling of the heart with crystalloids and colloids to optimise cardiovascular function , decrease postoperative morbidity , and shorten the length of hospitalisation [ 51 , 52 ] . in cardiac surgery patients and many intensive care patients with pao2 > 95% , optimisation of arterial saturation only adds little to oxygen delivery , leaving only increasing co or hb as possible means to increase oxygen delivery . infusion of colloids reduces the hb level and thus , despite unchanged total haemoglobin content , oxygen delivery in theory . however , the impact on co normally compensates for that and in hypovolaemic patients especially the overall impact may be substantially higher co and thus oxygen delivery . following infusion of rbc in this type of patients may have a double effect on oxygen delivery as both hb and co are increased . in contrast , in severely overloaded patients where further compensation in cardiac output is compromised or even deteriorated after infusion of colloid or rbc , there might be no effect on the oxygen delivery . in these cases many cardiac or critically ill patients are not monitored with pulmonary artery catheters and hence information of co and svo2 is not available . central venous oxygen saturation ( scvo2 ) obtained from the superior vena cava has been proposed as a surrogate for svo2 and may reflect the balance between oxygen supply and demand . studies have shown that the difference between scvo2 and svo2 is consistently about 5% across a wide range of cardiorespiratory conditions in both animals and humans [ 53 , 54 ] . however , scvo2 obtained from a central venous catheter is not fully interchangeable with mixed svo2 , but it can give some indication of oxygen balance in patients with peripheral saturation , co and hct within normal limits . one of the principal differences in cardiac surgery compared to other types of major surgery is the use of cardiopulmonary bypass ( cpb ) . this leads to a 2030% haemodilution and when weaning from cpb a relatively big portion of red blood cells ( rbc ) are left in the heart lung machine ( hlm ) ( figure 6 ) . although the surgeon is very careful during the procedure , a smaller portion of blood is always lost . using either cell - saver or retransfusing of suctioned blood will , however , limit the total loss . if using autotransfusion in the postoperative phase the perioperative blood loss during cardiac surgery can be diminished to approximately one unit of rbc ( scenario a ) and there is thus no need for transfusion . however , if none of these procedures are used , the overall result will be a considerable blood loss and a low postoperative haematocrit level ( scenario b ) . consequently , the patient will inevitably receive 13 units of rbc . if the patient continuously has problems with bleeding or haemodynamic instability , the risk of adding plasma or platelets is reasonably high . some studies have found low haemoglobin levels during cpb to be associated with increased postoperative morbidity and mortality [ 56 , 57 ] while other studies have not been able to find such a correlation [ 43 , 58 ] . patients with coronary artery disease ( cad ) may have an increased risk of myocardial ischemia in combination with low haemoglobin levels . however , experimental and clinical data indicate that persons with cad tolerate moderate normovolaemic haemodilution well [ 5962 ] . interestingly , data from our database show that patients with cabg received less blood and blood products ( table 2 ) . regional changes in myocardial systolic and diastolic contractile dysfunction [ 63 , 64 ] and ecg signs of myocardial ischemia [ 6568 ] may develop in areas supplied by a compromised coronary artery at low haemoglobin levels . additionally , haemoglobin levels below 6.0 g / dl ( 3.7 mmol / l ) have been associated with increased postoperative mortality in patients with coexisting cardiovascular disease , including cad . during both haemodilution and blood transfusion patients with a left ventricular ejection fraction between 25% and 85% responded similarly . this indicates that also patients with a reduced left ventricular ejection fraction are able to compensate with increased cardiac output in response to haemodilution if normovolaemia is maintained . however , the number of patients with low ejection fractions was limited and the findings should not necessarily be extrapolated to the low range of patients with left ventricular ejection fraction . patients above 65 years without known cardiac disease tolerated haemodilution to a haemoglobin of 8.8 g / dl ( 5.4 beta - blocked cad patients between 35 and 81 years responded adequately during haemodilution with an increase in cardiac output and oxygen extraction . similarly , during blood transfusion the compensatory changes in cardiac output , oxygen delivery , and oxygen consumption seemed to be independent of age ( 32 to 81 years ) . however , this study had relatively few patients above 75 years , stressing that these findings may not apply to the very old patients . patients with significant mitral insufficiency , including those with atrial fibrillation , tolerate moderate haemodilution to a haemoglobin level of 10 g / dl ( 6.1 mmol / l ) . tolerance of acute haemodilution or anaemia in patients with other valve abnormalities is less well known . theoretically , patients with aortic and pulmonary stenosis may be less tolerant to haemodilution as the increase in cardiac output during haemodilution may be limited due to the valvular stenosis . however , in the postoperative phase these patients may most likely be treated as patients without coexisting cardiovascular diseases . in patients with coexisting cardiovascular diseases refusing rbc transfusions for religious reasons , postoperative haemoglobin levels below 6.0 g / dl ( 3.7 mmol / l ) were associated with increased mortality and morbidity and an increasingly greater difference in mortality and morbidity between patients with and without coexisting cardiovascular diseases . in order to avoid or minimize transfusions in patients with low haemoglobin it is evident that the 2030% haemodilution during cpb might be the primary concern if the patient does not have cardiac capacity to tolerate very low post - cpb haemoglobin or that a needed high htlm cardiac output during cpb may increase perioperative complications . the overall conclusion is that even patients with coexisting cardiac diseases tolerate moderate haemodilution or acute anaemia well if normovolaemia is maintained . however , an excessive and aggressive haemodilution may cause myocardial ischemia which is reversible with a blood transfusion , even in normovolaemic patients [ 64 , 68 , 73 ] , and the lowest acceptable haemoglobin in patients with coexisting cardiovascular diseases is approximately 6.0 g / dl ( 3.7 mmol / l , haematocrit 0.180.20 ) . patients where pre - cpb calculations indicate a lower level during or right after cpb might be the target for preoperative haemoglobin adjustment with erythropoietin with or without autologous blood transfusion . the haemoglobin level at which regional cardiac ischemia may occur varies considerably and depends on both the degree of coronary stenosis and whether it is a single- or a multivessel cad . patients with or at risk of cad should thus not automatically receive a transfusion at a specific haemoglobin level but only if oxygenation is inadequate as suggested by the american society of anaesthesiologists ( asa ) . the question is whether anaemia - related myocardial ischemia is reversible by blood transfusion , which seems to be the case in some experimental and clinical [ 68 , 73 ] settings . patients with progressively lower haemoglobin levels also have other risk factors such as diabetes , preoperative congestive heart failure , prior coronary artery bypass operation , low left ventricular ejection fraction , and a higher frequency of emergency surgery . a high correlation has been found between such risk factors and a low haemoglobin level and the independent contribution of a low perioperative haemoglobin level to mortality is difficult to assess . after adjusting for other risk factors , only a haematocrit below or equal to 14% during cpb remained an independent predictor for increased mortality . only a prospective randomised study design can definitively determine whether varying haemoglobin transfusion triggers affect morbidity and mortality . in a study of icu patients it was demonstrated that a haemoglobin transfusion trigger of 7 g / dl ( 4.3 mmol / l ) did not negatively impact on mortality and morbidity , neither in general icu patients nor in patients with coexisting cardiac diseases [ 1 , 45 ] . several studies have shown the importance of controlling haemodynamics during cardiac [ 76 , 77 ] as well as noncardiac surgery . therefore , episodes of tachycardia may occur , which may be associated with st - segment depression , in particular in patients with t low haemoglobin levels . preventing tachycardia and following st - segment depression is thus imperative in patients with or at risk of cad . in addition , postoperative oxygenation is generally lower than during peroperative mechanical ventilation with a high inspiratory oxygen concentration . therefore , higher postoperative haemoglobin levels of 7 to 8 g / dl ( 4.34.9 mmol / l ) may be justified in patients with coexisting cardiac diseases . the question of when transfusion is appropriate in a patient with a coexisting cardiac disease thus remains unanswered . at a haemoglobin level lower than 6.0 g / dl ( 3.7 mmol / l ) blood transfusions may be indicated in most patients and in particular in patients with coexisting cardiac diseases [ 25 , 69 , 80 , 81 ] . furthermore , it has been shown in cardiac surgery patients that oxygen consumption only increased following a blood transfusion when oxygen consumption was very low before blood transfusion . as this remains impossible to determine also in patients with coexisting cardiac diseases , the haemoglobin level , at which a transfusion would generally be indicated , should follow the guidelines from the asa , stating that red blood cell transfusions should be based on the patient 's risk of developing complications of inadequate oxygenation . the immediate question is then , what are the signs of a beginning inadequate oxygenation in patients with coexisting cardiac diseases ? inadequate oxygenation may become globally manifested in the form of general haemodynamic instability with a tendency to hypotension and tachycardia despite normovolaemia , oxygen extraction higher than 50% [ 8082 ] , or myocardial ischemia detected by continuous 5-lead ecg monitoring , ideally with automatic st - segment analysis [ 65 , 68 , 83 ] and by new wall motion abnormalities in transoesophageal echocardiography . st - segment depressions above 0.1 mv or new st - segment elevations above 0.2 mv during more than one minute generally are regarded as a marker of myocardial ischemia . during progressive haemodilution , primarily st - segment depression is observed , suggesting subendocardial ischaemia . such anaemia - related ischaemia may , in case of tachycardia , be reversed by decreasing the heart rate and by increasing the haemoglobin level 1 - 2 g / dl ( 0.61.2 new wall motion abnormalities clinically detected by transoesophageal echocardiography may be the result of myocardial ischemia and can be treated by a minor increase in haemoglobin of 1 to 2 g / dl ( 0.61.2 early signs of inadequate circulation are general haemodynamic instability characterised by relative tachycardia and hypotension [ 81 , 85 ] together with an oxygen extraction fraction above 50% , a low mixed venous oxygen partial pressure ( pvo2 ) , and a decrease in oxygen consumption [ 80 , 81 ] . an oxygen extraction higher than 50% has been found to indicate exhaustion of compensatory mechanisms in several studies [ 8689 ] and might thus represent a transfusion indication . oxygen consumption decreases very late and at very low haemoglobin levels during progressive normovolaemic haemodilution [ 89 , 90 ] in conditions where oxygen extraction has increased and pvo2 has already decreased . therefore , any decrease in oxygen consumption of more than 10% at low haemoglobin levels should be viewed as a potential sign of a compromised oxygenation and a blood transfusion should be considered if normovolaemia has been achieved . in patients with coexisting cardiac diseases , the principles of rbc transfusion are not considerably different from healthy patients and must also largely be based on early signs of impaired oxygenation of specific organs or the entire organism . impaired oxygenation may be reached at higher haemoglobin values than in healthy patients and most patients have considerable variations in haemodynamic values . a previous study found considerable interpatient differences and intrapatient variation in patients monitored the night before cardiac surgery ( figure 7 ) . the most pronounced intrapatient variation was found in the cardiac index , ranging from 1.9 to 5.3 l / min / m . most of the patients had periodic spo2 values 92 and half of them in more than 15% of the observations . overall a svo2 < 70% was found in more than 40% of observations and less than 64% in more than 20% of the observations together with drops below 50% without obvious reasons . although the number of patients in the study was low the conclusion was that intrapatient variation was unexpectedly high in most hemodynamic variables . this demonstrated the challenge in using hemodynamic parameters to guide treatment and indicated that goal oriented therapy using currently accepted values may result in overtreatment in some patients . a restrictive rbc transfusion policy has proven equally effective and possibly even superior to a more liberal transfusion strategy . although there currently is no clear explanation for this effect , infections , immunosuppression , and the age of the rbc at the time of transfusion [ 5 , 93 , 94 ] have been suggested as possible impact factors . the clinical consequence of the storage lesions is a reduced survival time of red blood cells after the transfusion . whether stored rbc is also compromised in transporting oxygen to tissue is still controversial . several clinical [ 93 , 96 , 97 ] and experimental animal studies [ 98 , 99 ] support the notion that rbc older than 3 weeks have a reduced ability to transport oxygen to the tissues . animal studies have found a malperfused and underoxygenated microvasculature after haemodilution and a 26% decrease in micro vascular oxygen concentration after transfusion with washed human rbcs in haemodiluted rats . in human a recent study has shown increased risk of severe postoperative infections after cardiac surgery associated with the use of old rbc . however , the findings are not uniform as a recent clinical study could not demonstrate an effect of stored blood on oxygenation parameters and another did not observe an increase in morbidity in coronary artery bypass graft surgery patients receiving old rbcs or could not find a correlation between median ages of the rbc units and clinical outcomes . these differences in study outcomes may be explained by the inherent difficulty to measure tissue oxygen concentrations in the clinical setting . to estimate the effect of rbc transfusion on tissue oxygenation in patients , indirect measuring techniques have been used as surrogate endpoints . in addition , the diversity in the design of the performed studies complicates a proper comparison of their results . there seems to be an agreement that when a substantial part of the units issued for transfusion is stored for more than 3 weeks , a discussion about fresh and stored rbc could be clinically relevant . the evidence supports that each institution establishes its own patient blood management strategy to both conserve blood products and maximise outcome . newer transfusion guidelines in patients with coexisting cardiac diseases are similar to those in patients without such comorbidities . thus , rbc transfusions are most often indicated at haemoglobin levels below 6.0 g / dl ( 3.7 mmol / l ) and hardly ever at haemoglobin levels above 10 g / dl ( 6.1 mmol / l ) . in cardiac surgery , especially in cad patients , the lower limit should probably be elevated to 7 g / dl ( 4.3 mmol / l ) , though no available hard evidence . institutions should evaluate how they approach transfusion and each physician should evaluate each patient for signs and symptoms of ischemia or imbalance in oxygen supply and delivery , where the major challenge is how to decide when that imbalance is occurring in certain tissues . evidently such signs may be reached at higher haemoglobin values than in healthy patients , but further studies are needed to fully confirm that transfusing these patients at a higher haemoglobin level positively is making more good than harm . following this teams and institutions need to conduct open discussions about how they will approach patient blood management and instead of previous fixed haemoglobin levels the best practice would be individual haemoglobin / haematocrit triggers . strategy of transfusion : make local policy and guidelines for transfusion including preferably developing an algorithm for transfusioneducation and knowledge of physicians and nursespolicy on blood saving precautions , mechanical or medicalevaluation of cost of blood / blood products and cost alternativesavailability of blood and blood productstransfusion of only actual bleeding and/or unstable patientssafety related to needaffection of immune system acceptable ? preoperative evaluation deciding blood saving precautionscardiovascular reservedefining acceptable blood loss / haematocrit / absolute haemoglobin content . evaluate perioperative haematocrit look at it do not treat it butevaluate the absolute amount of haemoglobin . strategy of transfusion : make local policy and guidelines for transfusion including preferably developing an algorithm for transfusioneducation and knowledge of physicians and nursespolicy on blood saving precautions , mechanical or medicalevaluation of cost of blood / blood products and cost alternativesavailability of blood and blood productstransfusion of only actual bleeding and/or unstable patientssafety related to needaffection of immune system acceptable ? preferably developing an algorithm for transfusion education and knowledge of physicians and nurses policy on blood saving precautions , mechanical or medical evaluation of cost of blood / blood products and cost alternatives availability of blood and blood products transfusion of only actual bleeding and/or unstable patients safety related to need affection of immune system acceptable ? preoperative evaluation deciding blood saving precautionscardiovascular reservedefining acceptable blood loss / haematocrit / absolute haemoglobin content . deciding blood saving precautions cardiovascular reserve defining acceptable blood loss / haematocrit / absolute haemoglobin content . evaluate perioperative haematocrit look at it do not treat it butevaluate the absolute amount of haemoglobin . haematocrit look at it do not treat it but evaluate the absolute amount of haemoglobin . reevaluate cardiovascular capacity and decide transfusion need .

blood transfusion is associated with increased morbidity and mortality and numerous reports have emphasised the need for reduction . following this there is increased attention to the concept of patient blood management . however , bleeding is relatively common following cardiac surgery and is further enhanced by the continued antiplatelet therapy policy . another important issue is that cardiopulmonary bypass leads to haemodilution and a potential blood loss . the basic role of blood is oxygen transport to the organs . the determining factors of oxygen delivery are cardiac output , haemoglobin , and saturation . if oxygen delivery / consumption is out of balance , the compensation mechanisms are simple , as a decrease in one factor results in an increase in one or two other factors . patients with coexisting cardiac diseases may be of particular risk , but studies indicate that patients with coexisting cardiac diseases tolerate moderate anaemia and may even benefit from a restrictive transfusion regimen . further it has been shown that patients with reduced left ventricular function are able to compensate with increased cardiac output in response to bleeding and haemodilution if normovolaemia is maintained . in conclusion the evidence supports that each institution establishes its own patient blood management strategy to both conserve blood products and maximise outcome .

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this process requires proliferation , differentiation and migration of neurogenic stem cells into neurons . for many decades , it was believed that in mammals , neurogenesis occurs only during embryonic life , but a recent work has made it evident that neuronal regeneration occurs even after birth in specific tissues like the hippocampus , dentate gyrus , subventricular zone and olfactory epithelium ( 1 ) . damage to the olfactory periphery destroys the population of olfactory sensory neurons and , in the case of direct epithelial lesion , also eliminates other constituents of the epithelium . in marked contrast to other parts of the nervous system , there is substantial anatomical and functional recovery of the olfactory epithelium and its projection into the cns even in the face of overwhelming injury ( 2 ) . years , neuroscience researchers have searched for an ideal cell with the properties of absolute totipotency and regenerability in vitro and in vivo . science has come very close to the quest , but still many unanswered questions remain . recent studies indicate that transplantation of pure stem cell population is insufficient for attaining the maximum positive effect of neural regeneration . more complete recovery of the structure , sensory and motor functions of injured spinal cord can be attained using a complex of cells , including , apart from olfactory ensheathing cells ( oec ) , fibroblasts , astrocytes , schwann cells , and olfactory epithelial ( oe ) multipotent stem and progenitor cells . hence , the problem of the efficiency and the possibility of using various oe cells and tissues for transplantation therapy in cerebral and spinal injuries doubtlessly requires further experimental studies . nevertheless , consistent efforts with increasing optimistic outcome are being made to identify a candidate ( neuron- progenitor cell ) for a reliable therapeutic intervention of central nervous system ( cns ) injury . in this venture , recent years witnessed an increasing bang on the olfactory epithelium for suitable multipotent stem cells . multiple numbers of pluripotent stem cell candidates have been tried in the past for neuro - regenerative therapies with unconvincing results ( 3 ) . studies involving exposure of olfactory epithelium ( and causing its lesion ) to an irritant like methyl bromide ( mebr ) have shown that the olfactory epithelium is restored its normal status that is indistinguishable from unlesioned epithelium within 68 weeks after damage with no relation to the severity of the initial damage which could be up to 90% of the epithelium being destroyed . the first sign of regeneration of olfactory neurons appears on the 4 day after mebr exposure , the first mature neurons emerge during the 2 week after which there is an accelerated production of neurons which falls to normal around the 6 week after the lesion ( 2 ) . the reconstitution of the epithelium is sufficiently robust and precise so that the spatial distribution and the numbers of odorant receptors ( or ) are restored to normal ( 4 ) . transplantation of adult animal and human oe cells have been used in the past for experimental and clinical correction of spinal injuries ( 5 ) . the adult olfactory epithelium ( oe ) is a unique ( for its capacity to renew olfactory receptor neurons throughout adult life ) and a complex tissue containing heterogeneous population of epithelial cells . apart from the support cells and neruoreceptor cells , this complex is said to retain a kind of progenitor cells that are competent to make neurons ( neural stem cells ) and non- neural support cells like olfactory ensheathing cells , oligodendrocytes and schwann cells ( 6 ) . these support cells , play a role in the regeneration and myelination process of normal and of injured cns . recent studies identified stem like characteristics in a group of cells called globose basal cells ( gbcs ) residing in the basal compartment of the olfactory epithelium . the gbcs are the small , round , morphologically non - descript and cyto - keratin negative cells that sit between the horizontal basal cells ( hbcs ) below and the immature olfactory receptor neurons above , that proliferate at a high rate in the normal oe , are limited to the oe , and are poorly characterized at the level of their molecular phenotype . further , a second population of cells that are called the hbcs with stem - cell like properties has been described to reside among the gbcs . therefore , it appears that the olfactory epithelium contains at least two populations of cells with possible neuropotency ( 7 ) . some studies have shown that there is an increase in the population of gbcs and not the hbcs during the regenerative process of the olfactory epithelium ( 8) . gbcs function as broadly multipotent progenitors capable of giving rise to neurons and all of the cell types of the epithelium , and hence may be totipotent stem cells of the epithelium . marker studies using antibodies that are selective for gbcs in normal epithelium , during the acute phase in the recovery after mebr exposure , suggest that the gbcs are differentiating into non - neuronal cell types ( 9 ) . intranasal infusions of gbcs derived from retroviruses , give rise to neurons and multiple types of non - neuronal cells , while other stem cell progenitors give rise only to non - neuronal cells ( 10 ) . gbcs that are separated from the normal epithelium by cell cytometry using a gbc - selective primary antibody and are pure and free from other cells and contaminants , engraft easily into the mebr - lesioned epithelium after infusion into the nasal cavity and give rise to most of the neural and non - neural cells present in the oe ( 11 ) . embryonic stem cells ( 3 ) , haematopoetic stem cells and other mesenchymal stem cells have enormous ability to differentiate into different types of cells in the body ; but they are still the possibilities of immune rejection , adverse effects of immunosuppressive therapy and many ethical issues which exclude them as an ideal candidate for clinical practice ( 12 ) . moreover , these different varieties of stem cells do not reside in a niche similar to the neural stem cells and hence can not proliferate easily in the central nervous system . on the other hand , olfactory epithelial cells share the same environmental niche as the central nervous system and also have the same developmental hierarchy as the neural cells . olfactory mucosa is readily accessible , easily biopsied for autologous cell transplantation and regenerates completely without any loss of function . these properties make the olfactory epithelial stem cells the ideal candidate for use in clinical research and therapy . since the basal compartment contains two morphologically distinct cell types , horizontal basal cells ( hbcs ) and globose basal cells ( gbcs ) , there has been arguments regarding the identification of the neural stem cell population which can not be answered merely based on their location and mitotic activity and studies focusing on search for unique stem cell progenitor markers have to be undertaken ( 8 , 13 , 14 ) . researchers in the recent years have proposed the most likely lineage of the cells in the olfactory epithelium by using specific markers , determination of various transcription factor expressions and incorporation of analytical studies using h - thymidine and bromodeoxyuridine which suggest the proliferative activity of dividing cells ( 15 , 16 ) . it is now clear that immature orns are generated from one of the basal cell types and they in turn give rise to mature orns , but the identity of these cells has to be further characterised . only in limited experiments , it has been shown that gbcs are the cell population to multiply and regenerate vigorously after epithelial damage . there is still an uncertainty of the lineage relationship between the two basal cell populations . in this research , we study different neural progenitor markers and ionic channels that are present on the gbcs and hence characterize these cells as definitive mutipotent stem cells residing in the basal compartment of rat olfactory epithelium capable of transforming into neurons and non - neuronal elements . six albino wistar rats were purchased from the animal facility of christian medical college vellore after obtaining the clearance from the animal ethics committee . albino wistar rats are the most commonly used laboratory animals for genetic and cell line studies as many aspects of their behaviour and physiology are similar to humans and this similarity can be easily observed in them when compared to other animals ( 17 ) . this strain of rats was used in this study since their olfactory mucosa is easily accessible and their olfactory epithelium is histologically similar to that of the human . all animals were weighed prior to the experimental ( 94 -120 g , with an average of 105 g ) . the olfactory mucosa of the rats was obtained by surgical excision under complete , universal sterile conditions . the albino wistar rats were anesthetized by intraperitoneal administration of 90 mg / kg body weight of ketamine and 10 mg / kg body weight of xylazine . the nasal septum was completely excised along three lines : the arc of the perpendicular plate , the cribriform plate and the ceiling of the oral cavity to obtain the olfactory mucosa ( figure 1 ) . the nasal septum along with the olfactory mucosa was placed in frozen dulbeco s modified eagle s medium ( dmem ) containing antibiotics ( penicillin , streptomycin and amphotericin ) . the nasal septum along with the olfactory mucosa was then treated with 0.5 ml of dispase ii and was incubated at 37c for 45 min . the olfactory epithelium was separated from the lamina propria on both sides of the nasal septum using a micro spatula under a leica ez2hd dissecting microscope . the olfactory mucosa was mechanically dissociated with a scalpel blade and treated with 0.05% trypsin and an enzymatic cocktail containing collagenase 1 mg / ml , hyaluronidase 1.5 mg / ml and trypsin inhibitor 0.1 mg / ml to separate the cells . the oe cells were transferred to epithelial culture medium which was modified form of the previously described medium ( 18 - 20 ) . the culture medium was composed of dmem / f12 ( 1 : 1 ) 47.5 ml , 2% fetal bovine serum ( invitrogen , gibco , india ) 1.0 ml , n2 supplement ( invitrogen , gibco , india ) 0.5 ml , epidermal growth factor ( 25 ng / ml invitrogen , gibco , india ) 12.5 l and l - glutamine ( invitrogen , gibco , india ) 0.5 ml . a - arc of perpendicular plate , b - cribriform plate , c - ceiling of oral cavity . om - olfactory mucosa ( yellowish ) , rm - respiratory mucosa ( pinkish ) ten l of gbc iii antibody was added to the epithelial cell pellet under dark conditions and was left in ice for 45 min . gbc - iii is a mouse monoclonal igm antibody which recognizes a 40 kda surface antigen which is a laminin receptor surface protein . it is highly specific as a marker for gbcs , unlike the earlier antibodies used like gbc - i which were nonspecific markers for gbcs and showed positive reaction even with hbcs , sus and duct cells ( 21 ) . gbc - iii in powdered form was reconstituted into the recommended volume of 250 l . ten l of r - phycoerythrin - conjugated affinipure ( fab)2 fragment goat anti - mouse igm ( secondary antibody ) was added to the cell pellet and left under dark conditions in ice for 10 min . fluorescence assisted cell sorting ( facs ) was used to separate the gbcs from other epithelial cells . finally , 99% pure gbcs were obtained using facs and gbc - iii antibody and were plated in the epithelial medium . gbc cell suspension in the epithelial medium was washed 3 times with hanks balanced salt solution . cells were trypsinised using 100 l of 0.1% trypsin / edta and incubated at 37c for 5 min . epithelial medium was added immediately after incubation to neutralize the enzymes and the cell suspension was centrifuged at 1000 rpm for 8 min . a poly d - lysine plated cover slip was placed in a 35 mm petridish . the cell suspension was then plated on this cover slip and incubated for 2 hr . micropipettes were prepared using f-500 fine point microforge to have the tip diameter of 1 micron . the cover slip coated with the cells was washed with ecf ( extra cellular fluid ) and was placed in a 35 mm petridish filled with ecf . composition of the neuron solution ecf was nacl 135 mm , kcl 4 mm , nah2po4 1 mm , cacl2 1.2 mm , mgcl2 0.5 mm , hepes 10 mm and glucose 10 mm . all the micropipettes were filled with icf ( intra cellular fluid ) before being attached to the pipette electrode . composition of the neuron solution icf was kcl 140 mm , egta 1 mm , mgcl2 1 mm , hepes 10 mm and glucose - 10 mm . the petridish was placed on the stage of a phase contrast microscope and the bath electrode was introduced into the ecf . the micropipette tip was gently approximated to the cell membrane of the gbc and a negative suction was given to form a seal . the average seal resistance for the gbcs was between 1.2 m ohm and 2 m ohm . multiple sweeps were taken to calculate the current flow and analysed using the clampex scope episodic software . immunostaining of pure gbcs was done with multiple numbers of neural progenitor stem cell markers to characterize gbcs as neural progenitor cells . primary antibodies used for immunostaining were anti - ncam , anti - nestin , anti - sox-1 , anti - sox-2 , anti - cd29 , anti - cd54 and anti - cd73 . secondary antibodies used were goat anti - mouse igg 1 and goat ant - rabbit igg ( percp - conjugated affinipure f(ab)2 fragment ) . cover slips ( coated with poly d - lysine ) plated with the gbcs were washed in pbs 3 times . cover slips were placed on the slides and blocked using 2% goat serum ( blocking agent ) mixed with 0.1 % triton x ( permeating agent ) for 2 hr at room temperature . after blocking , the slides were washed 3 times with pbs and treated with the primary antibodies and incubated overnight at 4c . dilutions used for different primary antibodies were as follows ; anti ncam- 1:50 , anti nestin- 1:20 , anti sox 1- 1:100 , anti sox 2- 1:5 , anti cd29- 1:50 , anti cd54- 1:50 and anti cd73- 1:50 . six albino wistar rats were purchased from the animal facility of christian medical college vellore after obtaining the clearance from the animal ethics committee . albino wistar rats are the most commonly used laboratory animals for genetic and cell line studies as many aspects of their behaviour and physiology are similar to humans and this similarity can be easily observed in them when compared to other animals ( 17 ) . this strain of rats was used in this study since their olfactory mucosa is easily accessible and their olfactory epithelium is histologically similar to that of the human . all animals were weighed prior to the experimental ( 94 -120 g , with an average of 105 g ) . the olfactory mucosa of the rats was obtained by surgical excision under complete , universal sterile conditions . the albino wistar rats were anesthetized by intraperitoneal administration of 90 mg / kg body weight of ketamine and 10 mg / kg body weight of xylazine . the nasal septum was completely excised along three lines : the arc of the perpendicular plate , the cribriform plate and the ceiling of the oral cavity to obtain the olfactory mucosa ( figure 1 ) . the nasal septum along with the olfactory mucosa was placed in frozen dulbeco s modified eagle s medium ( dmem ) containing antibiotics ( penicillin , streptomycin and amphotericin ) . the nasal septum along with the olfactory mucosa was then treated with 0.5 ml of dispase ii and was incubated at 37c for 45 min . the olfactory epithelium was separated from the lamina propria on both sides of the nasal septum using a micro spatula under a leica ez2hd dissecting microscope . the olfactory mucosa was mechanically dissociated with a scalpel blade and treated with 0.05% trypsin and an enzymatic cocktail containing collagenase 1 mg / ml , hyaluronidase 1.5 mg / ml and trypsin inhibitor 0.1 mg / ml to separate the cells . the oe cells were transferred to epithelial culture medium which was modified form of the previously described medium ( 18 - 20 ) . the culture medium was composed of dmem / f12 ( 1 : 1 ) 47.5 ml , 2% fetal bovine serum ( invitrogen , gibco , india ) 1.0 ml , n2 supplement ( invitrogen , gibco , india ) 0.5 ml , epidermal growth factor ( 25 ng / ml invitrogen , gibco , india ) 12.5 l and l - glutamine ( invitrogen , gibco , india ) 0.5 ml . a - arc of perpendicular plate , b - cribriform plate , c - ceiling of oral cavity . ten l of gbc iii antibody was added to the epithelial cell pellet under dark conditions and was left in ice for 45 min . gbc - iii is a mouse monoclonal igm antibody which recognizes a 40 kda surface antigen which is a laminin receptor surface protein . it is highly specific as a marker for gbcs , unlike the earlier antibodies used like gbc - i which were nonspecific markers for gbcs and showed positive reaction even with hbcs , sus and duct cells ( 21 ) . gbc - iii in powdered form was reconstituted into the recommended volume of 250 l . ten l of r - phycoerythrin - conjugated affinipure ( fab)2 fragment goat anti - mouse igm ( secondary antibody ) was added to the cell pellet and left under dark conditions in ice for 10 min . fluorescence assisted cell sorting ( facs ) was used to separate the gbcs from other epithelial cells . finally , 99% pure gbcs were obtained using facs and gbc - iii antibody and were plated in the epithelial medium . gbc cell suspension in the epithelial medium was washed 3 times with hanks balanced salt solution . cells were trypsinised using 100 l of 0.1% trypsin / edta and incubated at 37c for 5 min . epithelial medium was added immediately after incubation to neutralize the enzymes and the cell suspension was centrifuged at 1000 rpm for 8 min . a poly d - lysine plated cover slip was placed in a 35 mm petridish . the cell suspension was then plated on this cover slip and incubated for 2 hr . micropipettes were prepared using f-500 fine point microforge to have the tip diameter of 1 micron . the cover slip coated with the cells was washed with ecf ( extra cellular fluid ) and was placed in a 35 mm petridish filled with ecf . composition of the neuron solution ecf was nacl 135 mm , kcl 4 mm , nah2po4 1 mm , cacl2 1.2 mm , mgcl2 0.5 mm , hepes 10 mm and glucose 10 mm . all the micropipettes were filled with icf ( intra cellular fluid ) before being attached to the pipette electrode . composition of the neuron solution icf was kcl 140 mm , egta 1 mm , mgcl2 1 mm , hepes 10 mm and glucose - 10 mm . the petridish was placed on the stage of a phase contrast microscope and the bath electrode was introduced into the ecf . the micropipette tip was gently approximated to the cell membrane of the gbc and a negative suction was given to form a seal . the average seal resistance for the gbcs was between 1.2 m ohm and 2 m ohm . multiple sweeps were taken to calculate the current flow and analysed using the clampex scope episodic software . immunostaining of pure gbcs was done with multiple numbers of neural progenitor stem cell markers to characterize gbcs as neural progenitor cells . primary antibodies used for immunostaining were anti - ncam , anti - nestin , anti - sox-1 , anti - sox-2 , anti - cd29 , anti - cd54 and anti - cd73 . secondary antibodies used were goat anti - mouse igg 1 and goat ant - rabbit igg ( percp - conjugated affinipure f(ab)2 fragment ) . cover slips ( coated with poly d - lysine ) plated with the gbcs were washed in pbs 3 times . cover slips were placed on the slides and blocked using 2% goat serum ( blocking agent ) mixed with 0.1 % triton x ( permeating agent ) for 2 hr at room temperature . after blocking , the slides were washed 3 times with pbs and treated with the primary antibodies and incubated overnight at 4c . dilutions used for different primary antibodies were as follows ; anti ncam- 1:50 , anti nestin- 1:20 , anti sox 1- 1:100 , anti sox 2- 1:5 , anti cd29- 1:50 , anti cd54- 1:50 and anti cd73- 1:50 . the basal cells in the olfactory epithelium are believed to be the easily accessible autologous stem cells in the adults . however selection and isolation of the specific basal cells which are progenitors for neural cells are still being studied . the present research work helped us to isolate and decipher numerous new facts about the globose basal stem cells in the olfactory epithelium of rats . culture medium used for growing and obtaining maximum confluency of the olfactory epithelial cells was standardized . the epithelial medium was composed of chemicals , enzymes and factors essential for epithelial cell proliferation and neurosphere formation . the desirable viable cell density for obtaining optimum growth of epithelial cells on the media was calculated to be 10 10 . phase contrast image showing 2 generation of neurospheres on the 14 day in epithelial medium as it is shown in figure 3 , 99% pure gbcs were obtained using facs and gbc - iii antibody . the highest number of gbcs was found in the area with the total cell density of 4 10 ( figures 4 and 5 ) . immunofluorescent microscopic picture showing gbc stained with gbc - iii antibody facs diva 6.0 graph showing p1 and p2 areas . green- gbc - iii positive cells ; red- gbc - iii negative cells facs diva 6.0 of unstained cells used as negative control . green- gbc - iii positive cells ; red- gbc - iii negative cells gbcs sorted by facs were immunohistochemically characterised using various neural progenitor cell markers . immunostaining of gbcs was positive with anti - ncam ( figure 6 , va ) , anti - nestin ( figure 6 , vb ) and anti - sox 2 ( figure 6 , vc ) , hence confirming that these cells contain intracellular proteins like ncam , nestin and sox 2 and are neural progenitor cells . gbcs staining was also positive for cd-29 ( figure 6 , vd ) , cd-54 ( figure 6 , ve ) and cd-73 ( figure 6 , vf ) which are cell surface markers present on a variety of cells including neural progenitor cells . marker study with sox-1 was negative probably because of reduced number of gbc in that respective well . electrophysiological characterisation of the isolated gbcs was done using patch clamping ( figure 7 ) . whole cell voltage - clamp recording from gbc was obtained using 60% series resistance compensation , following correction . also , + 1 mv increments were applied starting from -40 mv to -10 mv to check for opening of any voltage gated na ( sodium ) channels . voltage gated na channels were completely absent , hence proving the unexcitable nature of gbcs ( figure 8) . then , + 10mv increments were applied starting from -150 mv to + 150 mv to check for opening of any voltage gated k ( potassium ) channels . a smooth i - v curve was not obtained and only leaky k channels were found to be present on the gbc which was of no significance ( figure 9 ) . va : immunofluorescent image of a gbc stained with anti - ncam antibody ( 10x ) vb : immunofluorescent image showing nestin positive gbcs ( 10x ) vc : immunofluorescent image showing sox-2 positive gbcs ( 20x ) vd : immunofluorescent image of cd-29 positive gbcs ( 10x ) . nuclei is stained with dapi ve : immunofluorescent image of cd-54 positive gbcs ( 40x ) vf : immunofluorescent image of cd-73 positive gbcs ( 20x ) . 29 0f 30 sweeps are seen left arrow- na channels ( closed ) ; middle arrow- leaky k channels ; right arrow- holding voltage i - v curve of potassium current showing leaky channels . culture medium used for growing and obtaining maximum confluency of the olfactory epithelial cells was standardized . the epithelial medium was composed of chemicals , enzymes and factors essential for epithelial cell proliferation and neurosphere formation . it further enhanced the capability of the cells to propagate and form neurospheres . the desirable viable cell density for obtaining optimum growth of epithelial cells on the media was calculated to be 10 10 . as it is shown in figure 3 , 99% pure gbcs were obtained using facs and gbc - iii antibody . the highest number of gbcs was found in the area with the total cell density of 4 10 ( figures 4 and 5 ) . immunofluorescent microscopic picture showing gbc stained with gbc - iii antibody facs diva 6.0 graph showing p1 and p2 areas . green- gbc - iii positive cells ; red- gbc - iii negative cells facs diva 6.0 of unstained cells used as negative control . green- immunostaining of gbcs was positive with anti - ncam ( figure 6 , va ) , anti - nestin ( figure 6 , vb ) and anti - sox 2 ( figure 6 , vc ) , hence confirming that these cells contain intracellular proteins like ncam , nestin and sox 2 and are neural progenitor cells . gbcs staining was also positive for cd-29 ( figure 6 , vd ) , cd-54 ( figure 6 , ve ) and cd-73 ( figure 6 , vf ) which are cell surface markers present on a variety of cells including neural progenitor cells . marker study with sox-1 was negative probably because of reduced number of gbc in that respective well . electrophysiological characterisation of the isolated gbcs was done using patch clamping ( figure 7 ) . whole cell voltage - clamp recording from gbc was obtained using 60% series resistance compensation , following correction . also , + 1 mv increments were applied starting from -40 mv to -10 mv to check for opening of any voltage gated na ( sodium ) channels . voltage gated na channels were completely absent , hence proving the unexcitable nature of gbcs ( figure 8) . then , + 10mv increments were applied starting from -150 mv to + 150 mv to check for opening of any voltage gated k ( potassium ) channels . a smooth i - v curve was not obtained and only leaky k channels were found to be present on the gbc which was of no significance ( figure 9 ) . va : immunofluorescent image of a gbc stained with anti - ncam antibody ( 10x ) vb : immunofluorescent image showing nestin positive gbcs ( 10x ) vc : immunofluorescent image showing sox-2 positive gbcs ( 20x ) vd : immunofluorescent image of cd-29 positive gbcs ( 10x ) . nuclei is stained with dapi ve : immunofluorescent image of cd-54 positive gbcs ( 40x ) vf : immunofluorescent image of cd-73 positive gbcs ( 20x ) . 29 0f 30 sweeps are seen left arrow- na channels ( closed ) ; middle arrow- leaky k channels ; right arrow- holding voltage i - v curve of potassium current showing leaky channels . neurogenesis begins in the early embryonic period ( 4 week ) and continues 10 to 12 years after birth ( 22 ) . the nervous system development is completed only during the adolescent period when myelination of the neurons of major tracts is completed ( 23 ) . early researchers believed the nervous system to be fixed and incapable of proliferation and regeneration based on the their opinions that neurons and non - neuronal cells in the nervous system could not divide actively via mitosis and they lacked the potential to transform from simple to more complex forms of neuronal morphologies . another reason for such belief was inability of researchers in the past to demonstrate progenitor stem cells residing in the nervous system niche by specific markers . cells in the nervous system could be demonstrated only by negative staining which was very non - specific ( 18 , 22 , 23 ) . the earliest demonstration of neurogenesis in adults was done by joseph altman in 1962 in the cerebral cortex ( 24 ) followed by demonstration of neurogenesis in the dentate gyrus of the hippocampus in 1963 ( 25 ) . later , it was evident that neurogenesis also occurred in the subgranular zone and subventricular zone of the lateral ventricles ( 26 ) . now , researchers also believe that neurogenesis continues in the cerebellum during adulthood ( 27 ) . the most recent development in this field of research is the demonstration of neurogenesis in the olfactory system including the olfactory bulbs and more importantly in the olfactory mucosa . this research work studies one such population of stem cells residing in the olfactory epithelium which has the niche required to get accepted in the cns environment and is derived from the same germ line as the neuronal cells . this gives a hint that these might be the ideal neural progenitor cells which can be easily harvested and used for autologous transplantations . olfactory epithelial- mesenchymal stem cells have been isolated in the past from the olfactory mucosa using enzymatic dissociation with collagenase ia ( 28 ) in an impure form . isolated cultures of gbcs and horizontal basal cells have been grown in earlier experiments by immunoadhesion ( 29).this research work standardizes chemical methods to isolate these stem cells from the olfactory epithelium in a pure form . epithelial cells were harvested on the culture media after calculating the feasible cell densities for optimal growth and neurosphere formation . densities higher than this resulted in overcrowding of cells and loss of cells due to unwanted cell death . densities lower than this did not provide us with adequate number of cells and hindered neurosphere generation . the self renewal potential of olfactory stem cells was evident from the formation of multiple generations of neurospheres . egf and n-2 supplement seem to play a significant role in providing an optimum external niche for formation of multiple generations of neurospheres from epithelial stem cells . egf is a potential stem cell mitogen allowing cell proliferation through activating signalling pathway and inducing the production of beta fibroblast growth factor ( fgf ) from the progenitor cells . olfactory mucosa in cultures has been shown to produce certain soluble factors which help with proliferation , regeneration and neurosphere formation ( 30 ) . the area p1 was chosen with relatively higher percentage of gbc - iii stained cells and a graph was plotted showing the gbc density to be 65.3% of the total number of cells . p2 , a part of p1 , with the highest cell density was chosen and a similar graph was plotted which showed the gbc density to be 79.9% of the total ( figure 4 ) . . an insignificant number of 41 positive cells ( accounting for 0.4% of the total ) were sorted from the negative control proving the efficacy of the procedure ( figure 5 ) . gbcs have been isolated in the past from other progenitor cells of the olfactory epithelium using facs by tagging them with less sensitive and specific antibodies like gbc - i ( 9 ) and gbc - ii ( 29 ) . the complexities of the phenotypical stages of transformation of the gbcs into olfactory neurons upon neuroablation of the olfactory bulb have been explained using enhanced green[t6 ] fluorescent protein tagged with markers like sox2 and neurog1 ( 31 ) . further research on gbcs and other potential neural progenitor cells present in the olfactory mucosa will help us to understand their biology and effectiveness in neurogenesis . it will also help to apply these results to medical use for therapeutic treatment of different diseases . few areas on which special attention can be drawn in future are : isolation of gbcs from the oe using magnetic assisted cell sorting ( macs ) . hence , its usage will help us to compare its efficacy with facs and a better purity of cells can be obtained ( 32).electrophysiological studies to find out more about different ion channels present on the gbcs will help us to have a better understanding of the functional properties of these cells and their drug interactions.animal studies using gbcs to repair the spinal cord injuries and other nervous system disorders should be the ultimate aim to propagate and effectively utilize the present knowledge on these cells in human trials . hence , its usage will help us to compare its efficacy with facs and a better purity of cells can be obtained ( 32 ) . electrophysiological studies to find out more about different ion channels present on the gbcs will help us to have a better understanding of the functional properties of these cells and their drug interactions . animal studies using gbcs to repair the spinal cord injuries and other nervous system disorders should be the ultimate aim to propagate and effectively utilize the present knowledge on these cells in human trials . this research work has been able to throw some light on the prevailing ambiguity of the olfactory stem cells and has derived some definite conclusions about the nature and behaviour of these cells . non - excitable nature of gbcs has been documented in the present study . positive expression of all the neural progenitor cell markers has proved substantially about the neuro - transformation capability of the gbcs . now , after undertaking this study , we can conclude with concrete explanations that gbcs are the colony of neural stem cells residing in the basal compartment of the olfactory epithelium responsible for the ongoing neurogenesis in the oe throughout adult life .

objective(s):in the past few decades , variety of foetal , embryonic and adult stem and progenitor cells have been tried with conflicting outcome for cell therapy of central nervous system injury and diseases . cellular characteristics and functional plasticity of globose basal stem cells ( gbcs ) residing in the olfactory epithelium of rat olfactory mucosa have not been studied in the past by the neuroscientists due to unavailability of specific markers for gbcs . in the present research , we standardized some techniques to isolate gbcs from rat olfactory epithelium in pure form using a highly selective gbc - iii antibody passaged through fluorescence activated cell sorter ( facs ) . we also characterized these cells immunohistologically using various pluripotent stem cell markers . this work also throws some light on ionic channels present on these stem cells which are responsible for their neuron induction potential.materials and methods : globose basal stem cells were isolated from rat olfactory epithelium using gbc - iii antibody and were characterized as multipotent stem cells using various neural progenitor markers . ionic channels on gbcs were studied with voltage clamping.results:gbcs could be isolated in pure ( 99% purity ) form and were found to be stained positive for all neural progenitor cell markers . voltage gated na+ channels were completely absent , which proves the unexcitable nature of gbcs . leaky k+ channels were found to be present on the gbc which was of no significance.conclusion:this research work can be helpful in understanding the nature of these stem cells and utilising them in future as potent candidates for neuro - regenerative therapies .

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in the past , the central nervous system ( cns ) was considered a site of immune privilege . microglia , first described by achcarro and subsequently thoroughly characterized by his successor , rio - hortega , are now known to play a pivotal role in innate immunity within the cns . in addition , emerging evidence demonstrates the presence of a conduit that enables communication between the brain parenchyma and cervical lymph nodes via the glymphatic system ( an interstitial fluid ( isf)/cerebrospinal fluid ( csf ) exchange system located in the perivascular space ) ( figure 1 ) . along with brain microglia , this exchange system facilitates immune surveillance of brain isf . these findings suggest that the cns immune system is more constitutively active than previously envisioned . a currently supported view concerning the origin of microglia postulates that they are derived from erythromyeloid progenitors in yolk sac that enter the brain during different developmental stages in the brain . yolk sac erythromyeloid progenitors express the chemokine receptor cx3cr1 . to closely analyze the fate of erythromyeloid progenitors in the cns during embryogenesis , kierdorf et al . used cx3cr1gfp / wt mice , which contain a gfp knock - in on one allele of the cx3cr1 gene . they discovered that mouse microglia were derived from primitive c - kit erythromyeloid precursors present in the yolk sac of embryos at embryonic age of 7.5 to 8.0 days ( e7.5e8.0 ) ( figure 2(a ) ) . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . microglial maturation is accompanied by a reduction in cd31 expression and concomitant upregulation of the macrophage marker , f4/80 , and macrophage colony stimulating factor receptor ( mcsf - r ) . proliferating a2 cells develop into microglia and invade the developing brain at e9.5 , a process that requires activity of matrix metalloproteinase . after entering the cns , inhibition of microglia - mediated synaptic pruning results in aberrant brain circuitry with excessive dendritic spines and immature synapses . within the postnatal brain , microglia are maintained through cellular division or expansion of progenitors that colonized the brain during cns development . microglia form an autonomous population of cells within the brain that demonstrate activities independent of circulating monocyte activity . resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . figure 2.(a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . proliferating a2 cells develop into embryonic microglia . before completion of the blood brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . resident microglia are distributed in the brain parenchyma , and use motile processes to survey the microenvironment ( left ) . the brain parenchyma is equipped with the glymphatic system , which clears interstitial solutes through the exchange of isf and csf along the perivascular space between the wall of the veins and astrocytic endfeet . subsequently , csf is absorbed into the meningeal lymphatic vessels , which are located in the vicinity of the venous sinus . ( a ) schematic representation of microglial development : microglia are derived from c - kit - positive erythromyeloid precursors ( emps ) in the yolk sac at e7.5e8.0 . these precursors develop into cd45 c - kit cx3cr1 immature ( a1 ) cells and mature into cd45 c - kit cx3cr1 ( a2 ) cells . proliferating a2 cells develop into embryonic microglia . before completion of the blood brain barrier ( bbb ) , they invade the developing brain in a manner requiring matrix metalloproteinase ( mmp ) activity at e9.5 . unless the bbb is disrupted , microglia and monocytes are independent of each other in the postnatal stage . ( b ) multiple roles of microglia in the postnatal brain : microglia in the postnatal brain are multifunctional . primarily , they are involved in immune surveillance , and when necessary , they initiate an inflammatory response . although this contributes to the elimination of pathogens , an excessive inflammatory response may result in bystander tissue damage . microglia also phagocytose pathogens and apoptotic neurons and exert scavenging actions on tissue debris under pathological conditions . microglia recognizes apoptotic cells by phosphatidylserine and other apoptotic signals . with regard to synaptic refinement in addition , microglia - derived tnf can exert neuroprotective as well as neurodestructive action within different contexts . microglia also play a role in angiogenesis through by synthesizing vascular endothelial growth factor ( vegf ) . in the postnatal brain , microglia have multiple functions ( figure 2(b ) ) . as the powerhouse of local innate immunity , microglia protect the postnatal brain from infection . in response to pathogens invading the cns , microglia act as a front - line defense system by phagocytosing pathogens , secreting humoral substances , such as proinflammatory cytokines , and producing reactive oxygen species ( ros ) and reactive nitrogen species ( rns ) . microglia - producing proinflammatory cytokines include interleukin-1 ( il-1 ) , il-6 , and tumor necrosis factor ( tnf ) . the activation pattern of microglia in response to invading pathogens is typical of the classical proinflammatory phenotype of macrophages ( m1 polarization ) . for instance , cerebral ischemia and amyloid ( a ) stimulate il-1 synthesis in microglia through the activation of the inflammasome . they tend to retract their processes and under extreme conditions , assume an amoeboid shape . dead neurons expose phosphatidylserine and other apoptotic eat - me signals on their cell surface , which prompts microglia to carry out phagocytosis . such microglia - mediated clearance of unwanted neurons is an important process of proper brain tissue remodeling . with regard to microglia function under the physiological condition , recent in vivo imaging experiments have uncovered that these resident microglia constantly survey their surrounding microenvironment with their motile processes , which is likely relevant to immune surveillance . however , such motion has been observed even in the absence of offending microorganisms . increasing evidence has revealed that microglia engulf presynaptic and postsynaptic elements by extending their processes to contact dendritic spines , axons , and synapses as observed in the developing brain . microglia - mediated synaptic pruning is dependent on regional neuronal activity . in the visual cortex , visual experience causes microglial processes to change their morphology and motility ; these processes alter distributions of extracellular space , display phagocytic structures , appose synaptic clefts more frequently , and envelope synapse - associated elements more extensively . intriguingly , synaptic pruning occurs preferentially in response to less active inputs , which apparently contributes to activity - dependent synaptic plasticity and learning . microglia express cr3 , the high - affinity receptor for c3 , which is required for the precise execution of synaptic pruning . moreover , microglia are involved in synapse formation by secreting brain - derived neurotrophic factor ( bdnf ) . consistent with these data , genetic inhibition of microglia results in derangement of both synapse formation and elimination of dendritic spines . cortical spreading depression ( csd ) is defined as a slowly propagating ( 25 mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons ( figure 3 ) . by a strict definition , csd refers only to the electrical silence of brain electrical activity following spreading depolarizations . csd is accompanied by secondary changes of cerebral blood flow ( cbf ) , which comprised the following four distinct phases : ( i ) an initial , brief hypoperfusion , ( ii ) a marked , transient hyperemia , ( iii ) a later , smaller hyperemia , and ( iv ) a long - lasting oligemia . experimentally , csd can be elicited by chemical stimulation ( high potassium exposure , atpase inhibitors , endothelin-1 , n - methyl - d - aspartate ( nmda ) receptor agonists ) , pinprick stimulation , and electrical stimulation of the intact cerebral cortex . the most clinically convincing evidence for this came from an mri study by hadjikhani et al . , which demonstrated the clinico - radiological correlation of visual percept of aura symptoms and propagation of cortical blood oxygenation level - dependent ( bold ) signals in the visual cortex of a patient experiencing a migraine . in addition , csd has been recorded by electrocorticography in patients with ischemic and hemorrhagic stroke and brain trauma . in most cases , however , electrical activity of brain tissue is already compromised before the development of spreading depolarizations . as a result , the characteristic temporal profile of csd , in which rapidly evolving depolarizations of neural cells were followed by suppression of electrical activity , may not necessarily be observed . because of this , spreading depolarization is now regarded as a more precise and preferred term to describe such conditions than csd . spreading depolarization can be elicited by cerebral ischemia or traumatic brain injury experimentally . detailed electrophysiological analysis using hippocampal slices revealed that the apical dendrites of neurons were depolarized earlier than the somata during csd . as compared to action potentials , csd induces a greater magnitude of extracellular potential shift ( typically 1020 millivolts ) which continues for much longer ( at least several minutes ) . the initiation of csd entails a rapid increase in [ k]e from 4 mm to 3060 mm and a rapid decline in [ na]e and [ cl]e from 140 mm to 5070 mm and [ ca]e from 1.52.0 mm to 0.20.8 mm . for example , glutamate , the excitatory amino acid , is released into the extracellular space and binds to nmda - type glutamate receptors to further promote the influx of ca and na . in response to these environmental changes , consistent with these results , a loss - of - function mutation in the gene encoding the 2 isoform of the glial na , k - atpase ( atp1a2 ) causes familial hemiplegic migraine type 2 ( fhm2 ) , which is clinically characterized by prolonged aura episodes . hence , clearance of potassium and glutamate from the extracellular space appears to be a critical event that determines predisposition to csd inductions . activation of the glial na , k - atpase is required to establish neuronal resting membrane potential and correct the csd - induced derangement of the ionic environment . since this process is accompanied by elevated atp consumption . as expected , multiple consecutive csd episodes robustly increase the cerebral metabolic rate of glucose ( cmrglu ) , as compared to a single csd episode . csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . the initiation of csd entails a rapid increase in [ k]e from 4 mm to 3060 mm and a rapid decline in [ na]e from 140 mm to 5070 mm ( black arrows ) . na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . however , csd - associated oligemia may have a negative impact on na , k - atpase activity . csd - associated depolarizations occur first in the dendrites and then propagate to the soma in cortical neurons . the initiation of csd entails a rapid increase in [ k]e from 4 mm to 3060 mm and a rapid decline in [ na]e from 140 mm to 5070 mm ( black arrows ) . na , k - atpase activity then rectifies the resultant abnormal ion distribution ( red arrows ) . however , csd - associated oligemia may have a negative impact on na , k - atpase activity . microglia express voltage - sensitive ion channels , including nav1.1 , kv1.3 , and kv1.5 , and are thought to sense electrical activity pertaining to csd . csd initially elevates extracellular ph , which subsequently leads to a gradual decrease in tissue ph . accumulation of protons may be detected by microglial transient receptor potential cation channel subfamily v , member 1 ( trpv1 ) . a recent study revealed that microglia exhibit increased nmda - dependent inward rectifying potassium conductance after csd , which can be interpreted as a compensatory mechanism for elevated extracellular k concentration . reported that the number of major histocompatibility complex ( mhc ) class ii antigen - positive microglia significantly increased in the rat cerebral cortex between 16 and 24 h after csd . moreover , it has been shown that csd can increase the proliferation and migration of microglia , both of which are well - recognized essential features of the immune response . biochemically , there is evidence that csd induces ros production in microglia that are located in the affected brain tissues . in addition , csd stimulates microglial secretion of il-1 and tnf. collectively , these data suggest that csd can initiate inflammatory activation of microglia . interestingly , ros and tnf have been shown to lower the threshold for csd induction , thus forming a positive feedback mechanism that favors the perpetuation of csd induction . on the other hand , tnf has been shown to reduce csd amplitudes in a dose - dependent manner . in addition , il-1 is also known to attenuate csd amplitudes via the gabaa receptor activity at lower concentrations . however , at a high dose , il-1 did not alter the magnitude of csd amplitudes . since these cytokines promote vascular permeability , as a whole , they are likely to have a deleterious effect on cns tissue in csd pathophysiology . moreover , in hippocampal slice culture studies , selective depletion of microglial cells with clodronate conferred resistance to csd induction . conversely , restoration of microglial cells to previously depleted cultures restored the susceptibility to csd . a similar phenomenon has been reported in an in vivo neuroimaging study . an in vivo calcium imaging technique demonstrated that microglia depletion led to a perturbation of neuronal calcium response . hence , it is inferred that a certain microglia - regulated neuronal calcium response may be required for csd occurrence . furthermore , evidence shows that microglial csd - generating activity can be inhibited by insulin - like growth factor-1 ( igf-1 ) treatment and environmental enrichment , the latter of which promotes m2-polarization of microglia ( figure 4 ) . it has been demonstrated that igf-1 antagonizes tnf. these data were obtained in experimental settings where csd was induced in cluster . clinically , successive csd / spreading depolarizations have been demonstrated in patients with stroke and traumatic brain injury . experimental evidence demonstrates that csd / spreading depolarizations contribute to the expansion of infarct volume by several mechanisms . first , a limited supply of atp due to ischemia renders neurons unable to reestablish the resting membrane potential , which may lead up to the occurrence of terminal depolarization . igf-1 treatment and other therapeutic interventions that promote m2-polarization of microglia may ameliorate secondary brain damage resulting from cerebral ischemia . resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . resultant production of tnf and ros enhances the susceptibility of brain tissue to csd development . in addition , microglia polarized to the m2 activation mode by environment enrichment exhibit much less csd susceptibility . the action of environmental enrichment can be mimicked by nasal administration of interleukin-11 ( il-11 ) . as mentioned above , multiple csd inductions are likely to activate microglia . consequently , to determine if microglial activation occurs after a single csd event , as seen in the usual migraine aura , we compared morphological changes of microglia in response to single and multiple csd episodes . we found that a single csd induction led to only subtle morphological changes in microglia , whereas multiple csd inductions caused marked enlargement of microglia after 24 h. this morphological change normalized by 72 h after csd inductions . our data suggest that a single episode of csd is innocuous to the brain in terms of microglial activation . we did not detect activation of caspase-3 or dna fragmentation detectable by the tunel assay ( unpublished data ) . nedergaard and hansen reported that csd was not associated with neuronal injury in the normal brain . this is also consistent with the traditionally held belief that a single attack of migraine with aura is a benign condition that does not cause any clinically relevant structural brain damage . we sought to clarify the events upstream of the multiple csd - induced microglial enlargements . reported that csd causes the release of hmgb1 ( high - mobility group box 1 ) from neurons via pannexin 1 channels . although hmgb1 is primarily located in the nucleus , upon injurious stimuli , it is released into the extracellular space , where it serves as a damage - associated molecular pattern ( damp ) . we found that neuronal hmgb1 release was dependent on the number of csd inductions such that only multiple csd events were able to cause significant hmgb1 release from neurons ( figure 5 ) . in addition , transcriptional activity of the hmgb1 gene in cortical neurons was enhanced in response to multiple csd events , which may reflect an attempt to replenish the cellular pool of the hmgb1 protein . in general , damp molecules bind to their corresponding receptors , thus transmitting a danger signal to surrounding cells . the ligation of hmgb1 with these receptors initiates intracellular signaling cascades that involve myd88 and irak4 . we found that multiple csd inductions enhanced the transcriptional activity of the tlr2 , tlr4 , myd88 , and irak4 genes in brain tissue . moreover , application of anti - hmgb1 antibody to the cortical surface attenuated the morphological alterations of microglia caused by multiple csd inductions . these data indicate that the hmgb1tlr2/4 axis plays a crucial role in the microglial activation caused by multiple csd events . we observed that the majority of hypertrophic microglia displayed prominent immunoreactivity for cathepsin d , a lysosomal acid hydrolase ( figure 6(a ) ) . nevertheless , we did not observe any apoptotic changes in the brain tissue subjected to multiple csd episodes , making it unlikely that activated microglia are involved in the execution of apoptosis or phagocytosis of dead neurons . an alternative concept is that activated microglial phagocytotic activity is involved in synaptic pruning ( figure 6(b ) ) . a recent in vivo multiphoton microscopy study disclosed that csd causes dramatic structural alterations of synapses between axons and the dendritic spines of cortical neurons . several minutes after csd , axonal bouton density increased by 20% and bouton size decreased by 25 to 40% compared to the resting state . concomitantly , there was a morphological shift from predominantly stubby spines to thin or mushroom spines after csd , which implies enhancement of synaptic excitability . although single csd events cause abnormalities in synaptic morphology and functionality , these alterations are short - lived and reversible . however , successive csd episodes prevent the recovery of synaptic abnormalities , indicating that the accumulated stress of prior csd events lead to irreversible dendritic injury . thus , it is plausible that activated microglia engage in the repair of damaged synapses through synaptic pruning and formation after multiple csd episodes . figure 5.microglial activation is influenced by the number of csd episodes . a single csd event , as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . figure 6.upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . bar = 20 m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . microglial activation is influenced by the number of csd episodes . a single csd event , as seen in the usual aura of migraine patients , does not cause significant release of hmgb1 from cortical neurons . conversely , clustering of csd , as seen in stroke and brain trauma , induces a robust hmgb1 release from cortical neurons . subsequently , hmgb1 acts on the tlr2/4 on the surface of microglia , which is followed by microglial activation , as demonstrated by their hypertrophic morphological alterations . they may contribute to neuroprotection , for example , through the synaptic repair on the dendritic spines of multiple csd - affected cortical neurons . upregulation of cathepsin d , a representative lysosomal acid hydrolase , in activated microglia subjected to multiple csd episodes . ( a ) as compared to cortical microglia in untreated mice ( upper row ) , those subjected to multiple ( five times ) csd inductions ( lower row ) exhibit increased cathepsin d immunoreactivity ( arrow ) . bar = 20 m . ( b ) microglia activated by multiple csd events may be recruited to damaged dendritic spines for synaptic repair . although aforementioned data suggest that microglia are required for the occurrence of csd , the mechanisms by which microglia induce csd remain unknown . provided that microglia raise extracellular k concentration before csd occurrence , they would have to follow through in a voltage - independent manner . microglia have been shown to express potassium intermediate / small conductance calcium - activated channels , subfamily n , member 4 ( kcnn4/kca3.1 ) . this voltage - insensitive and calcium / calmodulin - regulated potassium channel has been implicated in lipopolysaccharide- and ischemia - induced brain damage . therefore , the involvement of kcnn4/kca3.1 in the initiation of csd may be worthy of investigation . another unresolved issue is whether multiple csd - induced microglial activation is an adaptive phenomenon or a harmful event to brain tissue . in - depth analysis of the molecules produced in these cells would be required to solve this problem . if microglia are indeed involved in synaptic pruning , the identification of synaptic components , like psd95 , within enlarged microglia should be an important confirmatory finding . lastly , it remains uncertain whether immunological and inflammatory activation of multiple csd - subjected microglia can influence systemic immunological activity . as mentioned above , the recently discovered lymphatic system makes possible immunological communication between the brain parenchyma and the peripheral lymphatic system . in this paradigm , fhm is characterized by prolonged migraine aura , and neuroimaging data support repeated inductions of csd . it has been reported that fhm attacks often cause high fever and in a fewer cases , csf pleocytosis . it may , therefore , be interesting to explore whether a potential crosstalk between activated microglia and the csf / lymphatic system is really relevant to csd pathophysiology . the publication of this article was supported by a jsps kakenhi ( grant number 26460706 to ms ) and a grant from nippon zoki pharmaceutical co. , ltd . the author(s ) declared no potential conflicts of interest with respect to the research , authorship , and/or publication of this article .

microglia play a pivotal role in innate immunity in the brain . during development , they mature from myeloerythroid progenitor cells in the yolk sac and colonize the brain to establish a resident population of tissue macrophages . in the postnatal brain , they exert phagocytosis and induce inflammatory response against invading pathogens . microglia also act as guardians of brain homeostasis by surveying the microenvironment using motile processes . cortical spreading depression ( csd ) is a slowly propagating ( 25 mm / min ) wave of rapid , near - complete depolarization of neurons and astrocytes followed by a period of electrical suppression of a distinct population of cortical neurons . not only has csd been implicated in brain migraine aura , but csd - like events have also been detected in stroke and traumatic injury . csd causes a considerable perturbation of the ionic environment in the brain , which may be readily detected by microglia . although csd is known to activate microglia , the role of microglial activation in csd - related neurological disorders remains poorly understood . in this article , we first provide an overview of microglial development and the multiple functions of microglia . then , we review existing data on the relationship between microglia and csd and discuss the relevance of csd - induced microglial activation in neurological disease .

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for a long time , transposable elements ( tes ) have been considered as pure selfish and junk elements parasiting the genome of living organism [ 1 , 2 ] . these sequences are able to move , that is , to insert into new locations within genomes . this phenomenon is called transposition . retroelements use retrotransposition , that is , the reverse transcription of an rna intermediate and integration of the cdna molecule produced , to generate new copies of themselves within genomes ( copy - and - paste mechanism ) . this mechanism directly increases the copy number of the element . among protein - coding autonomous retroelements , distinction is generally made between elements with long terminal repeats ( ltrs : ltr retrotransposons and retroviruses ) and retroelements without ltrs ( non - ltr retrotransposons or line elements ) . retroviruses and ltr retrotransposons are mainly distinguished by the presence versus absence of an envelope gene , which encodes a protein necessary for virus entry into the target cell . after germ line infection , reverse - transcribed retrovirus genomes can be integrated into the host genome and transmitted through vertical inheritance to the host progeny . gain or loss of the envelope gene can transform a retrotransposon into a retrovirus , and vice versa [ 4 , 5 ] . the second large category of tes , dna transposons , generally excises from their original insertion site and reintegrate into a new location ( cut - and - paste mechanism ) . for most dna transposons , transposition is catalyzed by an enzyme called transposase . finally , noncoding nonautonomous elements using for their transposition proteins encoded by autonomous sequences exist for both retroelements and dna transposons . despite the deep - rooted vision of junk dna , there is growing evidence that tes are more than simple genome parasites . particularly , they have been shown to serve as a genomic reservoir for new regulatory and coding sequences allowing genetic innovation and organismal evolution . a fascinating facet of the roles of tes in evolution is their ability to be molecularly domesticated to form new cellular protein - coding genes [ 7 , 8 ] . te - encoded proteins have properties that can be of interest for host cellular pathways . they can bind , copy , cut , process , and recombine nucleic acids , as well as modify and interact with host proteins . there are many cases of te - derived genes fulfilling important functions in plants , fungi , and animals , including vertebrates ( for review , [ 8 , 9 ] ) . we will present here several prominent examples of vertebrate genes formed from te - coding sequences during evolution , with more emphasis on gypsy integrase ( gin ) genes that we have analyzed in different fish species . several multigenic families have been formed from different events of molecular domestication of the gag gene of ty3/gypsy elements , a super family of ltr retrotransposons active in fish and amphibians but extinct in mammals [ 9 , 10 ] . the gag gene encodes a structural protein with three functional regions : the matrix ( ma ) domain playing a role in targeting cellular membranes , the capsid ( ca ) domain involved in interactions with other proteins during particle assembly , and the nucleocapsid ( nc ) , which binds to viral rna genomes through zinc fingers . most mart genes are found on mammalian x chromosome , suggesting an initial event of molecular domestication on the x , followed by serial local duplication events that subsequently extended this gene family . all mart genes have retained from the original gag sequence an intronless open reading frame . some of them still encode the ancestral gag zinc finger , suggesting nucleic acid binding properties for the protein . two autosomal mart genes , peg10 ( mart2 ) and peg11/rtl1 ( mart1 ) , are subject to genomic imprinting and are expressed from the paternal allele [ 12 , 13 ] . this epigenetic regulation has been proposed to be derived from a defence mechanism repressing the activity of the ancestral retrotransposon before domestication . at least two mart genes , peg11/rtl1 ( mart1 ) and peg10 ( mart2 ) , have essential but nonredundant functions in placenta development in the mouse [ 15 , 16 ] . peg10 and other mart genes might also control cell proliferation and apoptosis , with possible involvement in cancer ( and references therein ) . another mammalian gene family derived from a ltr retrotransposon gag gene is called ma or pnma ( paraneoplastic ma antigens ) . fifteen ma / pnma genes are present in the human genome , most of them being located on the x chromosome as observed for mart genes . some ma proteins are expressed by patients with paraneoplastic neurological disorders and might be targeted by autoimmune response leading to progressive neurological damage . several ma proteins are also involved in apoptosis , including ma4 ( pnma4/map1/maop1 ) and ma1/pnma1 [ 19 , 20 ] . this family is constituted of dna binding proteins with an n - terminus region called the scan domain , which is derived from the gag protein of a gmr1-like gypsy / ty3 retrotransposon [ 2124 ] . the scan family is vertebrate specific , with approximately 70 and 40 members in human and mouse , respectively . several scan proteins have been shown to be transcription factors regulating diverse biological processes such as hematopoiesis , stem cell properties , or cell proliferation and apoptosis ( for review ) . one of them , fv1 , is of retroviral origin and controls replication of the murine leukaemia virus in the mouse . during mammalian evolution , retroviral envelope genes have been domesticated several times independently to generate genes involved in placenta development . syncytins mediate the fusion of trophoblast cells to form the syncytiotrophoblast layer , a continuous structure with microvillar surfaces forming the outermost foetal component of the placenta . two syncytin genes of independent origins encoding placenta - specific fusogenic proteins are present in human and other simians ( syncytin-1 and -2 , ) as well as in rodents ( syncytin - a and syncytin - b , ) . independent syncytin genes are also found in rabbit , guinea pig , and carnivora , indicating multiple convergent domestication of env - derived syncytin genes in different mammalian sublineages . for example , human syncytin-1 plays a role in osteoclast fusion , neuroinflammation , and possibly multiple sclerosis [ 33 , 34 ] . other retroviral env - derived open reading frames are present in vertebrate genomes ; but intensive work is required to determine their functions . some of them might confer resistance to viral infection , as shown for the fv-4 locus . this locus , containing an entire ecotropic murine leukemia virus ( mulv ) env gene , controls susceptibility to infection by mulv . in mammals , a gene called cgin1 the integrase gene has been fused 125180 million years ago to a duplicate of the cellular gene kiaa0323 . one of them , a gene encoding a protein called saspase , is necessary for the texture and hydration of the stratum corneum , the outermost layer of the epidermis . finally , the telomerase , the reverse transcriptase extending the ends of linear chromosomes in vertebrates and other eukaryotes , might be derived from a retroelement . many examples of genes derived from transposase genes from diverse subfamilies of dna transposons have been described in vertebrates and other organisms [ 8 , 39 , 40 ] . one well - studied example is the recombination - activating protein rag1 , which together with rag2 catalyzes the v(d)j somatic site - specific recombination responsible for the formation and diversity of genes encoding immunoglobulins and t - cell receptors in jawed vertebrates . rag1 has been formed from the transposase of a transib dna transposon , and the v(d)j recombination signal sequences recognized by rag1 might be derived from the transposon ends bound by the ancestral transposase . the mammal - specific gene cenp - b encodes a pogo transposase - derived protein that controls centromere formation depending on the chromatin context . interestingly , an independent event of molecular domestication of pogo transposase also led to the formation of centromeric proteins in fission yeast . in yeast , cenp - b - like proteins restrict the activity of retrotransposons and promote replication progression at forks paused by retrotransposon ltrs [ 44 , 45 ] . one example is the jerky gene , which encodes a brain - specific mrna - binding protein that may regulate mrna use in neurons . similarly , several examples of genes derived from hat transposases have been found in mammals , some of them having been fused to zinc finger domains . indeed , it has been shown that mutation in a regulatory sequence prohibiting zebd6/mgr binding leads to igf2 upregulation and enhanced muscle growth in commercially bred pigs [ 48 , 49 ] . in primates , the gene encoding the metnase / setmar protein has been formed through fusion of the transposase gene of a mariner transposon with a set histone methyltransferase gene . metnase / setmar is a dna binding protein with endonuclease activity that promotes dna double - strand break repair through nonhomologous end joining ( nhej ) [ 50 , 51 ] . several genes derived from piggybac - like transposons have been detected in human and other vertebrates . one of them , pgbd3 , serves as an alternative 3 terminal exon for the cockayne syndrome b ( csb ) gene , leading to the expression of a csb - transposase fusion protein . at least one harbinger transposon - derived gene , harbi1 , encoding a predicted nuclease , is present in mammals , birds , amphibians , and fish . likewise , genes derived from a new type of dna transposon called zisupton have been identified in fish and other vertebrates . finally , mammalian and bird genomes possess at least one gene clearly derived from a p transposon ; additional vertebrate genes like thap9 encoding proteins with a thap domain might be also related to p - like transposases [ 8 , 40 , 5661 ] . two vertebrate genes with unknown functions , gin1 and gin2 ( gypsy integrase 1 and 2 ) , encode proteins showing significant homologies to integrases encoded by ltr retrotransposons [ 62 , 63 ] . further analyses showed that both genes have been formed from gin transposons , a new family of metazoan dna transposons with a transposase that shows strong similarities with ltr retrotransposon integrases . gin1 , which shows similarities with gino transposons from hydra magnipapillata , is present in mammals , birds , and reptiles , suggesting a molecular domestication event at the base of the amniota ca . mammalian gin1 proteins have conserved amino - acid residues necessary for integrase activity . using our own analyses we provide here updated gin2 structural and phylogenetic analyses using new vertebrate sequences and present first expression data for this gene in fish . gin2 is present in several fish species , as well as in cartilaginous fish ( elephant shark ) , coelacanth , amphibians , birds , reptiles , and marsupials , but neither in monotremes nor in placental mammals ( figures 1 , 2 , and 3 ) . hence , the molecular domestication event having led to the formation of gin2 might have taken place before the divergence between tetrapods / bony fish and cartilaginous fish around 500 million years ago , with subsequent loss in monotremes and placental mammals . the formation of gin2 might even be older , since potentially domesticated gin - like sequences related to gin2 have been detected in the urochordates ciona savignyi and c. intestinalis . phylogenetic analysis suggests that gin2 is derived from gina transposons , which are bona fide transposable elements in hydra magnipapillata ( figure 1 ) . this suggests that gin1 and gin2 have been formed through two independent molecular domestication events , one at the base of amniota and the other in a more ancient vertebrate ancestor ( figure 3 ) . after domestication , the hhcc zinc finger present in the ancestral integrase has been maintained , suggesting ability to bind to dna or rna ( figure 2 ) . conservation of the important catalytic triad ( dde , aspartic acid / aspartic acid / glutamic acid ) of the integrase is less obvious . while this motif has been proposed to be conserved in gin1 , this is not the case for gin2 based on a published alignment with sequences from gin - related transposases ( figure 2 ) . as shown in figure 2 , the first aspartic acid residue is present in most species but absent from amphibians and birds . however , multiple sequence alignment revealed an aspartate conserved in all gin2 and gin1 sequences ca . the second aspartic acid residue is not found in gin2 but an aspartate is conserved four amino acids away in all gin2 sequences except for opossum . finally , the glutamic acid residue is found only in several species and substituted by an aspartate in fish ; but a conserved glutamate is detected 16 amino acids away . hence , the question of the functionality of gin2 as an integrase remains open and should be definitely answered through functional analyses . a third domain with unknown function called gpy / f [ 64 , 67 ] is also detected in gin proteins , but in some cases the phenylalanine residue is replaced by a leucin . gin2 contains eight protein - coding exons , with an exon - intron structure well conserved in fish and other vertebrates ( figure 4 ) . some introns might be derived from the ancestral transposon ; others might be the result of events of intronization after molecular domestication . gin2 is located in the same orthologous genomic region between ogfod2 and abcb9 in marsupials , birds , reptiles , and fish , confirming that this gene does not correspond to a mobile sequence ( figure 5 ) . expressed sequence tag ( est ) analysis indicated that gin2 is expressed in different adult tissues and developmental stages in chicken : brain ( accession number : cn219658 ) , liver ( bg713188 ) , head ( bu225420 ) , embryonic tissue ( bu210425 ) , limb ( bu256599 ) , small intestine ( bu297502 ) , muscle ( bu437928 ) , and ovary ( bu447634 ) . only ests from the whole body are available for xenopus . few ests are also found in zebrafish : muscle ( ct684014 ) , gills ( eb908574 ) , reproductive system ( bi867074 ) , and eye ( bi879358 ) . to determine more precisely gin2 expression pattern in fish , quantitative real - time pcr was performed on different embryonic developmental stages in zebrafish ( danio rerio ) , as well as on adult tissues from zebrafish and platyfish ( xiphophorus maculatus ) ( figure 6 ) . during zebrafish embryogenesis , gin2 expression level strongly increases from the dome stage and progressively decreases until the end of somite stages . gastrulation , which is characterized by morphologic movements of involution and extension , starts at the beginning of the epiboly to finish at bud stage . in adult zebrafish , the higher level of expression for gin2 was observed in brain , followed by gonads and eyes . in contrast , gin2 expression was maximal in gonads in the platyfish ( figure 6 ) . to conclude , our analysis integrates data from several newly sequenced vertebrate genomes , particularly teleostean and cartilaginous fishes as well as coelacanth , in order to better understand the distribution and evolutionary history of gin genes . since gin2 is apparently not present in lamprey , we propose that gin2 was formed before the divergence between cartilaginous and ray - finned fish about 500 million years ago ( figure 3 ) . we also provide the first expression data for gin2 in fish particularly supporting a function in gastrulation during zebrafish embryogenesis . dna with no important functions for genomes and organisms . today , nobody can deny the importance of transposable elements during evolution in terms of innovation power , particularly through molecular domestication events . domesticated elements are bona fide cellular genes derived from transposable element sequences encoding for example integrases , transposases , gag proteins , or envelopes . after domestication , te - derived genes have lost their ability to transpose through the elimination of sequences such as long terminal repeats , terminal - inverted repeats , or other open reading frames and protein domains essential for transposition . elimination of such sequences might occur by genetic drift or might even be selected for transposition or retrotransposition of a domesticated sequence might change its copy number and pattern of expression . transposition of such a gene might have strongly deleterious consequences for the host , for instance cancer . it might , therefore , be important to immobilize te - derived genes at fixed position within a genome to control their expression . in vertebrates , many te - derived genes are mammal specific , suggesting that molecular domestication probably played an important role in the evolution of this specific sublineage . other te - derived genes like gin2 are present in some vertebrate sublineages but absent from mammals . in birds , reptiles , amphibians , and fish , domesticated sequences might be more difficult to identify due to the concomitant presence of active tes within genomes . availability of additional genome sequences will probably allow the identification of many te - derived genes specific of these sublineages that contribute to diversification within vertebrates . we focused on gin genes , a pair of ancient vertebrate domesticated genes for which no function has been identified so far . both gin1 and gin2 are derived from gin transposons that themselves gained their transposase from the integrase of ltr retrotransposons . gin1 was detected in mammals , birds , and reptiles , indicating that it was formed in a common ancestor of amniota ca . gin2 might be even older , since it was detected in tetrapods , bony fish , and sharks , and possibly in urochordates . the presence of both genes over such long periods of evolution is suggestive of important , so far unknown conserved functions in vertebrates . gin2 was lost in a common ancestor of monotremes and placental mammals , suggesting that either gin2 function was not essential anymore , or that this function is fulfilled now by gin1 in these sublineages . presence of conserved intron positions suggests a unique origin followed by duplication and intron gain in a common ancestor of gin1 and gin2 ( paralogy ) . in this case , gin1 would have been lost among others in fish . alternatively , gin1 and gin2 might have been generated from two independent events of molecular domestication , as suggested by the close phylogenetic relationship of bona fide gin transposons with each of both genes ( figure 1 ) . presence of introns at conserved positions might in this case reflect intron conservation between ancestral gin transposons at the origin of both molecular domestication events . gin1 and gin2 functions might be related to the binding to dna or rna , since both proteins have conserved the hhcc zinc finger present in the ancestral integrase . conservation of the integrase activity appears possible but must be tested through functional assays . in fish , gin2 is particularly expressed in brain and gonads ; its expression pattern during zebrafish embryogenesis suggests a role during gastrulation . functional analysis in fish taken together , data on gin and other te - derived genes support the important role of molecular domestication as a driver of genetic innovation during evolution . there is no doubt that future genome comparisons and functional gene analyses will uncover new domesticated genes and novel biological functions essential for the diversification of vertebrates and other living organisms .

due to their ability to drive dna rearrangements and to serve as a source of new coding and regulatory sequences , transposable elements ( tes ) are considered as powerful evolutionary agents within genomes . in this paper , we review the mechanism of molecular domestication , which corresponds to the formation of new genes derived from te sequences . many genes derived from retroelements and dna transposons have been identified in mammals and other vertebrates , some of them fulfilling essential functions for the development and survival of their host organisms . we will particularly focus on the evolution and expression of gypsy integrase ( gin ) genes , which have been formed from ancient event(s ) of molecular domestication and have evolved differentially in some vertebrate sublineages . what we describe here is probably only the tip of the evolutionary iceberg , and future genome analyses will certainly uncover new te - derived genes and biological functions driving genetic innovation in vertebrates and other organisms .

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these progressive diseases affect increasing number of people ( presently 45 million people world wide , expected to reach 76 millions in 2030 ) . these devastating diseases have high societal costs and lead , directly or indirectly , to a dramatic loss in life expectancy and quality of life . these diseases have in common the aggregation after misfolding and/or incorrect elimination of distinct polypeptides . most important however is their ability to amplify by recruiting the soluble form of homologous polypeptides and to propagate from one cell to another . thus , through their template - driven misfolding of similar proteins over time , e.g. amplification , spread from one cell from the central nervous system to another , e.g. transmissibility and accumulation within the central nervous system , misfolded protein aggregates create a self - sustaining , exponential vicious circle leading to disease . within our cells , nascent proteins fold spontaneously or following interaction with molecular chaperones in order to be functional . at any given time a fraction of newly synthesized polypeptides populate non - native conformers [ 24 ] , with a sub - fraction corresponding to mis - processed peptides ( for example a after app cleavage by and secretases instead of the and secretase product p3 ) or polypeptides where translation errors occurred . a fraction of these conformers is given another chance to fold correctly after full / partial unfolding by molecular chaperones while another , never reaches the native state and is cleared or exported outside the cell . thus , by opposing to protein misfolding , unfolding and aggregation , molecular chaperones , play a central role in the maintenance of normal cellular proteostasis . upon cellular stress the amounts of misfolded / unfolded proteins and/or mis - processed and mutant polypeptides increase . this triggers heat shock response via the activation of the heat shock transcription factor 1 leading to partial correction of misfolding / unfolding and incorrect processing [ 5 , 7 , 8 ] . as sustained activation of the heat shock response is detrimental , normal cellular proteostasis is imbalanced with saturation of proteostatic networks [ 1 , 5 , 6 ] . as a consequence , misfolded proteins aggregate into intra- or extracellular protein deposits , depending on whether they are within the cytosol of in the extracellular milieu . it is widely accepted that the ubiquitin proteasome system degrades efficiently misfolded / unfolded or incorrectly processed proteins as long as the monomeric and aggregated forms are in dynamic equilibrium . in contrast , high molecular weight irreversible assemblies are cleared by autophagy . when the latter process fails either because of defects in autophagosome formation , transport , fusion with lysosomes or recognition of the substrate , protein aggregates accumulate with deleterious consequences . misfolded proteins aggregation can cause both loss of function and gain of pathologic function [ 1214 ] . this is the consequence of either i - misfolded protein aggregates - mediated permeabilization of plasma membrane and/or membranous compartments , ii - perturbation in membrane protein dynamics and distribution they may cause , iii - the formation of novel pathogenic signaling platforms they may mediate , iv - the trapping of significant amounts of molecular chaperones and other partner proteins within the aggregates and v - the ability of the misfolded protein aggregates to recruit the soluble form of the proteins they are formed of and amplify . protein aggregates made of fibrillar material , in some cases straight as needles , in others curved and twisted , are the hallmarks of a variety of diseases affecting the central nervous system , in particular , alzheimer s ( ad ) , parkinson s ( pd ) , huntington s ( hd ) and creutzfeldt - jacob ( cjd ) diseases . the number of deposits within the affected tissues correlates most often with disease severity [ 1517 ] . as the probability of occurrence of events such as i - misfolding and aggregation events of newly synthesized polypeptide chains , ii - unfolding of native polypeptide chains following a variety of stresses , iii - errors during translation and iv - incorrect processing and elimination of proteins targeted to degradation , is dependent on time , misfolding diseases are associated to ageing . until recently , the spread and transmission of disease via misfolded protein aggregates was thought to be restricted to the prion protein ( prp ) . a wide range of recent experimental evidence suggest that other protein aggregates that are the hallmarks of major neurodegenerative diseases spread in a prion - like manner [ 20 , 21 ] . thus , misfolded protein aggregates propagation and amplification appears to be a widespread phenomenon in neurodegenerative diseases as opposed to a curiosity limited to a unique protein : prp . indications for such a process originated from the work of heiko braak and co - workers who described distinctive patterns of pathological changes over time throughout the brain for the major neurodegenerative diseases in man . indeed , based on autopsy cases , braak and co - workers noticed that protein aggregates associated to pathology initiate in circumscribed areas of the brain specific to each disease and progress in a topographically predictable manner following anatomical connections [ 1517 ] . these observations led them to establish a disease progression scale and to hypothesize that neurotropic pathogens were spreading through defined pathways to interconnected regions within the central nervous system . as for prp , these pathogens were first thought to be of viral nature . the first evidence for the transmission of misfolded protein aggregates not involving prp in man came from the observation that lewy bodies in pd brains contaminate grafted fetal mesencephalic progenitor neurons decades after transplantation [ 22 , 23 ] . injection of brain extracts containing amyloid plaques , which main constituent is the aggregated form of the amyloid -peptide ( a ) , neurofibrillary tangles , that are mainly made of the microtubule associated proteintau , and lewy bodies , which main constituent is the protein alpha - synuclein ( -syn ) , into the brain of model animal induced lesions characteristic of ad and pd [ 2434 ] . the induced lesions , that were initially confined to the injected brain region , propagated to neighboring and/or axonally connected areas over time , suggesting directed spreading and amplification through neuronal transport processes [ 3235 ] . the intraperitoneal injection of brain extracts containing amyloid plaques and neurofibrillary tangles also induced cerebral lesions characteristic of ad ( a and tau deposits ) [ 36 , 37 ] . similarly , the injection of brain extracts and lewy bodies enriched fractions from patients developing pd into the brain of non - human primates , wild - type and model mice , induced several months after injection the formation of lesions characteristic of pd [ 3135 ] . finally , lewy bodies of human nature appeared in rodents neural grafts implanted in the brains of mice expressing human -syn that develop lewy bodies over time and aggregated huntingtin ( htt ) with a polyq stretch length associated to hd and huntingtin n - terminal moiety ( htt - exon1 ) were shown to move between cultured cells , in organotypic slice cultures of a htt mouse model and between host and grafted tissues [ 3840 ] . luckily , we can reproduce in test tubes the aggregation of a , tau , -syn , htt - exon1 with a polyq stretch length associated to hd , and cu / zn superoxide dismutase 1 ( sod1 ) point mutants associated to motor neuron death in familial cases of amyotrophic lateral sclerosis ( als ) . pathologic htt - exon1 , only in its fibrillar form , binds to cultured cells , is internalized through yet an unknown mechanism , and induces the aggregation of sub - pathologic htt - exon1 within the cytosol of the recipient cells [ 38 , 41 ] . -syn mega - dalton protein assemblies were also shown to be taken up and to trigger the aggregation of endogenous cytosolic -syn in cultured primary cortical neurons and in neuronal cell lines [ 4245 ] . they were shown to be transported anterogradely and retrogradely following their uptake in primary neurons and to be transmitted to secondary neurons . in vitro generated tau assemblies were also shown to bind and enter cells and to transmit a misfolded state to intracellular tau [ 44 , 4749 ] . finally , mutant sod1 aggregates made in vitro were shown to penetrate inside neurons by macropinocytosis and induce the aggregation of soluble mutant sod1 in the cytoplasm . they were taken up by neurons and glial cells and shown to transfer from neurons to oligodendrocytes . fibrillar assemblies induced several months after injection the formation of lesions characteristic of pd and ad [ 32 , 37 ] . under physiological ph , salt and temperature conditions , a , tau , -syn , htt - exon1 with a pathologic polyq stretch and mutant sod1 , self - associate into assemblies that can elongate in a cooperative manner by incorporation of monomers . growth is unlimited as the incorporation of a monomer creates a new binding site for another monomer ( fig metastable , prefibrillar oligomeric intermediates , considered as the precursors of mature fibrils , are populated prior or concomitant to fibrils formation [ 52 , 53 ] . in some cases , it is the binding of ligands to aggregation prone polypeptides or their modifications that yield oligomeric species that are off - assembly pathway as for example upon the interaction of -syn with dopamine . the nature of the assemblies that exhibit the highest toxicity and/or seeding capacity is subject to intense debate . fibrillar assemblies made under physiological conditions have been shown to be the only entities capable of recruiting the soluble form of homolog proteins in vitro , in cell cultures and in vivo , i.e. with seeding capacities , and a body of evidence demonstrates their cytotoxic potential [ 30 , 33 , 38 , 41 , 4345 , 4850 , 5558 ] . nonetheless , they are considered as storage , inert form of misfolded proteins by those who consider the oligomers that form during the early stages of assembly as toxic [ 5963 ] . toxicity in the latter case is thought to be the consequence of impairment of membrane integrity and permeability [ 59 , 64 , 65 ] . the conflict is certainly due in some cases to non - physiological assembly conditions such as assembly after polypeptide dehydration , under high or low ph , at very low ionic strength ( e.g. distilled water ) , extreme salt conditions and temperatures that are suitable for extremophiles . it is also and mostly due to i - comparison of the properties of protein particles at different concentrations , ii - difficulties in defining what is precisely an oligomer and iii- oligomers - fibrils inter - conversion . at a given concentration of monomeric a , tau , -syn , htt - exon1 with a pathologic polyq stretch or mutant sod1 the concentrations of on - assembly pathway oligomers and fibrils diverge by several orders of magnitude as the molecular masses , number of constituting molecules and sizes of oligomeric and fibrillar species differ very significantly . indeed , oligomeric species are made of less than 10 to 50 monomeric proteins at most while fibrils that are over 0.1 m in length are made of thousands of polypeptide chains . thus , it is critical to compare the toxicity of the different assemblies at identical particle concentrations as cells interact with the particles as a whole , not the precursor monomeric protein within the assemblies . measurement of the molecular weight of defined on - assembly pathway oligomeric assemblies and fibrils with homogeneous length allowed calculating the particle concentration of assemblies at any given concentration of precursor monomeric polypeptide . when the relative toxicities of monomeric , oligomeric , and fibrillar assemblies were compared , the fibrillar assemblies , with homolog polypeptide seeding and spreading propensity , were found most toxic . as fibrils and oligomers have distinct molecular weights they are frequently separated by centrifugation . however as the solutions are often viscous , a significant amount of short fibrils may remain in the supernatant . it is therefore important to bear in mind that one can not exclude that observations derived from such fractionation procedures are due to the presence of short fibrils . finally as fibrils and precursor oligomeric species are in equilibrium one needs to keep in mind that oligomers can form fibrils in the time course of the experiment either in solution or when associated to the cell membranes as their concentrations increase when moving from a three- ( test tube ) to a bi- ( cell membrane ) dimensional space . similarly , while fibrils most often exhibit some resistance to detergents , one can not exclude their possible disassembly by cellular factors . the toxicity of fibrillar and oligomeric assemblies is in part the consequence of their binding to and permeabilization of the cell membrane . the binding of assemblies , besides inducing lipid rafts and the monosialoganglioside gm1 clustering , certainly impinge membrane curvature , thus further affecting cell survival . furthermore , the lateral diffusion of assemblies in the plane of the plasma membrane together or independently of membranous components such as receptors , channels , adhesion molecules , integrins certainly contributes to the redistribution and/or coalescence of protein receptors at the surface of the cell . misfolded protein assemblies may even selectively recruit membranous proteins thus creating novel signaling platform with undesirable properties , thus contributing to cell apoptosis . once internalized , they appear to leak out of the endosomal - lysosomal compartments as fibrils in particular reach the cytosol and seed the aggregation of their soluble cytosolic counterparts [ 38 , 42 , 45 , 47 ] . by crossing these membranous compartments , through yet an unknown mechanism , they may cause leakage of the lysosomal content or rupture of lysozomes , with cathepsin b - dependent increase in reactive oxygen species within the cytosol , thus triggering pathogenesis . the latter may account for the mitochondrial dysfunction and inflammation described in synucleinopathies . upon reaching the cytosol , exogenous misfolded protein assemblies certainly imbalance proteostasis following the titration of the cytosolic molecular chaperones and/or saturation of the clearance machinery of the cell leading to additional deleterious effects [ 5 , 76 ] . finally , toxicity may also be the consequence of the seeding capacity of the protein assemblies , e.g. the recruitment of fibrils soluble counterpart and exhaustion of the functional form of the protein . as exogenous a , tau , -syn etc ... assemblies bind and penetrate cells , amplify by recruiting their endogenous soluble counterparts through a seeding process and propagate to other cells they can be considered as infectious in accord with the modifications that have been proposed to koch s postulates . the observation that the aggregation of non - mutant prp give rise to distinct diseases ( creutzfeldt jakob s disease ( cjd ) , new variant cjd ( vcjd ) and kuru in man , classical and nor98 scrapie in sheep , classical and atypical ( h - bse and l - bse ) bovine spongiform encephalopathies ( bse ) ) that are distinguishable by the disease incubation time , lesions type in the brain and proteolytic cleavage patterns has led to the notion that prp can adopt different conformations into the mega - dalton assemblies associated to disease . indeed , each conformation an amino acid stretch adopts can establish a defined set of inter - molecular interactions . as a consequence alternative packing is possible yielding structurally unlike assemblies that expose distinct surfaces ( fig . 1 ) . the lateral surfaces of these different assemblies rule their ability to interact with receptors and lipids at the surface of the cells and protein partners within the cell . these distinct conformations are indeed imprinted to the newly incorporated prp upon assemblies growth either through a templating process or the selective recruitment of defined prp conformers that can establish intermolecular interactions with the well defined assemblies tips thus leading to the faithful amplification of distinct prion strains . in a manner similar to prp , the deposition of -syn within the central nervous system is not only tightly associated to pd but also to distinct pathological phenotypes : multiple system atrophy ( msa ) and dementia with lewy bodies ( dlb ) . , the aggregation of tau also leads to distinct tauopathies : argyrophilic grain disease , tangle - only dementia , pick disease , progressive supra - nuclear palsy and cortico - basal degeneration . the involvement of the deposits made of the same protein in diseases exhibiting different phenotypic traits can be accounted for either by the existence of distinct i - -syn and tau strains , ii - areas within the central nervous system affected by neurodegeneration or iii - neuroinflammatory responses , given that neuroinflammation has been repeatedly linked to neurodegeneration . indeed , mounting evidence from epidemiological , postmortem , brain imaging and animal studies indicate that neuroinflammatoryprocesses are tightly linked to neurodegeneration [ 7982 ] . nonetheless , it is still unclear whether the inflammatory process is involved in disease etiology or a secondary consequence of neurodegeneration and whether neuroinflammation affects to different extents distinct brain regions . differences in the biochemical properties of aggregated -syn in different genetic backgrounds in humans have been reported suggesting that -syn aggregates may be unlike . the existence of -syn strains was established upon the generation of pure -syn fibrillar polymorphs under stringent and tighly controlled assembly conditions in vitro . distinct fibrillar polymorphs made of wild - type , full - length -syn , exhibited different shapes in the electron microscope , limited proteolytic and x - ray fiber diffraction patterns and were distinguishable by conformational antibodies . assessment of their intrinsic structures by solid - state nuclear magnetic resonance ( nmr ) revealed that although they have high beta - sheet content , -syn molecules within the fibrillar polymorphs are packed in distinct manner . as a consequence they recruit soluble -syn with different efficacy , elongate at distinguishable rates and must have different surface properties . functional studies revealed that the distinct polymorphs bind and permeabilize cells to different extents ; they exhibit different toxicities , efficacy of recruitment of cellular -syn and persistence / propagation propensity . they were also shown to imprint their distinct intrinsic structures to the recruited cellular -syn ( fig . 2 ) . the finding that distinct -syn strains bind to cells and are taken up with different efficiencies suggest that they may exhibit different tropism for cells from the central nervous system , in other words , bind with different affinities to neurons , glial cells or astrocytes . by targeting different cells from the central nervous system or circuits , they may be at the origin of distinct synucleinopathies . as distinct strains differ by their surface properties they also may trigger the inflammatory response to different extents . finally , the progression of distinct synucleinopathies may also reflect the different seeding propensity , resistance to clearance and persistence in cell cultures of distinct -syn strains . baffling evidence for the existence of -syn strains in vivo were brought by the groups of s. prusiner , e. bezard and m. vila who demonstrated that semi - purified aggregated -syn from patients developing msa or pd faithfully led to msa or pd - like neuropathological disorders in recipient animals [ 34 , 84 ] . additional evidence for the existence of putative -syn strains came from the lesion profiles observed after injection of either newly formed -syn fibrils orrepeatedly seeded -syn fibrils and the differential induction of tau aggregation by the two kinds of assemblies . it also came from comparing the proteinase k degradation profiles of sarkosyl - insoluble protein fractions isolated from patients diagnosed pd or pd and ad because of abundant tau neurofibrillary tangles and senile plaque . while one can not rule out that the differences in the degradation profile of aggregated -syn are due to the abundance of tau neurofibrillary tangles and a - beta these findings remain striking . recently , amyloid plaques extracted from the brain of two patients developing ad , with distinct neuropathology and clinical history , were used to seed the aggregation of a in vitro . although a aggregation in control brain extracts from healthy aged individuals and patients developing unrelated neurodegenerative diseases was not assessed , these observations suggest that a within the initial seeds recruit soluble a in vitro and imprint their structures to the synthetic , isotopically labeled , a. further evidence for the existence of a strains came from the use of tightly controlled assembly conditions in vitro of synthetic a140 and 142 . starting from the observation that synthetic fibrillar a140 recruits when injected into the brain of recipient animals both a140 and a142 while synthetic fibrillar a142 induce the aggregation of a142 , it was shown that slightly different assembly conditions lead synthetic fibrillar a142 to stably acquire the seeding properties characteristic of fibrillar a140 . evidence for the existence of tau strains have been brought through the use of truncated tau . when exposed to the fibrillar form of truncated tau , cells expressing the same truncated tau fused to a reporter fluorescent protein develop truncated tau clusters of different shapes . the pattern of these clusters is stably inherited over generations and transmissible to nave cells . when lysates from cells exhibiting distinct clusters were injected into the brain of recipient model animals , they induced distinguishable neuropathological patterns that could be stably propagated in mice . finally , brain homogenate from patients developing different tauopathies induced the clustering of reporter tau in cultured cell lines . reporter tau inclusion morphology and limited degradation patterns revealed homogeneous for most pathological cases with exceptions suggesting that a number of patients may suffer from more than one taustrain . symptomatic treatments are the only therapeutic tools available for patients developing ad and pd . in pd , for example drugs aiming at restoring dopamine levels , such as l - dopa , dopamine agonists , mao - b and comt inhibitors , although powerful , lose efficiency and occasion side effects with time [ 8890 ] . invasive interventions , such as deep - brain stimulation , are implemented when drugs fail to relieve the patients . they are used when neurodegeneration has become sufficiently severe to induce a clinical phenotype . the discovery that a , tau and -syn fibrillar assemblies transit between cells within the central nervous system and amplify by recruitment of their soluble counterparts in recipient cells , thus contributing to disease progression , suggests that pathogenesis starts many years before the first symptoms become apparent . targeting early enough protein assemblies involved in disease formation and propagation may therefore represent a novel and effective preventive therapeutic avenue . thus , in compliment to the classical approaches aimed at either inhibiting the initial aggregation of proteins or favoring their clearance by boosting the level of molecular chaperones within the cells and/or the ubiquitin - proteasome clearance machinery , or both , therapeutic strategies targeting disease - associated protein assemblies propagation and seeding capacities become highly relevant . small molecules such as the polyphenols rottlerin , curcumin or epigallocatechin-3-gallate have been shown to either interfere with the aggregation of -syn , stimulate proteostasis , activate the ubiquitin - proteasome clearance machinery or counteract the neuro - inflammatory response associated to -syn aggregation [ 9193 ] . a novel class of small molecules that repair proteostasis networks specifically through hsf-1 , foxo and nrf-2 and restore proteome balance has been identified . these molecules have a promising therapeutic potential in a variety of protein conformational diseases as they enhance cell stress pathways and chaperone activity . they protect therefore the cells in a generic manner against misfolding and/or aggregation and the resulting pleiotropic damages . in addition , strategies aimed at protecting and/or stimulating neuronal cells through the use of neurotrophic factors such as brain - derived neurotrophic factor or clearing the polypeptides associated to neurodegeneration prior to their aggregation / deposition through immunotherapy are explored [ 97 , 98 ] . finally , neural stem cell - based treatments for neurodegenerative diseases are considered . in the case of pd , innovative strategies aimed at interfering specifically with -syn assemblies propagation and seeding capacities have not yet been developed to a stage where they can be used for therapeutic purposes . the key step in this process is the docking of -syn assemblies to the cell . one can interfere with this process either by i - perturbing the binding capacity of extrinsic membrane proteins that interact with -syn assemblies or ii - changing the surface properties of -syn assemblies so that they become incapable of binding to their receptors . as no specific receptors of -syn assemblies have been identified so far , the strategy consisting of rendering those receptors unavailable for binding -syn assemblies is impossible . furthermore changing the binding capacities of these receptors may cause undesirable pleiotropic effects . designing -syn assemblies binders that change their surface properties in such a way that they either become unable to bind the cell surface or incapable of elongating by recruitment of the endogenous protein because their ends are capped appears more promising . two classes of binders , molecular chaperones and antibodies , have been shown to interfere with -syn assemblies uptake by cells and/or toxicity [ 100 , 101 ] . thus , identifying within these molecules what is necessary and sufficient for binding is critical for the design of smaller molecules with therapeutic potential . the interaction interfaces between the molecular chaperone hsc70 client protein binding site and -syn have been identified through the use of chemical cross - linkers and mass spectrometry [ 102 , 103 ] . the same strategy can be employed to identify paratopes within antibodies exhibiting high affinity for -syn . peptides reproducing hsc70 or anti--syn paratopes that bind with the highest affinity -syn assemblies represent potential therapeuticcandidates .

abstractmisfolded protein aggregates are the hallmark of several neurodegenerative diseases in humans . the main protein constituent of these aggregates and the regions within the brain that are affected differ from one neurodegenerative disorder to another . a plethora of reports suggest that distinct diseases have in common the ability of protein aggregates to spread and amplify within the central nervous system . this review summarizes briefly what is known about the nature of the protein aggregates that are infectious and the reason they are toxic to cells . the chameleon property of polypeptides which aggregation into distinct high - molecular weight assemblies is associated to different diseases , in particular , that of alpha - synuclein which aggregation is the hallmark of distinct synucleinopathies , is discussed . finally , strategies targeting the formation and propagation of structurally distinct alpha - synuclein assemblies associated to different synucleinopathies are presented and their therapeutic and diagnostic potential is discussed .

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hepatitis c virus ( hcv ) related morbidity and mortality risk is significant in patients with chronic hepatitis c ( chc ) , which affects approximately 3.2 million people in the united states ( 1% of the population).15 in patients treated with interferon ( ifn ) and ribavirin , 2560% achieved sustained virus response ( svr ) , and with the addition of first generation direct acting antivirals ( daas ) , svr was achieved in almost 80%.69 treatment with new daas led to svr rates greater than 90%.1012 african americans ( aa ) are twice as likely to be infected with hcv relative to the nonhispanic - caucasian us population and have historically had lower response rates to approved ifn based therapies.420 although fibrosis progression is less rapidly in aa compared to caucasians , it is unlikely to be a good clinical strategy to counsel aa that they are at lower risk for rapidly developing advanced liver disease.1921 thus , more specific information about the aa population is needed to better understand the progression of liver disease , especially after achieving viral clearance , and to identify optimal treatment strategies . a number of publications have compared the response and outcomes in patients treated with ifn - based therapies.2227 all studies have demonstrated that patients who achieve an svr have better outcomes with respect to cirrhosis , development of hcc , and all - cause mortality . a report by cozen et al compared outcomes for treated and nontreated patients in two representative diverse populations . the results suggested that patients who received ifn - based therapies but failed to clear hcv may be at increased risk for developing cirrhosis compared to untreated patients . overall survival was not significantly different.28 additional studies provided conflicting results , but these did not contain a significant aa population . finally , a study utilizing electronic medical records ( emrs ) from a department of veterans affairs clinical registry confirmed the value of achieving svr and found that aa were at lower risk than caucasians for all liver events.29 the aim of this prospective study was to evaluate the effect of ifn based therapies on outcomes in aa patients . the predominant population in our urban clinic is aa , and we previously described the demographics and hcv relevant disease parameters in this patient group.30 here , we identified 346 aa first seen in 19952008 and followed them for 8 years on average . our findings further the understanding of liver disease progression in aa and provide information that will specifically guide clinical management and therapy decisions in this underserved and under - investigated population . in this prospective study for outcomes , we started with screened a database of 3,800 chc patients who were seen at the wayne state university physician group ( upg ) gastroenterology clinic between 1995 and 2008 . all patients signed an institutional review board ( irb ) approved consent form . using the emr and an irb approved health insurance portability and accountability act ( hipaa ) exemption from upg and detroit medical center , we identified patients who were diagnosed with hcc between 2009 and 2013 and were in the original database . we also identified patients were seen again between january 2012 and july 2013 in order to identify patients with a significantly long follow up time . in total , we identified 346 aa patients with hepatitis c genotype 1 that were followed for 4 to 18 years . available patient information included demographic information , laboratory values , biopsies , imaging , and response to treatment . first visit and early visit indicate that the information was relevant to the initial assessment of the patients although some data was order and obtained later as part of the initial assessment . cirrhosis was diagnosed with a combination of liver biopsy , ultrasound , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and/or esophagogastroduodenoscopy evidence of portal hypertension . since repeat biopsies were rarely performed , two well - studied markers of fibrosis were used to determine changes in fibrosis over time.3134 the aspartate amino transferase ( ast ) platelet ratio index ( apri ) was defined as ( ( ast(iu / ml)/40 iu / ml)/platelets ( count10/ml))100.33 fibrosis-4 ( fib-4 ) was defined as ( ( age(years))(ast(iu / ml)))/((platelets(count10/ml))(square root alt(iu / ml)).34 both assays are continuous variables and the literature cutoffs to define minimal vs significant fibrosis were 0.7 for apri and 1.45 for fib-4 . for cirrhosis , the response of aa to therapy was based on intent to treat ( itt ) . the sas based statistical program jmp was used for statistical analysis . to compare different groups , student s t test , analysis of variance ( anova ) , or chi - square changes between early visit and recent visit were evaluated using pairwise analysis . in order to determine the relationship between multiple factors defined in univariate analysis and outcomes , multivariate analysis ( logistic regression ) was used . in total , 346 aa patients , followed for an average of 8 years , were included in our study . the study group was 57% male , and the mean age was 54 years with a range consistent with known infection rates of individuals born between 1945 and 1965 . the mean levels of alanine aminotransferase ( alt ) and ast were elevated , but albumin and platelet levels were within normal range . the majority of patients had liver biopsy ( 257 out of 346 ( 74% ) ) , and the biopsy mean fibrosis score , as defined by the metavir , system was 1.88 . significance defined by pair wise analysis ; ns , not significant , p>0.01 ; * p<0.01 ; * * p<0.001 . all treated patients received ifn based therapies , and 147 patients ( 43% ) received at least one dose of treatment . the majority of treated patients ( 86% ) were given dual therapy of pegylated - ifn and ribavirin . the svr rate was 15% based on itt and 23% based on patients who adhered to the protocol . when patients who relapsed following treatment were included , the end of treatment response ( etr ) was 24% . when patients were stratified by treatment , there were minimal differences between various groups of patients at the first visit ( table 1 and fig . 1 ) . the largest difference , although statistically insignificant , was a reduction in hcv ribonucleic acid ( rna ) levels in patients with svr relative to other groups . in addition , the values before treatment and after treatment / no treatment for liver related laboratory values as a function of response to treatment were compared . the most improved patients were those who achieved an svr , had reversal of alt and ast elevation , and had no decline in albumin and platelet counts . apri and fib-4 were used to assess changes in fibrosis / cirrhosis.3134 we used biopsy data to validate both the apri and fib-4 in our study population . there was a strong correlation between the degree of fibrosis defined by biopsy and fibrosis as calculated by apri and fib-4 ( p<0.001 data not shown ) . also the receiver operating characteristic and the area under the receiver operating characteristic curve ( auroc ) values for the aa population were between 0.56 and 0.75 ( data not shown ) . these results are consistent with the literature , and confirm that both calculations are sensitive and specific for measuring differences in fibrosis in aa.3134 the fibrosis scores by apri ( a ) and fib-4 ( b ) are plotted as the average for paired patients between the initial visit ( 1 ) and follow - up visit ( 2 ) . n treat , nontreated ; dis , discontinued ; nonr , nonresponders ; rel , relapsed ; svr , sustained virus response . significance was defined using the paired difference test . when the apri and fib-4 were evaluated in patients at the initial visit and follow - up , there was no significant change in fibrosis in patients that were not treated ( fig . only the patients who achieved svr had a significant improvement in these fibrosis scores relative to the other groups . statistical significance was assessed using a pair - wise analysis , and there were differences in individual patients but not group averages . patients who achieved svr did not develop new cirrhosis or hcc in the follow - up period ( fig . patients who were not treated , who discontinued treatment , or who were nonresponsive to treatment developed hcc and newly identified cirrhosis . the nonresponding patients , however , had a lower incidence of hcc than untreated or discontinued treatment patients . patients achieving an etr but not svr did not develop hcc but exhibited a similar incidence of new cirrhosis as the other groups . patients were evaluated for the development of hepatocellular carcinoma ( hcc ) and new cases of cirrhosis at visits following the initial visit . the majority of patients were treated with peg - ifn + ribavirin ( 86% of treated patients ) . etr ( svr + relapse patients ) was 24% by itt and 36% by protocol . n treat , nontreated ; dis , discontinued ; nonr , nonresponders , rel , relapsed ; svr , sustained virus response . since males are more likely to develop hcc , we compared hcc development and response to therapy by gender ( fig . the response to treatment was similar for both genders ( 13% f vs. 18% m ) , although the percentage of females treated ( 46% ) was greater than males ( 38% ) . 3 , aa males were more likely to develop hcc than females , but patients achieving an svr or etr with subsequent relapse did not develop hcc regardless of gender . in addition , although males had a higher incidence of hcc , the time to development of hcc was not statistically significant between the genders ( f=9 years vs. m=8 years ) . the % of patients who developed hcc was plotted by response to treatment and gender . similar numbers of aa patients were treated ( 46% f and 38% m ) , and response rates based on intent to treat were also similar ( 15% ) . although males were more likely to develop hcc , hcc did not develop in either the svr or etr ( relapse ) patients , regardless of gender . the average time to hcc was 9 years for females and 8 years for male ( nonsignificant by chi - square ) . in order to define the natural history of the disease in aa patients the time to hcc onset ranged from 1 year to 14 years after the first visit , with 4 to 6 years being the most common timeframe ( n=24 , fig . 4 ) . there was no difference in time to hcc diagnosis between aa patients with or without cirrhosis at the time of the first visit . all patients ( n=51 ) who developed hcc were stratified by those who had cirrhosis at first visit ( hcc from cirrhotic ; n=12 ) and those who did not ( hcc - noncirrhotic ; n=39 ) . almost half ( 24/51=48% ) of patients developed hcc 47 years after the first visit but a significant number of patients ( 25% ) did not receive a diagnosis of hcc for more than 11 years . the risk of developing of hcc at the first visit was not predicted by the presence of cirrhosis , since the time to hcc distribution was similar for those with and without cirrhosis at the first visit . untreated patients who did not develop hcc were compared to those who did develop hcc in order to determine whether particular parameters at the first visit were useful predictors of hcc development . alt , ast , albumin , platelet counts , and alpha fetoprotein ( afp ) all correlated / inversely correlated with hcc development in aa ( p<0.005 , data not shown ) . fig . 5 presents the data related to fibrosis ( liver biopsy metavir scores , apri , and fib-4 ) , and all were significant predictors of hcc . fibrosis but not cirrhosis at the first visit was a significant risk factor for hcc . there was a wide range and considerable overlap in all patient parameters among patients who developed hcc and those who did not . therefore , the values could not be used to predict individual risk for developing hcc . when using multivariate analysis for all risk factors ( alt , ast , albumin , platelets , afp , stage of fibrosis , apri , fib-4 , and cirrhosis ) , no parameters were identified that were independent predictors of risk for developing hcc when subjected to logistic regression analysis . when the multivariate analysis was restricted to only fibrosis relevant independent predictors ( metavir stage , apri , and fib-4 ) , the only independent predictor of hcc was stage of fibrosis ( p<0.002 ) . individual patients were stratified by those who developed hcc or those who did not and their individual values for potential risk factors at first visit are plotted . for continuous variables , the graphs have the mean and sem and significance was defined with a student s t test ( a , b ) . for discontinuous values ( fibrosis by metavir and presence / absence of cirrhoses ) the data are presented as mosaic plots and significance was calculated using pierson chi - square ( c , d ) . the width of the plots reflect the total numbers of patients , and the various shadings represent the % of patients with each parameter ( scores of 0 - 4 or cirrhosis or no cirrhosis ) . the bar to the right of each graph reflects the % of total patients . similar results were obtained for ast , albumin , platelet counts , and afp ; and the difference at entry between hcc and no hcc was significant ( p<0.001 for all , data not shown ) . the wide variability and overlap between individuals in those assays was also similar to that shown in the figure . although patients who developed hcc were more likely to have cirrhosis at first visit , the difference was not statistically different . in this study , we followed aa patients with chc for an average of 8 years who were infected with hcv genotype 1 . chc outcomes were defined by either the development of cirrhosis , hcc , or fibrosis from the initial visit to the most recent follow up in 2012 - 2013 . as part of this study , we confirmed that a significant number of patients with early visit liver biopsy results had a good correlation with both apri and fib-4 , thus confirming them as useful surrogate markers for fibrosis in aa patients . since repeat biopsies were rarely performed , fibrosis was detected using apri and fib-4.7,3134 the study demonstrated that permanent viral elimination ( defined as svr ) with ifn based treatment in aa patients with chc protected them from cirrhosis and hcc and reversed fibrosis . although chc patients with etr who relapsed developed new cases of cirrhosis at rates similar to those of nontreated or treatment failure patients , they did not develop hcc . in contrast , patients who failed treatment or were not treated were at high risk for the development of hcc and new onset cirrhosis . this study also confirmed that aa males are at higher risk for developing hcc than aa females . the response to therapy was similar and achieving an svr reduced the risk of hcc in both genders . since a significant number of patients developed hcc , we were able to determine whether hcc risk in aa was predictable based on the first clinical visit . unlike many studies , we plotted individual patients rather than an average in order to assess the clinical value per patient . regardless of whether serum assays ( alt , ast , afp , albumin , platelets ) or fibrosis assays ( biopsy , apri , fib-4 ) were compared between patients who did or did not develop hcc , there was no reliable predictor on an individual basis . somewhat unexpected , we found that cirrhosis at first visit in aa was not predictive of hcc development . when multivariate analysis was used to determine whether there was a single independent factor that was predictive of development of hcc in aa , only fibrosis , as defined by biopsy at the first visit , was even moderately predictive . this lack of positive predictive value for factors was further confirmed by the finding that when patients who developed hcc were stratified by cirrhosis ( or no cirrhosis ) at first visit , there was no relationship between time to hcc and cirrhosis . although the number of patients achieving an svr was sufficient for analysis , we could not rule out the possibility of the development of hcc in patients who achieved an svr . there are a number of publications suggesting that the rate of hcc may be as high as 5% in patients experiencing an svr.3537 those studies are , however , primarily in asian and european populations and do not contain significant numbers of aa . the primary risk factors for developing hcc in patients with svr are males with genotype 1 and cirrhosis at the time of viral clearance . the majority of hcc cases occurred within 3 years , suggesting the presence of small precancerous lesions at the time of viral clearance . since our population contains patients with these significant risk factors and a high incidence of hcc was present in the untreated and nonresponding population , it is possible that the development of hcc in aa after clearance of the virus is considerably less likely than in the other populations studied . to a large extent , this study is prospective in nature since patients were first seen prior to 2008 and subsequently seen until 2013 or the development of hcc . patients who developed hcc were followed for 114 years , while those who did not , were seen for a minimum of 4 years and a maximum of 18 years after initial presentation . this study did not compare caucasians to aa , as the number of caucasians seen in our clinic has continued to decline and there were insufficient numbers available for statistical analyses . the incidence of hcc in aa in our study was 20% in untreated patients , which was sufficient to provide statistical and clinical relevance to our study . perhaps the most important observation of the study was that aa who did achieve svr were similar to other groups of svr patients who also had improved outcomes relative to untreated or nonresponding patients . with the advent of more effective daa therapies , possible differences in response rates between aa and caucasians are no longer an issue , and more patients should achieve an svr.12 although there are many risk factors for the development of hcc in aa patients , if patients are not successfully treated , it remains difficult to predict the development of hcc . since identification of cirrhosis at an early visit may not be a significant risk factor in aa , surveillance in patients with significant fibrosis , rather than just cirrhosis , is warranted . unfortunately in many studies to date , it is often a challenge to provide effective hcc surveillance of this population ; and thus , elimination of the virus remains the optimal goal of therapy .

background and aims : african americans ( aa ) historically have a low response rate to hepatitis c therapies , and there is limited information available for this patient population regarding the development and treatment of chronic hepatitis c ( chc ) . the aim of this study was to evaluate liver disease progression and hepatocellular carcinoma ( hcc ) development in aa with chc . methods : between 1995 and 2008 , 246 aa patients with chc were identified from a database of patients and followed until 2012 - 2013 ( average 8 years ) or the development of hcc after 2008 . results : viral clearance ( intent to treat ; sustained virus response ( svr ) ) was achieved in 15% of patients with interferon based therapies with or without ribavirin . aa patients who achieved an svr ( n=22 ) did not develop hcc or new onset cirrhosis , whereas the hcc incidence in untreated aa patients was 23% ( 51/203 ) . patients who achieved an svr also had improved fibrosis , as defined by the ast platelet ratio index ( apri ) and fibrosis-4 ( fib-4 ) score , relative to nonresponders and untreated patients . conclusions : the severity of liver disease at the first visit ( except for cirrhosis ) correlated with the development of hcc , but because of the overlap in values between patients , these measurements were not useful for predicting individual risk . since cirrhosis at the first visit was not a predictive factor , treatment with newer antiviral therapies is the best option for reducing the incidence of advanced liver disease and its harmful outcomes in the aa population .

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stroke is the second common cause of death following ischemic heart disease.1 stroke has accounted for nearly 5.7 million deaths globally and 87% of these deaths take place in low and middle income nations.2 stroke is a multi - factorial disease and epidemiological and animal studies have robustly recommended genetic influences in the pathogenesis of ischemic stroke.3 the genetic influences are probably polygenic whereby multiple genes exert a small influence or risk on phenotype . beta adrenergic receptors are members of a family of receptors known as g - protein coupled receptors ( gpcr ) and have a seven membrane spanning domain structure , an extracellular amino terminus , three intracellular and three extracellular loops , and an intracellular carboxyl terminus.4 these are receptors for neurohormone epinephrine and nor - epinephrine . several mechanisms contribute to loss of receptor activity including uncoupling of the receptor from adenylyl cyclase activity , internalization of the receptor and phosphorylation of internalized receptors.5 several studies support the role of cyclic adenosine monophosphate ( camp ) in atherogenesis by modulating the function of vascular endothelium , the production of reactive oxygen species , the recruitment of circulating monocytes to the artery wall and their differentiation into macrophages - foam cells , by controlling the expression of pro - and anti - inflammatory interleukin , and regulating serum level of triglycerides and cholesterol.6,7 camp is also a possible target for prevention and treatment of atherosclerosis.6 the major non - synonymous snps of 2ar have been recognized at nucleotides 46 ( a > g ) ( rs1042713 ) and 79 ( c > g ) ( rs1042714 ) causing changes in amino acid residues at position 16 ( arg > gly ) and 27 ( gln > glu ) of the amino terminus respectively of the fourth intracellular loop . an enhanced agonist - mediated receptor down - regulation for the gly16 variant of 2ar and a resistance to down - regulation for the glu27 variant of 2ar has been observed.8 it is hypothesized that due to the polymorphism in 2ar , functional alteration in the receptor function occurs which influences a certain intermediate mechanism for the predisposition of cardiovascular and cerebrovascular diseases.9 we conducted a meta - analysis with available published studies to precisely determine the association of 2ar with ischemic stroke in order to offer early diagnosis of the susceptible subjects . the following search keywords were applied : ' bar ' or ' bar gene variant ' or 2ar , ' beta - adrenergic receptor ' or ' beta adrenergic receptor polymorphism ' and ' cerebral infarction ' or ' cerebrovascular accident ' or ' ischemic stroke ' or ' brain infarction ' . inclusion criteria : ( i ) case control study studying the association between 2ar polymorphism and stroke ; ( ii ) studies published in english language with full text ; ( iii ) stroke confirmed by mri or ct . exclusion criteria : ( i ) studies other than case control design ; ( ii ) studies did not report genotypic and allelic frequencies ; ( iii ) duplicate publication . two investigators ( ak and mp ) independently evaluated the title , abstracts and search terms for eligibility based on the predetermined selection criteria . all discrepancies were resolved after rechecking the source papers and further discussion among the two authors . the relevant data from each study were independently extracted by two reviewers ( ak and mp ) using a standardized , structured form including first author 's name , year of publication , country , genotyping method , no . of cases and controls and frequency distribution of genotype and allele . the following search keywords were applied : ' bar ' or ' bar gene variant ' or 2ar , ' beta - adrenergic receptor ' or ' beta adrenergic receptor polymorphism ' and ' cerebral infarction ' or ' cerebrovascular accident ' or ' ischemic stroke ' or ' brain infarction ' . inclusion criteria : ( i ) case control study studying the association between 2ar polymorphism and stroke ; ( ii ) studies published in english language with full text ; ( iii ) stroke confirmed by mri or ct . exclusion criteria : ( i ) studies other than case control design ; ( ii ) studies did not report genotypic and allelic frequencies ; ( iii ) duplicate publication . two investigators ( ak and mp ) independently evaluated the title , abstracts and search terms for eligibility based on the predetermined selection criteria . all discrepancies were resolved after rechecking the source papers and further discussion among the two authors . the relevant data from each study were independently extracted by two reviewers ( ak and mp ) using a standardized , structured form including first author 's name , year of publication , country , genotyping method , no . of cases and controls and frequency distribution of genotype and allele . two studies were excluded because they did not meet the inclusion criteria for study design ( they were cohort studies ) . two more studies were excluded because they studied another gene polymorphism and one study was excluded for being a pharmacogenetic study . two studies11,12 reported the genotypic data , and therefore were included for genotypic association meta - analysis . allelic data was reported in another study.10 a total three studies were included for allelic association meta - analysis . the study characteristics , which are included in the present meta - analysis , are described in table 1 . a total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . meta - analysis results did not show a significant association between arg16gly polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or 1.20 ; 95% ci , 0.92 to 1.56 ) ( figure 2a ) or dominant model of inheritance ( or 1.14 ; 95% ci , 0.68 to 1.91 ) ( figure 2b ) . the allelic association also did not show statistically significant association between arg16gly polymorphism of 2ar and the risk of ischemic stroke ( or 1.04 ; 95% ci , 0.85 to 1.28 ) ( figure 2c ) . meta - analysis results did show a significant association between gln27glu polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or , 2.09 ; 95% ci , 1.20 to 3.64 ) ( figure 3a ) or the dominant model of inheritance ( or , 1.47 ; 95% ci , 1.14 to 1.90 ) ( figure 3b ) . a significant allelic association was observed between gln27glu polymorphism of 2ar and ischemic stroke ( or , 1.58 ; 95% ci , 1.38 to 1.81 ) ( figure 3c ) . two studies were excluded because they did not meet the inclusion criteria for study design ( they were cohort studies ) . two more studies were excluded because they studied another gene polymorphism and one study was excluded for being a pharmacogenetic study . two studies11,12 reported the genotypic data , and therefore were included for genotypic association meta - analysis . allelic data was reported in another study.10 a total three studies were included for allelic association meta - analysis . the study characteristics , which are included in the present meta - analysis , are described in table 1 . a total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . meta - analysis results did not show a significant association between arg16gly polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or 1.20 ; 95% ci , 0.92 to 1.56 ) ( figure 2a ) or dominant model of inheritance ( or 1.14 ; 95% ci , 0.68 to 1.91 ) ( figure 2b ) . the allelic association also did not show statistically significant association between arg16gly polymorphism of 2ar and the risk of ischemic stroke ( or 1.04 ; 95% ci , 0.85 to 1.28 ) ( figure 2c ) . meta - analysis results did show a significant association between gln27glu polymorphism of 2ar and ischemic stroke when assuming either recessive model of inheritance ( or , 2.09 ; 95% ci , 1.20 to 3.64 ) ( figure 3a ) or the dominant model of inheritance ( or , 1.47 ; 95% ci , 1.14 to 1.90 ) ( figure 3b ) . a significant allelic association was observed between gln27glu polymorphism of 2ar and ischemic stroke ( or , 1.58 ; 95% ci , 1.38 to 1.81 ) ( figure 3c ) . the present meta - analysis was conducted to determine the precise estimation of association between polymorphism of 2ar and risk of ischemic stroke . several studies have shown an independent association of gln27glu polymorphism of 2ar gene number of diseases like obesity,13,14 dyslipidemia,14 myocardial infarction,15 and diabetes.16 thus , gln27glu polymorphism of 2ar has been suggested to be an independent risk factor for cardiovascular diseases and cerebrovascular diseases.17,18 our meta - analysis suggests a significant association of gln27glu polymorphism of 2ar with ischemic stroke . our results are in agreement with earlier published study.12 however , two prospective cohort studies19,20 failed to show a significant association of 2ar variant with the incidence of stroke . a prospective cohort study including 25,225 women showed that the different haplotypic combination of beta receptor gene variant did not affect the incidence of ischemic stroke in women.20 another cohort study reported from same population which included a total of 808 black and 4,441 white participants failed to find significant association of 2ar genotypic with risk of ischemic stroke and combined cardiovascular outcome.19 these two prospective cohort studies in which significant associations were not observed , were reported from the american population19,20 while the other two studies,11,12 which showed the significant genotypic association , were reported from india and italy . as the prevalence of genetics variants often varies among populations,19 this could explain the discrepancy of association of gln27glu polymorphism of 2ar and ischemic stroke across the studies . a case control study reported by zhao et al.10 in chinese population showed significant allelic association between glu allele of 2ar and risk of ischemic stroke ( figure 2c ) . in all the published case control studies ( three),10,11,12 frequency of the risk allele ( glu27 ) was higher in cases as compared to controls . our meta - analysis of allelic association including three studies with the risk allele versus protective allele suggested significantly higher risk of ischemic stroke with glu27 allele . the meta - analysis for genotypic association including two studies also suggested a significant association between gln27glu polymorphism and risk of ischemic stroke under both dominant model and recessive model of inheritance . some limitations exist in the present study , which may have affected the results of meta - analysis . the haplotype analysis was not done which plays a crucial role for association studies of complex diseases . the interaction between gene and environment and gene - gene interaction was not studied in this meta - analysis . controls were selected from the hospital , which may have lead to the selection bias . multivariate analysis for adjusting the several confounding factors that could have an effect on results was not performed as individual data from each study was not available . studies were reported from different ethnic population and deviation from hwe equilibrium in control subjects in one study included in this meta - analysis may account for the heterogeneity . some limitations exist in the present study , which may have affected the results of meta - analysis . the haplotype analysis was not done which plays a crucial role for association studies of complex diseases . the interaction between gene and environment and gene - gene interaction was not studied in this meta - analysis . controls were selected from the hospital , which may have lead to the selection bias . multivariate analysis for adjusting the several confounding factors that could have an effect on results was not performed as individual data from each study was not available . studies were reported from different ethnic population and deviation from hwe equilibrium in control subjects in one study included in this meta - analysis may account for the heterogeneity . the present study provides preliminary evidence to support the view that carrier of gln27glu polymorphism of 2ar demonstrates an increase in risk of ischemic stroke on the basis of meta - analysis of case control studies . furthermore , well designed larger prospective cohort studies are required to validate this finding and to provide a higher level of evidence . the underlying molecular causal pathways that confer susceptibility to ischemic stroke are warranted to be established .

background and purposethe purpose of this meta - analysis was to determine the precise association between beta-2 adrenergic receptor ( 2ar ) polymorphism and ischemic stroke.methodspublished case control studies on association between 2ar and ischemic stroke were searched from electronic databases . pooled odds ratio and 95% confidence interval were calculated by using software revman version 5.2.resultsa total of three studies involving 1,642 cases and 1,673 controls , which were published from 2007 to 2014 , were subjected to meta - analysis for allelic association and 518 cases and 510 controls for genotypic association . pooled analysis of two studies for genotypic association suggested that subjects carrying gln27glu polymorphism of 2ar had an increased risk for ischemic stroke under recessive model ( or 2.09 ; 95% ci ; 1.20 to 3.64 ) and under dominant model ( or 1.47 ; 95% ci 1.14 to 1.90 ) . pooled analysis of three studies for allelic association showed a significantly higher glu27 allele of 2ar in the patients with ischemic stroke ( or 1.58 ; 95% ci ; 1.38 to 1.81).conclusionsthe present meta - analysis suggests that gln27glu polymorphism of 2ar gene is associated with increased risk for ischemic stroke .

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methadone ( d , l - methadone hydrochloride ) is frequently used in different therapies including opioid addiction , long - lasting analgesics in cancer and neuropathic pain syndromes [ 13 ] . however , numerous reports indicate a negative impact on human cognition by chronic exposure to opioid drugs . patients subjected to methadone maintenance programs show impaired cognitive abilities in aspects such as psychomotor performance , information processing , attention , problem solving , memory , decision making , reaction time , and emotional facial expression recognition [ 410 ] . changes in the cytosolic free - calcium concentration ( [ ca]cyt ) are involved in control of a large number of cellular and physiological processes including neuronal excitability , synaptic plasticity , and gene transcription [ 11 , 12 ] . however , the physiological ca signal can switch to a death signal when the [ ca]cyt increases dramatically . for example , excitotoxic high glutamate concentrations result in an initial transient increase in [ ca]cyt that is followed by a delayed , irreversible rise in [ ca]cyt known as delayed calcium deregulation ( dcd ) . although several steps preceding dcd remain to be clarified , there is evidence that dcd is the irreversible end point of a sequence involving mitochondrial ca overloading . they buffer variations in ca concentrations by taking up ca when and where [ ca]cyt levels are passing a threshold level above which the mitochondrial ca uniporter is activated , and slowly release ca back to the cytosol when [ ca]cyt drop below this point . mitochondrial ca overload , if large and sustained enough , may contribute to mitochondria permeability transition pore ( mptp ) formation and ultimately lead to cell death [ 11 , 15 ] . because , mitochondria may accumulate a considerable amount of ca during neurotoxic exposure , a possibility is that dcd may represent the final consequence of mitochondrial ca overload . mptp is a large , proteinaceous , ca - activated , proton- and adp - inhibited voltage - dependent pore . it spans the inner and outer mitochondrial membrane allowing the passage of ions and substrates less than 1.5 kda . characteristically , opening of the mptp is inhibited by cyclosporin a [ 16 , 17 ] . sh - sy5y cells are considered a suitable model for investigating opioid - mediated responses in neurons . , we showed that sh - sy5y cells exposed to high concentrations of methadone ( 0.5 mm ) died through a necrotic - like cell death mechanism and that methadone may induce changes in the [ ca]cyt [ 19 , 20 ] . however , the underlying mechanisms causing alterations of the [ ca]cyt in sh - sy5y cells in the presence of methadone remained unknown . therefore , the aim of the present study was to investigate those mechanisms . a clear understanding of the factors that mediate this phenomenon might help to resolve the mechanisms that promote neuronal cell death during methadone - induced cognitive damage . sh - sy5y cells ( atcc ) were plated at a density of 5.3 10 cells / cm on -dish 35 mm high ibitreat ( ibidi gmbh , martinsried , mnchen , germany ) as previously reported . changes in [ ca]cyt in sh - sy5y cells were measured by loading cells with the calcium probe fura2/am and using an inverted fluorescence microscope ( nikon eclipse te2000-s ) as described elsewhere . cells were perfused with a medium containing 140 mm nacl , 5 mm kcl , 1 mm mgcl2 , 2.5 mm cacl2 , 10 mm hepes , and 11 mm glucose , ph 7.35 . ratios of fluorescence emission excited at 340 and 380 nm were captured every 5 seconds . methadone or other compounds were added from 1000x stock solutions to reach the appropriate final concentrations . the effects of selected compounds were tested on [ ca]cyt in the absence and the presence of 0.5 mm methadone in different sets of cells . cells were pooled according to the dynamics of the rises in [ ca ] . to directly visualize mitochondrial morphology changes in intact cells overexpressing pdsred2-mito plasmid ( clontech laboratories , inc . mountain view , ca , usa ) a leica sp2 confocal microscope ( 63 1.4 na objective ) was used . for transfection of cells the reagent lipofectamine was used ( invitrogen , carlsbad , ca , usa ) . rat liver mitochondria were isolated in msh / edta and msh media containing 210 mm mannitol , 70 mm sucrose , 5 mm hepes , with or without 1 mm edta , ph 7.4 , by differential centrifugation according to the standard procedure . the mitochondrial suspensions were kept on ice and immediately used for measurements of oxygen - uptake rate . the rate of oxygen uptake of isolated rat liver mitochondria was measured at 37c in a water - thermostatized incubation chamber with a computer - controlled clark - type o2 electrode ( oxygraph , hansatech , uk ) in 0.5 ml incubation buffer ( 145 mm kcl , 30 mm hepes , 5 mm kh2po4 , 3 mm mgcl2 , 0.1 mm egta , 0.1% defatted bsa , ph 7.4 ) . the respiratory substrates used were complex i- or complex ii - linked , 2.5 mm glutamate/2.5 mm malate or 5 mm succinate in the presence of 2 m rotenone . the following additions were applied : 250 m adp , 200 m cacl2 , 0.4 m fccp , and 0.5 mm methadone . for estimating mitochondrial ca uptake in isolated mitochondria a ca index was calculated , which denotes the ratio of oxygen - uptake rate triggered by addition of 200 m ca to previous oxygen uptake rate in this study , we have investigated the mechanisms involved in methadone - induced rises in [ ca]cyt in sh - sy5y cells . consistent with previous observations from our laboratory and other data , methadone induced a rise in [ ca]cyt in most of the sh - sy5y cells . analysis of the dynamics of the [ ca]cyt recordings in the absence and the presence of methadone suggested that four different types of calcium recordings can be observed in sh - sy5y cells . for the first group of cells no rise in [ ca]cyt was observed during the entire period ( 30 min ) of measurements ( type 1 ) . a second group of cells was unable to regulate [ ca]cyt homeostasis shortly after the addition of methadone ( type 2 ) and [ ca]cyt increased continuously . a third group displayed [ ca]cyt deregulation with a delay of 1520 minutes ( type 3 ) . finally , the fourth group was able to control [ ca]cyt after an initial increase ( type 4 ) . groups 1 and 4 represent , respectively , nonresponsive of cells and cells responding with transient rise in [ ca]cyt and amount 20.82 and 31.38% of the cells . however , types 2 and 3 displayed short or delayed continuous rise in [ ca]cyt that may be considered as early or delayed ca deregulation according to the literature [ 2629 ] . the abundance of cells showing deregulation ( type 2 and type 3 ) amount to 32.83 and 14.92% , of the total , respectively we have analyzed the relative abundance of the four types of recordings described above in the absence ( control ) and the presence ( methadone ) of 0.5 mm methadone . clearly , methadone decreased type 4 ( transient responsive cells ) and increased types 2 and 3 ( deregulated cells ) . these results suggest that methadone may induce a short or delayed ca deregulation in sh - sy5y cells . the mechanisms underlying the observed quantitative changes in the types of ca responses mediated by methadone are unknown . therefore , we performed a comprehensive pharmacological survey to study the possible contribution of different carelated mechanism to the response to methadone . as illustrated in figures 1(b ) and 1(c ) and discussed below , a 5 min pretreatment of sh - sy5y cells with different conditions and drugs affected the methadone - induced [ ca]cyt response , monitored as changed frequencies of the different types of the responses . we found that in the absence of extracellular calcium ( 0 ca ) no change in responses to methadone was observed ( n = 291 cells ) . these results suggest that ca entry does not contribute to the reported changes in [ ca]cyt . to confirm that extracellular ca was not involved in these responses we tested the effect of methadone in the presence of nifedipine , a specific isopropyl l - type ca channel blocker . nifedipine ( 2 m ) did not modify the effects of methadone on the [ ca]cyt in sh - sy5y cells ( n = 226 cells ) . so , we ruled out the involvement of this voltage - dependent channel family in methadone - induced [ ca]cyt variations . cells were treated with thapsigargin ( 1100 m ) . to deplete intracellular ca stores before methadone treatment . as expected , thapsigargin induced a significant release in ca from the endoplasmic reticulum , causing a transient increase in the [ ca]i that failed to returned to the basal level within a 10-minute period . a detailed observation of the methadone induced rise in [ ca]i shows that cell responses were partially affected by this treatment , suggesting that the rise in calcium is partially due to release from thapsigargin - sensitive , intracellular ca stores ( n = 146 cells ) . we found that in untreated cells addition of thapsigargin induced a transient increase in [ ca ] that has been attributed to a leakage of ca from the endoplasmic reticulum . this effect of thapsigargin alone interferes the interpretation of the type 2 and 3 responses after methadone addition ( data not shown ) . , we treated the cells with 50 m naloxone ) , the competitive antagonist of the -opioid receptor . we found that this treatment did not modify the relative abundance of any of the four types of cell populations ( n = 80 cells , 4 exp ) . this result suggests that the changes in [ ca]cyt and dcd induced by methadone in sh - sy5y cells are independent of opioid receptors . in agreement with the lack of -opioid receptor participation as found here , it has been reported that methadone - toxic pathways are not mediated by receptors [ 19 , 3032 ] . to test the possible contribution of mitochondria we used the mitochondrial uncoupler carbonyl cyanide p - trifluoromethoxyphenylhydrazone ( fccp ; 1 m ) it is well established that fccp collapses mitochondrial potential and abolishes the ability of mitochondria to take up ca . we found that in untreated cells , addition of fccp induced a transient increase in [ ca ] that has been attributed to leakage of ca from depolarized mitochondria . we found that fccp induced a 44% decrease in the appearance of type 2 cells whereas increased the relative abundance of cells showing a type 3 response by 2.7-fold in fccp ( figures 1(b)-1(c ) ) . nevertheless , it must be taken into account that as the simple usage of protonophores does not allow a clear - cut study of the role of mitochondrial ca transport as they also may cause a lowering of the atp / adp ratio , thereby affecting atp - dependent ca pumps . an approach that has been previously exploited to investigate the role of mitochondria in synaptosomal ca homeostasis involves inhibition of mitochondrial atp synthesis by oligomycin and application of glycolysis as the source of atp and independent manipulation of m with specific respiratory chain inhibitors [ 33 , 34 ] . inhibition of atp synthase by oligomycin prevents mitochondrial oxidative phosphorylation , but unlike protonophore addition , it does not cause hydrolysis of cytoplasmically generated atp . it has been reported that dcd might result from a failure in ca extrusion caused by cytoplasmic atp depletion . therefore , we tested the effects of a short , 5 min , incubation with 10 g / ml oligomycin on ca responses . under these conditions , oligomycin alone did not modify the [ ca]cyt responses during the 30 min recording period . specifically , the abundance of sh - sy5y cells showing a type 2 response were increased whereas the pool of cells showing a dcd - related type 3 response were lost . additionally , oligomycin induced a 3.3-fold increase in cells showing a type 4 response . to test further the contribution of mitochondria we investigated the possible role of the mitochondrial ca/2na exchanger . mitochondrial ca efflux is normally primarily regulated by a ca/2na exchanger . to block mitochondrial ca exit we used cgp37157 ( 50 m ) , an inhibitor of the ca/2na - exchanger . a five min exposure of cells to cgp37157 significantly increased the proportion of cells showing no rise in [ ca]cyt in response to methadone during the entire [ ca]cyt measurement period ( type 1 ) ( data not shown ) because cgp37157 resulted in a drastic decrease in the population of cells showing either type 2 ( 74% ) or type 3 ( 85% ) responses . in addition , the relative abundance of cells showing a type 1 response returned to the value obtained in untreated cells . these results support data suggesting the relevance of mitochondria in methadone - induced dcd ( figures 1(b)-1(c ) ) . to test contribution of reactive oxygen species we used the cell - permeable , small molecule compound tempol ( 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy ) to mimic superoxide dismutase activity . in the presence of tempol ( 0.2 m ; n = 80 cells ) , cells responded to methadone in a different way . specifically , tempol decreased the number of cells showing a type 2 response by ~70% , whereas the abundance of cells showing a type 3 response was largely increased ( 3.5 fold ) . suggested that enhanced ros is a consequence rather than a cause of dcd . in their studies , they applied a novel technique to monitor the bioenergetic status of in situ mitochondria in cultured neurons in a model of glutamate excitotoxicity . in agreement with this , a general ineffectiveness of antioxidants to decrease dcd in the presence of glutamate has been observed . finally , we tested the contribution of the mitochondrial permeability transition ( mptp ) . additional efflux of mitochondrial ca can occur by induction of mptp formation , which is dependent on the mitochondrial matrix ca concentration and can be inhibited by cyclosporine a [ 16 , 17 ] . to evaluate mptp participation we administered csa ( 1 m ) . csa diminished the occurrence of response type 2 by 36% ( n = 89 cells ) in cell cultures challenged with 0.5 mm methadone . moreover , consistent with the hypothesized role of the pore in dcd , csa induced a 2-fold increase in cells showing type 4 response . however , interpretation of these results is difficult because csa may also inhibit the mitochondrial ca uniporter in some instances . taken together , our data indicate that only drugs affecting mitochondrial handling of ca , such as oligomycin , fccp , cgp 37157 , and cyclosporine a , were able to modulate methadone - induced delayed calcium deregulation in sh - sy5y cells . we therefore conclude that methadone - induced dyshomeostasis is caused by improper functioning of mechanisms that directly control mitochondrial activity rather than a participation of plasma membrane ca channels or opioid receptors . next , the effect of methadone on mitochondrial morphology was studied in sh - sy5y cells transfected with pdsred2-mito . in untreated cultures , mitochondria presented a long and tubular morphology ( figure 2(a ) ) , which became dramatically shorter and rounder upon three hours of methadone treatment ( figures 2(b)2(d ) ) . cell counting of the different mitochondrial morphologies ( filamentous , mixed and fragmented ) indicated that methadone , in a dose - dependent manner , induced mitochondrial fragmentation ( figure 2(d ) ) . this effect seems contradictory to our earlier observations that methadone failed to induce mitochondrial swelling in isolated rat liver mitochondria . in fact , our data support this hypothesis , and indicate a role of dcd in methadone - induced toxicity . in agreement with this , it has been reported that the mptp opens under pseudopathological conditions with relatively high ca and low atp concentrations as was the case in our previous experiments with sh - sy5y cells . the rupture of the mitochondrial membrane caused by ca overload reduces the number of healthy mitochondria and this will affect crucial neuronal functions including synaptic transmission and axonal transport . finally , the effect of methadone on mitochondrial ca uptake was studied ( figure 3 ) . we used a clark electrode and applied different respiratory substrates , namely succinate and glutamate / malate . the respiratory chain is less dependent on the presence of m for succinate than for glutamate / malate . therefore , as a control , 0.4 m fccp was used because this concentration of fccp resulted in an increase of oxygen - uptake rate comparable with the uptake rate calculated for 0.5 mm methadone ( figure 3(a ) ) . the calculated values of the state u ( uncoupled state ) to state 4 ( resting state ) ratios ( u/4 ratios ) were as follows : for succinate 2.8 0.5 ( + fccp ) and 2.3 0.4 ( + methadone ) , and for glutamate / malate 2.1 0.5 ( + fccp ) and 2.5 0.4 ( + methadone ) . then , we checked the effect of fccp and methadone on ca uptake by mitochondria ( figures 3(b ) and 3(c ) ) . for this purpose a ca index was calculated , which denotes the ratio of oxygen - uptake rate triggered by addition of 200 m ca to previous oxygen uptake rate . the values of the ca index were as follows : for succinate 3.0 0.7 ( control ) , 1.7 0.3 ( + fccp ) , and 1.7 0.4 ( + methadone ) , and for glutamate / malate 4.8 0.3 ( control ) , 1.8 0.4 ( + fccp ) , and 1.9 0.5 ( + methadone ) . thus , the effects of fccp and methadone on ca uptake are comparable , although in the presence of glutamate / malate the effect appears to be much more pronounced . this probably results from a stronger uncoupling effect of methadone on glutamate / malate access to the respiratory chain and a consecutive additional impairment of ca uptake . therefore , the effect of methadone on ca uptake by mitochondria may be dependent on the respiratory substrates . on the other hand , when the values of the u/4 ratios calculated in the absence and presence of ca uptake were compared ( figures 3(a ) and 3(c ) ) , a distinctive decrease was observed in traces recorded in the presence of ca uptake ( figure 3(c ) ) . the calculated values of u/4 ratio decreased as follows : for succinate from 2.8 0.5 to 2.0 0.2 ( + fccp ) and 2.3 0.4 to 2.1 0.3 ( + methadone ) and for glutamate / malate 2.1 0.5 to 1.9 0.3 ( + fccp ) and 2.5 0.4 to 2.1 0.2 ( + methadone ) . this could be caused by the reduction of a component of the protomotive force as a result of ca uptake , leading to a decrease of uncoupling capacity by fccp and methadone . the data presented indicate that methadone induces dcd in sh - sy5y cells by altering the capacity of mitochondria to handle calcium , and correlates with distinct changes of mitochondrial morphology . these morphological changes , in turn , can be associated with mitochondrial damage and cell death . interestingly , swollen mitochondria have been observed in the context of neurodegenerative diseases [ 36 , 37 ] . an imbalance in mitochondrial ca homeostasis might be important for both early and late stages of the observed side effects and , perhaps account for some of the observed clinical symptoms , for example , memory impairment .

previously , we have shown that sh - sy5y cells exposed to high concentrations of methadone died due to a necrotic - like cell death mechanism related to delayed calcium deregulation ( dcd ) . in this study , we show that , in terms of their ca2 + responses to 0.5 mm methadone , sh - sy5y cells can be pooled into four different groups . in a broad pharmacological survey , the relevance of different ca2 + -related mechanisms on methadone - induced dcd was investigated including extracellular calcium , l - type ca2 + channels , -opioid receptor , mitochondrial inner membrane potential , mitochondrial atp synthesis , mitochondrial ca2+/2na+-exchanger , reactive oxygen species , and mitochondrial permeability transition . only those compounds targeting mitochondria such as oligomycin , fccp , cgp 37157 , and cyclosporine a were able to amend methadone - induced ca2 + dyshomeostasis suggesting that methadone induces dcd by modulating the ability of mitochondria to handle ca2 + . consistently , mitochondria became dramatically shorter and rounder in the presence of methadone . furthermore , analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial ca2 + uptake in a respiratory substrate - dependent way . we conclude that methadone causes failure of intracellular ca2 + homeostasis , and this effect is associated with morphological and functional changes of mitochondria . likely , this mechanism contributes to degenerative side effects associated with methadone treatment .

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risk - reducing salpingo - oophorectomy ( rrso)the removal of the fallopian tubes and ovaries to reduce the risks of breast and ovarian cancer is a cancer prevention strategy used by many women who carry germline mutations in the brca1 and/or brca2 genes ( brca1/2 ) . however , the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not is unclear . a fixed - effects meta - analysis of pooled results from 10 published reports of rrso and the risks of breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers . rrso was found to be strongly associated with substantial reductions in the risks of breast , ovarian , and fallopian tube cancers among women who carry mutations in brca1 or brca2 . the summary risk reduction estimates should provide guidance to women in planning cancer risk reduction strategies . data were not available to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . risk - reducing salpingo - oophorectomy ( rrso)the removal of the fallopian tubes and ovaries to reduce the risks of breast and ovarian cancer is a cancer prevention strategy used by many women who carry germline mutations in the brca1 and/or brca2 genes ( brca1/2 ) . however , the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not is unclear . a fixed - effects meta - analysis of pooled results from 10 published reports of rrso and the risks of breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers . rrso was found to be strongly associated with substantial reductions in the risks of breast , ovarian , and fallopian tube cancers among women who carry mutations in brca1 or brca2 . the summary risk reduction estimates should provide guidance to women in planning cancer risk reduction strategies . data were not available to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . to identify all reports of rrso in brca1/2 mutation carriers , we searched the pubmed database using the search terms oophorectomy and brca1 or brca2 . this search yielded 346 studies that were published between january 1999 and december 2007 : 309 that included the term brca1 in the title and 267 that included the term we then evaluated the full text of these citations to identify articles presenting primary data that provided estimates of risk reduction due to rrso . no publications were excluded based on quality , sample size , language of publication , or other objective criteria related to study design and analysis . however , some publications that reported rrso in brca1/2 mutation carriers were not included because they did not estimate risk reduction . these included case reports , psychosocial or behavioral studies , commentaries , and clinical recommendations . because the number of brca1/2 mutation carriers is relatively limited and most research groups studying these women are in routine communication and collaborate with one another , we also undertook personal communications with all of the researchers or consortia that have large series of brca1/2 mutation carriers and were known to have data that could have been used to report data on this topic . we undertook a fixed - effects meta - analysis using the hazard ratios ( hrs ) and/or odds ratios ( as published in the original reports ) to estimate the pooled relative risks and 95% confidence intervals ( cis ) . when two or more studies had overlapping study samples , we included only one published report from each group . of the studies identified here , sample overlaps were noted in the studies of rebbeck et al . ( 16 ) , which had the largest sample size of these five studies , was chosen for inclusion in the meta - analysis . there were no apparent overlaps among the other datasets , although we can not rule out the possibility that a few individuals had participated in more than one study . we carried out separate meta - analyses in brca1 mutation carriers , brca2 mutation carriers , and among women who carried either brca1 or brca2 mutations ( denoted brca1/2 ) . a chi - square test of homogeneity among the individual risk ratio estimates of the identified studies was also performed . to evaluate potential for publication bias , we used the adjusted rank correlation test of begg and mazumdar ( 20 ) . all analyses were conducted using stata / se v9.0 ( statcorp , college station , tx ) . to identify all reports of rrso in brca1/2 mutation carriers , we searched the pubmed database using the search terms oophorectomy and brca1 or brca2 . this search yielded 346 studies that were published between january 1999 and december 2007 : 309 that included the term brca1 in the title and 267 that included the term we then evaluated the full text of these citations to identify articles presenting primary data that provided estimates of risk reduction due to rrso . no publications were excluded based on quality , sample size , language of publication , or other objective criteria related to study design and analysis . however , some publications that reported rrso in brca1/2 mutation carriers were not included because they did not estimate risk reduction . these included case reports , psychosocial or behavioral studies , commentaries , and clinical recommendations . because the number of brca1/2 mutation carriers is relatively limited and most research groups studying these women are in routine communication and collaborate with one another , we also undertook personal communications with all of the researchers or consortia that have large series of brca1/2 mutation carriers and were known to have data that could have been used to report data on this topic . we undertook a fixed - effects meta - analysis using the hazard ratios ( hrs ) and/or odds ratios ( as published in the original reports ) to estimate the pooled relative risks and 95% confidence intervals ( cis ) . when two or more studies had overlapping study samples , we included only one published report from each group . of the studies identified here , sample overlaps were noted in the studies of rebbeck et al . ( 16 ) , which had the largest sample size of these five studies , was chosen for inclusion in the meta - analysis . there were no apparent overlaps among the other datasets , although we can not rule out the possibility that a few individuals had participated in more than one study . we carried out separate meta - analyses in brca1 mutation carriers , brca2 mutation carriers , and among women who carried either brca1 or brca2 mutations ( denoted brca1/2 ) . a chi - square test of homogeneity among the individual risk ratio estimates of the identified studies was also performed . to evaluate potential for publication bias , we used the adjusted rank correlation test of begg and mazumdar ( 20 ) . all analyses were conducted using stata / se v9.0 ( statcorp , college station , tx ) . the studies that formed the basis of this meta - analysis included case control studies as well as prospective and retrospective cohort studies ( table 1 ) . as can be seen in this summary , limitations of the currently available data regarding rrso in brca1/2 mutation carriers include variable study designs , small sample sizes for individual studies , many of which are retrospective in nature , and short post - rrso follow - up times in prospective studies . eight studies ( 8 - 10,12 - 16 ) estimated the risk of breast cancer in brca1/2 mutation carriers who were treated with rrso relative to brca1/2 mutation carriers who did not receive this treatment ( table 2 ) . as summarized in table 3 and figure 1 , three nonoverlapping studies ( 1416 ) , which included 5703 participants , estimated the risk of breast cancer in brca1/2 mutation carriers who received rrso relative to brca1/2 mutation carriers who did not receive the procedure , giving a summary hr estimate of 0.49 ( 95% ci = 0.37 to 0.65 ) . four nonoverlapping studies ( 12,1416 ) estimated the risk reduction associated with rrso for breast cancer in brca1 mutation carriers , giving a summary hr estimate of 0.47 ( 95% ci = 0.35 to 0.64 ) . finally , three nonoverlapping studies ( 1416 ) estimated the relative risk for breast cancer in brca2 mutation carriers , giving a summary hr estimate of 0.47 ( 95% ci = 0.26 to 0.84 ) ( table 3 , figure 1 ) . published studies of risk - reducing salpingo - oophorectomy and cancer risk in brca1/2 mutation carriers * in all but one of the prospective cohort studies , women with a prior diagnosis of breast cancer were included for the ovarian endpoint . in domchek et al . ( 13 ) all patients were unaffected with breast or ovarian cancer at the start of follow - up , as the study was designed to evaluate the impact of rrso on cancer incidence as well as disease - specific and overall survival . b1 = brca1 mutation carriers ; b2 = brca2 mutation carriers ; b1/2 = combined brca1 or brca2 group ; pc = prospective cohort ; rc = retrospective cohort ; cc = case control ; namc = north american multicenter cohort ; sis = single - institution study ; imc = international multicenter cohort ; ismc = israeli multicenter cohort ; emc = european multicenter cohort ; myfu = mean years of follow - up ; py = person - years ; nr = not reported ; and na = not applicable ; rrso = risk - reducing salpingo - oophorectomy . in the original publications , the prospective and retrospective cohorts were analyzed by survival / failure time analysis methods ; the case control studies were analyzed by logistic regression . six studies ( 8,10,11,13,16,17 ) ( table 2 ) estimated the risk of gynecologic cancer in brca1/2 mutation carriers treated with rrso relative to brca1/2 mutation carriers who did not receive this treatment . based on data from the three nonoverlapping datasets ( 11,16,17 ) , which included 2840 participants , the summary hr was 0.21 ( 95% ci = 0.12 to 0.39 ) ( table 3 , figure 1 ) . only one study ( 16 ) estimated the risk of gynecologic cancer in brca1 mutation carriers treated with rrso relative to untreated brca1 carriers ( hr = 0.15 , 95% ci = 0.04 to 0.56 ) ( table 2 ) . ( 16 ) did investigate risk reduction in 294 women with brca2 mutations , but observed no post - rrso gynecologic cancers in this sample . published studies of risk - reducing salpingo - oophorectomy and cancer risk in brca1/2 mutation carriers * hazard ratios ( hrs ) , odds ratios ( ors ) ( with 95% confidence intervals ) , and sample size ( n ) are presented . na = not applicable . not included in summary hr estimate because the sample set overlaps with that of other reports . studies included in the summary estimate were chosen to maximize the sample size ( power ) of the meta - analysis . we found no evidence of publication bias of any of our estimates based on the begg and majumder test statistics presented in table 3 . no evidence of study heterogeneity was found based on the x test ( table 3 ) . summary estimates for ovarian / fallopian tube cancer and breast cancer risk reduction associated with salpingo - oophorectomy in brca1/2 mutation carriers * na = not applicable ; hr = hazard ratio ; ci = confidence interval . according to begg and mazumder ( 20 ) . the clinical management of cancer risk in brca1 and brca2 mutation carriers is complex and should consider patient preferences ; these preferences can be informed by accurate knowledge of the risks and benefits of the interventions considered ( table 4 ) . the results of our meta - analysis suggest an 80% reduction in ovarian / fallopian tube cancer risk and a 50% reduction in breast cancer risk associated with rrso in women who carry mutations in brca1 or brca2 . the consistency of these findings across the included studies confirms the strong association of rrso with reduced risks of breast and ovarian cancer in brca1 or brca2 mutation carriers . in addition , modeling studies have also demonstrated that salpingo - oophorectomy has a large effect on years of life added , particularly when adjusted for quality of life ( 31 ) . furthermore , in a prospective study with short - term follow - up , rrso was associated with a 90% reduction in breast cancer specific mortality , a 95% reduction in gynecologic cancer specific mortality , and a 76% reduction in overall mortality ( 13 ) . therefore , all of the available data demonstrate the utility of salpingo - oophorectomy in this population of patients . synopsis of management strategies available to brca1 and brca2 mutation carriers * rrso = risk - reducing salpingo - oophorectomy ; er = estrogen receptor ; mri = magnetic resonance imaging . despite the consistent evidence favoring rrso in women with mutations in brca1 or brca2 , first , the influence of cohort effects on cancer risk over time remain unclear , despite evidence that differences in risk over time may reflect changing exposures , lifestyle , reproductive history , and use of screening or preventive surgeries ( 32 ) . we lacked the data necessary to evaluate the effects of birth cohort , timing of surgery , or other factors that may influence the risk reduction estimates associated with rrso . therefore , at this time it is difficult to infer whether specific cohorts , exposure groups , or other strata may experience different risk reduction effects than others . to limit the possibility that reporting bias influenced our findings however , we did not include any studies that reported the association of rrso with cancer risk without providing estimates of risk reduction because these data would not contribute to pooled estimates of risk reduction . because some studies included in the this analysis were limited in sample size and statistical power , their effect estimates for rrso were large but not statistically significant , suggesting that a meta - analysis and presentation of summary statistics was appropriate . two studies ( 15,17 ) were included in the summary estimates even though they used case control designs , and therefore they yielded odds ratios rather than hazard ratios . although odds ratios may slightly overestimate the risk reduction associated with rrso , the annual incidence of ovarian and breast cancer in brca1/2 mutation carriers is no more than 2%4% , with the result that odds ratios are likely to be similar to hazard ratios in this setting . some of the variability in the individual study estimates reported may reflect study design differences , including the use of retrospective vs prospective samples and poorly characterized selection biases . despite these differences , we noted no statistically significant heterogeneity in the estimates of risk reduction after rrso . in addition , cohort studies estimated a greater reduction in cancer risk associated with rrso ( particularly ovarian / fallopian tube cancers ) compared with the case control studies ( table 1 ) . as a result , there is some variability in the estimates obtained using case control and cohort studies ; nonetheless , the estimates all consistently reflect risk reduction associated with rrso . we have included all of the large collaborative group studies that addressed the question of reduced risk conferred by rrso and whose study populations come from and are representative of mutation carriers in north america and europe . no studies of rrso in nonwhite populations have been reported , and additional data may be needed to understand the role of rrso in these groups . finally , the samples of women with brca1/2 mutations reported here represent those who have generally been identified through high - risk clinics . however , they do represent the population of women who receive genetic testing and may be candidates for rrso . therefore , the populations summarized here represent the most relevant group in whom rrso may be applied at this time . despite the strength and consistency of the data in the literature as reflected in our meta - analysis , a number of questions remain . there are only a few estimates of the association of rrso with cancer risk in populations composed exclusively of brca1 mutation carriers or brca2 mutation carriers ( 12,1416 ) , and it is critical to understand how risk reduction may differ by gene . using a prospective cohort approach and a large consortium dataset , we recently estimated gene - specific risks and found that hormonal modulation by rrso may be associated with a greater reduction in breast cancer risk in brca2 mutation carriers than in brca1 mutation carriers ( 16 ) . in contrast , the studies that used retrospective cohort ( 14 ) or case control approaches ( 14,15 ) did not observe this difference , and therefore , there was no difference in the pooled estimates of breast cancer risk reduction reported in table 1 . thus , differences in study design may influence the inferences we can make about the differences in risk reduction associated with rrso in brca1 vs brca2 mutation carriers . the potentially larger risk reduction associated with rrso in brca2 vs brca1 mutation carriers is of interest , given the high proportion of estrogen receptor ( er)negative breast tumors in brca1 mutation carriers compared with brca2 mutation carriers ( 33 ) . our observation of a higher risk in brca2 mutation carriers should be followed up in larger studies that specifically evaluate tumor markers . in addition , attention needs to be given to the time interval between rrso and breast cancer diagnosis . for example , it is possible that there is greater breast cancer risk reduction in brca2 mutation carriers , in whom the majority of tumors are er positive , given that rrso may treat some subclinical breast tumors . in contrast , in brca1 mutation carriers , who have predominantly er - negative breast cancer , it is unclear whether a treatment effect may exist , and any primary prevention effect may require more time to emerge . eisen et al . ( 15 ) reported that the breast cancer risk reduction with rrso was greater in brca1/2 mutation carriers who underwent surgery before age 50 than in women who underwent surgery after age 50 . among brca1 mutation carriers older than age 50 , no statistically significant association of rrso at any age with risk reduction was observed in brca2 mutation carriers . although these findings are consistent with effects of removal of hormone exposures in premenopausal women and not in postmenopausal women , the sample sizes in this analysis ( 15 ) were relatively small . the importance of understanding the optimal age at which a woman should consider rrso is underscored by a recent study ( 34 ) conducted in the general population that suggests that rrso in women younger than age 45 is associated with an increased mortality , particularly if hormone replacement therapy ( hrt ) is not used . an initial report of hrt use after rrso suggests that women can undergo rrso and take hrt for a short time if needed after surgery because breast cancer risk is not substantially elevated in hrt users after rrso ( 35 ) . although data on postmenopausal women do not demonstrate a cardiovascular benefit from hrt ( 36 ) , an important limitation of this study ( 36 ) was the older age of the participants . more recent data have suggested that younger women going through natural menopause may indeed derive a cardiovascular benefit from hrt ( 36,37 ) , and it is possible that brca1/2 mutation carriers undergoing abrupt surgical menopause to reduce ovarian cancer risk who receive hrt may in fact derive important cardiovascular , bone health , and quality - of - life benefits . although the risk benefit ratio of rrso is very different in brca1/2 mutation carriers than in the general population , and rrso in brca1/2 mutation carriers has been associated with improved overall survival in the short term , these studies pointing to the potentially complex relationship of rrso and hrt exposure raise important and difficult questions . for example , it is not yet clear whether the long - term effects of long - term hrt in unaffected mutation brca1/2 carriers will ultimately be more beneficial in preventing noncancer mortality in these women or more harmful by increasing their risk of breast cancer ( or potentially increasing cardiovascular events ) compared with the general population . given this possibility , studies that address the type , timing , and length of administration of hrt as well as its long - term effects on the association between rrso and cancer risk and on other health factors in brca1/2 mutation carriers , we provide a summary of clinical recommendations related to the detection and prevention of cancer in brca1/2 mutation carriers ( table 4 ) . finally , although rrso has become the standard of care for cancer risk reduction in women who have inherited brca1/2 mutations , other options for risk reduction also exist . women with brca1/2 mutations who have been treated with risk - reducing mastectomy have a substantially reduced breast cancer risk ( 30 ) . furthermore , a study of breast cancer screening that added yearly magnetic resonance imaging to screening mammography suggested that combination of these modalities may also have benefit in the early detection of breast cancer in this group of women ( 28 ) . in conclusion , the summary risk reduction estimates presented here confirm that brca1/2 mutation carriers who have been treated with rrso have a substantially reduced risk of both breast and ovarian cancer . therefore , additional cancer risk reduction and screening strategies are required to maximally reduce cancer incidence and mortality in this high - risk population . forest plots of relative risk ( rr ) estimates for risk reduction associated with risk - reducing salpingo - oophorectomy ( rrso ) . the box sizes reflect the relative sample sizes of the individual studies ; horizontal lines represent 95% confidence intervals ( cis ) . estimates less than a value of 1.0 suggest a favorable reduction in cancer risk associated with rrso . national institutes of health ( r01-ca083855 and r01-ca102776 to trr ) ; the department of defense breast cancer research program ( damd17 - 03 - 1 - 0375 to ndk ) ; the cancer genetics network ( hhsn21620074400c to smd ) ; the eileen genet fund and the project hope fund for ovarian cancer research and education .

backgroundrisk - reducing salpingo - oophorectomy ( rrso ) is widely used by carriers of brca1 or brca2 ( brca1/2 ) mutations to reduce their risks of breast and ovarian cancer . to guide women and their clinicians in optimizing cancer prevention strategies , we summarized the magnitude of the risk reductions in women with brca1/2 mutations who have undergone rrso compared with those who have not.methodsall reports of rrso and breast and/or ovarian or fallopian tube cancer in brca1/2 mutation carriers published between 1999 and 2007 were obtained from a pubmed search . hazard ratio ( hr ) estimates were identified directly from the original articles . pooled results were computed from nonoverlapping studies by fixed - effects meta-analysis.resultsten studies investigated breast or gynecologic cancer outcomes in brca1/2 mutation carriers who had undergone rrso . breast cancer outcomes were investigated in three nonoverlapping studies of brca1/2 mutation carriers , four of brca1 mutation carriers , and three of brca2 mutation carriers . gynecologic cancer outcomes were investigated in three nonoverlapping studies of brca1/2 mutation carriers and one of brca1 mutation carriers . rrso was associated with a statistically significant reduction in risk of breast cancer in brca1/2 mutation carriers ( hr = 0.49 ; 95% confidence interval [ ci ] = 0.37 to 0.65 ) . similar risk reductions were observed in brca1 mutation carriers ( hr = 0.47 ; 95% ci = 0.35 to 0.64 ) and in brca2 mutation carriers ( hr = 0.47 ; 95% ci = 0.26 to 0.84 ) . rrso was also associated with a statistically significant reduction in the risk of brca1/2-associated ovarian or fallopian tube cancer ( hr = 0.21 ; 95% ci = 0.12 to 0.39 ) . data were insufficient to obtain separate estimates for ovarian or fallopian tube cancer risk reduction with rrso in brca1 or brca2 mutation carriers.conclusionthe summary estimates presented here indicate that rrso is strongly associated with reductions in the risk of breast , ovarian , and fallopian tube cancers and should provide guidance to women in planning cancer risk reduction strategies .

pubmed

oral lichen planus ( olp ) is a chronic inflammatory disease affecting the oral mucosa , with a prevalence of about 1~2% of the population [ 1 , 2 ] . once established , the lesions rarely undergo self - remission and , in some cases , have a malignant potency . histologically , olp is characterized by a dense subepithelial infiltration of lymphocytes , increased numbers of intraepithelial lymphocytes , and the degeneration of basal keratinocytes . the lymphocytic infiltrate consists predominantly of t cells [ 2 , 4 , 5 ] . although the etiology of olp remains unclear , accumulating evidence supports a role of immune dysregulation in the pathogenesis of olp , especially involving a t - cell - mediated immune response and the abnormal production of various inflammatory molecules [ 2 , 4 , 6 ] . previous studies have identified that a variety of proinflammatory molecules are aberrantly synthesized and secreted by both infiltrated t cells and altered keratinocytes in the lesions of olp , which contribute to the onset and persistence of the inflammatory responses [ 712 ] . these findings indicated that olp lesion development is closely associated with the activation , production , and functions of these inflammatory molecules . the interleukin- ( il- ) 23/il-17 axis is a newly discovered proinflammatory signaling pathway , in which il-23 and il-17 are two pivotal cytokines [ 13 , 14 ] . il-23 is a heterodimeric proinflammatory cytokine , which is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . secreted by various cell types , such as activated dentritic cells , macrophages , and epithelium , il-23 functions as an important driving factor in the immune response . notably , il-23 serves an essential role in induction and maintenance of a novel subset of cd4+th cells , namely , th17 [ 16 , 17 ] . in addition , il-23 also induces th17 to produce its distinctive cytokine il-17 , which is composed of two il-17a subunits [ 18 , 19 ] . subsequently , il-17 functions as a proinflammatory cytokine , which can activate different cells such as epithelial , endothelial , fibroblast , chondrocyte , and osteoblast to produce numerous inflammatory molecules including cytokines , chemokines , defensins , and mmps [ 20 , 21 ] . together with these downstream proinflammatory products , the il-23/il-17 axis is extensively involved in the processes of chronic inflammation within various pathological states . the il-23/il-17 axis has recently been described to play a major role in the pathogenesis of different chronic inflammatory and immune diseases , such as rheumatoid arthritis ( ra ) , psoriasis , atopic dermatitis , inflammatory bowel disease ( ibd ) , and periodontitis [ 2229 ] . in our previous study , we have detected the serum and saliva levels of il-17 in olp patients but found no significant difference compared to healthy groups . in others ' investigation , the presence of il-17 has been found in the lesions of olp , suggesting its role in the local environments . however , till now , the role of il-23/il-17 axis in the pathogenesis of olp is still unclear . the aim of the present study was to investigate the expression patterns and regulatory roles of il-23/il-17 axis in olp . all tissue and blood specimens of olp patients and healthy volunteers were obtained from west china school and hospital of stomatology , sichuan university . the olp cases were clinically diagnosed , pathologically confirmed , and subdivided as reticular or erosive forms based on the modified who diagnostic criteria of olp . subjects with other oral or systematic diseases or taking corticosteroids or immunosuppressive drugs within 3 months prior to the specimen collection were excluded from the recruitment . for immunohistochemistry ( ihc ) staining , 27 olp lesion specimens , including 13 with erosive form and 14 with reticular form , were recruited from the archives of the department of pathology , and 10 normal oral mucosa ( nom ) tissues were collected from healthy volunteers receiving orthognathic surgery . for quantitative pcr analyses , 14 reticular olp and 10 nom tissues were obtained during the biopsy or orthognathic surgery , respectively , and subsequently snap - frozen in liquid nitrogen for the following experiment procedure . for studying the effect of il-23 on the cd4+t cells , written informed consent was obtained from each subject , and the whole experiment procedure was conducted in accordance with the declaration of helsinki and approved by the scientific and ethical committee board of sichuan university . paraffin - embedded sections of olp lesion specimens and nom tissues were treated in xylene and hydrated in graded ethanol , followed by blocking the activity of endogenous peroxidase with 3% hydrogen peroxide . after antigen retrieval by heat and press , the sections were incubated with goat anti - human il-17 monoclonal antibody ( 1 : 100 , r&d systems , usa ) or rabbit anti - human il-23p19 monoclonal antibody ( 1 : 100 , abcam , uk ) overnight at 4c . subsequently , sections were incubated with the rabbit anti - goat lgg antibody - hrp polymer or goat anti - rabbit lgg antibody - hrp polymer detection reagent ( zsgb biotechnology , beijing , china ) for 15 minutes at room temperature and then 3,3-diaminobenzidine tetrahydrochloride ( dab ) for 1 - 2 minutes . the expression of il-23p19 subunit was assessed using an arbitrary scoring system as follows : 0 , no staining ; 1 , very weak staining ( 15 cells per section ) ; 2 , weak staining ( 530 cells per section ) ; 3 , moderate staining ( 30100 cells per section ) ; 4 , strong staining ( 100400 cells per section ) ; and 5 , strong staining ( > 400 cells per section ) . the expression activities were assessed as previously described [ 33 , 34 ] . to evaluate the expression of il-17 , five high power fields ( hpf ) at a magnification of 400 were randomly picked up and the numbers of il-17 + cells were counted separately . the average number of il-17 + cells per field was then calculated and compared . five milliliters of heparinized blood was obtained from 10 olp patients , respectively . the peripheral blood mononuclear cells ( pbmcs ) subsequently , cd4+t helper ( th ) cells were purified by anti - human cd4 magnetic particles ( bd biosciences , usa ) on a cell separation magnet ( bd biosciences , usa ) according to the manufacturer 's instructions . the purified cd4+th cells were resuspended at a density of 1 10 cells / ml rpml 1640 medium ( thermo scientific hyclone , beijing , china ) supplemented with 10% fetal bovine serum ( fbs ; gibco , grand island , ny , usa ) . for activation of cd4+th cells , 10 g / ml of purified mouse anti - human cd3 antibody ( bd pharmingen , usa ) and 5 g / ml of purified mouse anti - human cd28 antibody ( bd pharmingen , usa ) were added . to study the effect of il-23 on the cd4+th cells , the cells were cultured with or without recombinant human il-23 ( ril-23 , 20 ng / ml ; r&d systems , minneapolis , mn , usa ) for 36 hours . subsequently , cells and culture supernatant were collected separately for the following intracellular cytokine staining and elisa detection . for intracellular cytokine staining , collected peripheral blood cd4+th cells were resuspended at a density of 1 10 cells / ml and stimulated in rpml 1640 medium containing phorbol myristate acetate ( ( pma ) , 50 ng / ml , sigma ) , ionomycin ( 1 g / ml , sigma ) , and monensin ( 0.67 g / ml , goldistop , bd pharmingen , usa ) for 4 hours . after that , cells were stained using a cytofix / cytoperm fixation / permeabilization solution kit ( bd pharmingen , usa ) , anti - human cd4 percp - cy5.5 antibody ( bd pharmingen , usa ) , and anti - human il-17 ape antibody ( bd pharmingen , usa ) , successively , according to the manufacturer 's instructions . stained cells were analyzed using the facsaria flow cytometer and bd facsdiva software ( bd biosciences , san diego , ca , usa ) . the concentration of il-17 in the culture supernatants of peripheral blood cd4+th cells was measured using a human il-17 elisa kit ( boster , wuhan , china ) . according to the manufacturer 's instructions , the detectable range of il-17 content was from 31.2 to 2000 pg / ml . hok16e6e7 , a human immortalized oral keratinocyte cell line , was plated in 6-well plates ( 2 10/well ) in keratinocyte serum - free medium ( ksfm , gibco brl life technologies , grand island , ny , usa ) containing supplemented with epidermal growth factor and calcium . after 24 hours ' growth , medium with different doses of recombinant human il-17 ( rhil-17 , 020 ng / ml ; r&d systems , minneapolis , mn , usa ) or medium only was placed . after another 24 hours ' culture , the cells were collected and kept in 70c until the rna isolation . total rna was isolated from 14 reticular olp and 10 nom tissue specimens or the cultured cells using trizol reagent ( invitrogen , carlsbad , ca , usa ) . cdna was synthesized from 400 ng of rna using the primescript rt reagent kit ( takara biotechnology , dalian , china ) . primers purchased from takara biotechnology were listed in table 2 . to avoid the amplification of genome dna , all the primers the real - time quantitative pcr on 10 ng of cdna was performed using the sybr green detection assay on an applied biosystems 7300 real - time pcr system . the amplification procedure consisted of 10 s at 95c , followed by 40 cycles of the amplification procedure composed of 95c for 5 s and 62c for 40 s. each sample was run in triplicate . the relative gene expression normalized to the expression of gapdh housekeeping gene and control sample were analyzed by the ct method . kruskal - wallis test and mann - whitney test were used to determine differences between groups , paired t - test was used for the pair comparison , and spearman 's test was used to analyze the correlations . to identify whether il-23/il-17 is involved in the local pathogenesis of olp , we firstly detected the expression and distribution of il-23 p19 , a unique subunit of il-23 , and il-17 in olp lesions and nom tissues . using ihc detection , we observed diffuse and strong expressions of il-23p19 in both erosive and reticular olp lesions . the positive staining of il-23p19 predominantly concentrated on the epithelium of olp lesions and also on the extracellular matrix of the lamina propria ( figures 1(a)1(d ) ) . in contrast , only a few keratinocytes in the epidermis layer of the nom tissues showed weak stain of il-23p19 ( figures 1(e ) and 1(f ) ) . moreover , we found abundant il-17 positive stainings on the cytoplasm of the infiltrated lymphocytes in the lesions of both erosive and reticular olp , but only a few sporadic il-17 + cells in the normal oral mucosa ( figures 1(g)1(l ) ) . the statistical data showed that both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 , as well as the numbers of il-17 + cells , compared to the normal oral mucosa . in addition , erosive olp lesions contained a significantly increased number of il-17 + cells compared to the reticular olp lesions . however , there is no significant difference in il-23p19 staining score between erosive and the reticular olp lesions ( figures 2(a ) and 2(b ) ) . to verify the ihc results , we also detected the mrna expressions of both subunits of il-23 ( il-23p19 and il-12p40 ) and il-17 in 14 reticular olp lesional tissues and 10 nom tissues and found that the mrna expressions of all the three genes in olp lesions were significantly increased compared to nom tissues ( figures 2(c ) and 2(d ) ) . these data demonstrated overexpression of il-23 and il-17 in the olp lesions , indicating that the il-23/il-17 axis may be involved in the local immune network of olp . considering il-23 as an important upstream inducing cytokine of il-17 , we next investigated whether the upregulation of il-23 in the progress of olp lesion is associated with the increased expression of il-17 . analyzing based on the data above , we found no correlation between the il-23p19 staining scores and the numbers of il-17 + cells in the olp lesions ( figure 3(a ) ) . however , in reticular olp subgroup , there was a positive correlation between the il-23p19 staining scores and the numbers of il-17 + cells ( figure 3(c ) ) , whereas no correlation was found in erosive olp group ( figure 3(b ) ) . moreover , we also found that the mrna expressions of both il-23 subunits , il-23p19 ( figure 3(d ) ) and il-12p40 ( figure 3(e ) ) , are positively correlated with mrna expression of il-17 in reticular olp samples . these results showed that overexpressions of il-23 and il-17 are positively correlated in the reticular olp lesion , indicating a potential regulatory role of il-23 to the expression of il-17 in the early stage of olp lesion . next , we explored the potential role of il-23 in the production of il-17 in olp . although il-17 has been recently reported to be produced by various cell types , a cd4+th cell subset , namely , th17 cell , is one of its main sources . on the other hand , in the local lesion of olp , therefore , here we focused on the effect of il-23 to the il-17 production in cd4+th cells from 10 olp patients . we observed that compared to the control group , the stimulation of ril-23 significantly increased the percentage of cd4+il-17 + cells ( identified as th17 ) in cd4+th cells from olp patients ( figures 4(a)4(c ) ) . in addition , the il-17 content in the culture supernatant of cd4+th cells also increased under the stimulation of il-23 than the control group ( figure 4(d ) ) . these data suggest that the overexpression of il-23 in olp lesions probably contributes to the induction of th17 and the production of il-17 . we next explored the potential biological effects of il-17 , the essence effector of il-23/il-17 axis , in olp lesions . it is well known that oral keratinocyte is an important component in the oral mucosa immunity and plays an important role in the pathogenesis of many chronic inflammatory oral diseases , including olp , by producing various inflammatory mediators , such as cytokines , chemokines , and defensins . therefore , we investigated the effect of il-17 on the expressions of inflammatory mediators by hok16e6e7 , a human oral keratinocyte cell line . first we detected effect of il-17 on the mrna expression of human -defensins ( hbd ) in hok cells . we observed that il-17 significantly increased mrna expressions of hbd-2 and -3 in a dose - dependent manner , but not hbd-1 ( figure 5(a ) ) . we next detected the mrna expression of 6 chemokines and found that il-17 could significantly increase the mrna expressions of il-8 , ccl-20 , but not cxcl-9 , -10 , -11 or ccl-5 , in hok cells ( figure 5(b ) ) . furthermore , we found that il-17 significantly increased mrna expressions of tnf- , an important proinflammatory cytokine in the pathogenesis of olp , in hok . however , mrna expression of il-6 exhibited no significant difference with or without the effect of il-17 ( figure 5(c ) ) . the data revealed that the il-17 could selectively regulate the expressions of some , but not all , inflammatory mediators in oral keratinocytes , indicating the selectively regulatory role of il-23/il-17 axis in the immune network in olp lesions . in recent years , the il-23/il-17 axis has been widely described to play a pivotal role in the pathogenesis of different chronic inflammatory disorders . however , study about the expression and regulatory role of this new axis in olp is just at its beginning . in a recent study of our group , we found no difference in the serum levels of il-23 and il-17 between the olp patients and controls . the present study is focused on the expression and regulatory role of il-23/il-17 axis in the local environment of olp lesions . il-23 is the upstream driving cytokine in the il-23/il-17 axis ; its importance in inflammation and autoimmunity has been widely demonstrated . to determine whether il-23 is involved in the development of olp , we first detected its expression in the olp lesions compared with the nom tissues . structurally , il-23 is composed of a unique p19 subunit and a common p40 subunit shared with il-12 . to avoid confusion however , in the quantitative pcr assay , we examined the mrna expressions of both the il-23p19 and il-12p40 , for the expression of each gene could influence the amount of the il-23 . our results revealed an upregulation of il-23p19 in both erosive and reticular olp lesions compared to the nom , indicating its involvement in the disease progress . as a driving factor of immune response , il-23 is predominantly produced by the antigen presenting cells ( apcs ) including dentritic cells and macrophages , but its expression was also found in the keratinocytes from normal and psoriatic skin . in addition , the epidermis in lesional psoriatic skin revealed markedly stronger il-23p19 stain than the epidermis in normal skin . similarly , here we also observed a diffuse and strong expression pattern of il-23p19 in the epithelial layer of olp lesions , where keratinocytes are the major cell type . these findings suggested that keratinocytes may be a major source of il-23 production under the chronic inflammatory condition in the mucocutaneous system . although both the erosive and reticular olp lesions had significantly increased immunostaining scores of il-23p19 than the normal oral mucosa , the expressions of il-23p19 between the erosive and reticular olp lesions are similar . this observation suggests that the overexpression of il-23 may be an early event in the pathogenesis of olp lesion and be maintained at a high level in the later progress . il-17 is another key component in the il-23/il-17 axis and primarily functions as a downstream effector . overexpressions of il-17 have been observed in many autoimmune and inflammatory diseases , and its pivotal roles in the pathogenesis have been profoundly identified [ 20 , 35 ] . although il-17 can be secreted by a variety of innate and adaptive immune cells , t cells are still its major sources , especially the new subset of cd4+th cells , namely , th17 . considering the involvement of a t - cell - mediated immune response in the pathogenesis of olp , it is not surprising that il-17 and th17 cells may be present and play a regulatory role in the local environment of the disease . indeed , we observed a large number of il-17 + cells located in the subepithelial lymphocytic infiltrate in the olp lesions . besides , our data showed increased numbers of il-17 + cells and higher mrna expressions of il-17 in the olp lesions compared to the nom tissues . in addition , compared to the reticular olp lesions , erosive olp lesions contained much more il-17 + cells . these findings are consistent with the recent published study by piccinni and his colleagues , who also found an elevated mrna expression of il-17 , together with other th17 type molecules in the olp lesions compared to the healthy mucosa . moreover , the presence of th17 was also identified in another recent study conducted by xie et al . . we must admit that the overexpression of il-17 in olp lesions is attributed partly to the infiltration of lymphocytes in the local environment . however , the large amount of il-17 cytokine in the local lesions of olp can not be overlooked , for its potent proinflammatory properties may induce profound biological effects and play an important role in the formation and progress of the disease . on the other hand , piccinni et al . also found that the cd4+t cell clones generated from olp lesions produced significant higher levels of il-17 than those generated from the healthy oral mucosa . this phenomenon indicated that the overexpression of il-17 in olp lesions attributes not only to the lymphocytic infiltration , but also to other unknown regulatory mechanisms , which is needed to be further explored . since il-23 is a major upstream inducer of il-17 production , it is reasonable to further explore whether the upregulation of il-23 has any regulatory role in the il-17 production in the local environment of olp . based on the data , we conducted correlation analysis of the expressions of il-23 and il-17 . although no correlation between il-23p19 staining scores and il-17 + cell numbers was found in the total olp group or erosive olp subgroup , there were positive correlations between the expressions of il-23 and il-17 at both protein and mrna levels in reticular olp subgroup , indicating that the upregulation of il-23 is associated with increased levels of il-17 in the early stage of olp lesion . on the other hand , the lacking of correlation between il-23 and il-17 expressions in erosive olp lesions may be due to the persistent high levels of il-23 but various levels of il-17 , indicating the existence of other potentially regulatory mechanisms , other than il-23 , in the il-17 expressions in the erosive stage of olp . according to the recent published data by xie et al . , the il-17 in the olp lesion is mainly expressed in cd4+t cells , which is identified as th17 , in the subepithelial lymphocytic infiltration , as observations by double immunofluorescence staining . in addition , cd4+t cell clones generated from olp lesions exhibited an elevated activity in the il-17 production . thus , we investigated the effect of il-23 on the cd4+t cells from olp patients . our results showed that the stimulation of il-23 could significantly increase the percentage of th17 and the il-17 production in cd4+t cells from olp patients . recent immunological findings have supported the opinion that , although il-23 has no effect on the initiation of th17 differentiation , it is crucially required for the proliferation and stability of precommitted th17 cells , their further migration into the pathological tissues , and the production of il-17 , in which way to promote the pathogenic function of th17 cells [ 37 , 38 ] . based on these findings and our data in the present study , it can be suggested that the overexpression of il-23 may be functional and contributes , at least in part , to the accumulation of th17 cells and the increased level of il-17 in the olp lesion . therefore , the il-23/il-17 axis may represent a new signaling pathway in the crosstalk between the keratinocytes and cd4+t cells , which may be involved in the immunopathogenesis of olp . to investigate the potential biological effects of il-23/il17 in the olp lesion , we further explored the effects of il-17 , the major effector of il-23/il-17 axis , on the production of different inflammatory mediators by the oral keratinocytes . accumulated studies have demonstrated that oral keratinocyte is one of the major sources of the aberrant expressions of various inflammatory mediators in olp lesions , including cytokines , chemokines , and defensins , which interact with each other and compose the complex immune network in the olp environment [ 712 , 3941 ] . however , the mechanism of the functional alterations in the keratinocyte from olp lesions is still unclear . our results showed that the stimulation of il-17 could significantly increase the mrna expressions of hbd-2 , 3 , il-8 , ccl-20 , and tnf- , but not the expression of hbd-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in hok cells . these data revealed that the effects of il-17 on the oral keratinocyte are selective but not extensive , suggesting a unique regulatory role of il-23/il-17 axis in the local environment of olp lesions . in summary , based on the findings in the present study , we propose a novel model of interaction between the t cells and keratinocytes in the pathogenesis of olp , in which the il-23/il-17 axis is involved ( figure 6 ) . firstly , keratinocytes in olp lesion produce a large amount of il-23 via an unknown mechanism . next , keratinocyte - derived il-23 may contribute to the accumulation of th17 cells and the overproduction of il-17 in the local lesion of olp . subsequently , il-17 reversely induces the keratinocytes to selectively produce various inflammatory mediators , which compose the complex immune network in the inflammatory environment of olp lesions . our results warrant further explorations on the intrinsic mechanisms of the overexpression and the regulatory effects of il-23/il-17 axis , as well as its interaction with other signaling pathways , in the olp lesions . notably , il-23/il-17 axis has been recently considered as a relevant therapeutic target in chronic inflammatory and autoimmune diseases , and several biological agents blocking il-23 or il-17 have been currently developed [ 22 , 42 ] . therefore , further understanding of the role of il-23/il-17 axis in the pathogenesis may contribute to the development of novel therapeutic strategies for the prevention and management of olp in the future .

interleukin- ( il- ) 23/il-17 axis is a newly discovered proinflammatory signaling pathway and has been implicated in the pathogenesis of many chronic inflammatory and immune disorders . here we investigated whether the il-23/il-17 axis was present and functional in the lesions of oral lichen planus ( olp ) , a chronic inflammatory disease affecting the oral mucosa . using immunohistochemistry and quantitative pcr , we found that the subunits of il-23 and il-17 were overexpressed in olp lesions than in normal oral mucosa tissues . in addition , the expressions of il-23 and il-17 are positively correlated in reticular olp tissues . results from in vitro studies revealed that exogenous il-23 could increase the percentage of th17 cells and il-17 production in the cd4+t cells from reticular olp patients . furthermore , we also found that exogenous il-17 could significantly enhance the mrna expressions of -defensin-2 , -3 , ccl-20 , il-8 , and tnf- , but not -defensin-1 , cxcl-9 , -10 , -11 , ccl-5 , and il-6 in human oral keratinocytes . taken together , our results revealed an overexpression pattern and selectively regulatory roles of il-23/il-17 axis in the olp lesions , suggesting that it may be a pivotal regulatory pathway in the complex immune network of olp lesions .

pubmed

the isolation of pure populations of cells from heterogeneous cell suspensions is an essential part of clinical as well as basic research . the diagnostic test for hiv infection , for example , relies on the separation of human lymphocytes from whole blood . isolation of pure cell populations is an essential part of research efforts to understand fundamental aspects of the body s response to injury ( feezor et al 2004 ; cobb et al 2005 ) . more recently , with major advances in stem cell biology , the isolation of rare cells has become an active area of research . for example , islet 1 + cells that appear to be resident cardiac progenitor cells have been identified in mouse and human hearts ( laugwitz et al 2005 ) . these cells are present in very small numbers , ~100 islet 1 + cells among 10 heart cells . the progenitors are currently isolated by pre - plating of the heart cell suspension for 1 hour , after which fibroblasts and some islet 1 + cells remain attached to the tissue culture plastic . after 46 days in culture , the progenitors make up only 0.5% of the total cell population found in the plates . development of robust and efficient methods for isolating rare cells such as these would clearly represent a major contribution to stem cell biology . miniaturized cell separation devices offer many advantages over conventional separation techniques ( eg , density gradient centrifugation ) , such as small sample volumes , portability , low cost , improved sterility , and potential for integration with analytical techniques . many of these technologies are derived from the semiconductor industry or from advances in synthetic chemistry . cell separation techniques can be broadly classified into two categories : techniques based on size and density , and techniques based on affinity ( chemical , electrical , or magnetic ) . in this review , rather than providing a comprehensive review of all cell separation techniques , we provide representative examples from each category with emphasis on micro- and nanotechnology . since cells are of the order of microns in size and usually handled in suspensions , nearly all of the methods described here are microfluidic . however , some of these techniques , in particular the magnetic separation techniques , also incorporate nanoscale elements . purity and throughput are important metrics for any separation process , and it is often impossible to attain high levels of both . several novel size - based separation processes are being employed in micro- and nanoscale devices . these devices are compact , simple , and usually do not require additional energy sources or additional equipment . most of the devices force a fluid containing a heterogeneous particle population through a series of channels or obstacles of varied size . unlike macroscale size - based separation approaches such as the use of cell strainers , the microscale geometry of the flow channels in these devices ensures that fluid flow is laminar . the main advantage of the size - based approach is that it does not require the presence of cell - specific markers or proteins to achieve separation . hence this approach can be used to isolate stem cells and other rare cells that do not express known markers . furthermore , since the devices do not contain any biological markers or proteins , they have long shelf - life and are easily transportable . however , one of the major concerns with this approach is maintaining cell viability as the cells pass through the microfluidic device . huang et al ( 2004 ) describe a technique that allows for separation of micron - sized particles with a resolution of 0.1 m . in this approach , individual fluid streams can be made to flow along confined lanes in a deterministic manner simply by allowing them to pass through an array of obstacles . the assumption is that a fluid stream will bifurcate when it encounters an obstacle , and thereby sort the particles it brings with it ( figure 1a ) . as indicated by the arrows in the figure , the fluid streams travel around the obstacles and become confined to one of three lanes . depending on the size of the particles in the stream and the obstacle parameters , the stream can enter one of two modes of displacement . if the particles are smaller than the width of the lane they are flowing in , the particles zigzag back and forth around the obstacles ( zigzag mode ) , whereas if they are larger than the lane width , they collide with the obstacles and keep displacing in one direction ( displacement mode ) . by manipulating the obstacle parameters , such as their spacing and diameter , as well as the amount by which they are staggered in each subsequent row of the patterned array , micron - sized particles can be sorted rapidly ( in a few minutes ) with high resolution and with an uncertainty less than 1% of the particle size , or , of the order of tens of nanometers . such resolution is similar to that of other sorting techniques such as hydrodynamic chromatography and quasi - elastic laser light scattering , and considerably better than that of techniques such as cell straining and density gradient centrifugation . as a proof of concept , the authors used the technique to sort fluorescent polystyrene spheres , as well as circular plasmid dna . some limitations of the technique include the requirement of laminar flow and the possibility of random diffusion between adjacent lanes . nanofabrication may allow for improved resolution and sorting of supramacromolecules such as viral particles and protein complexes . ( a ) a stream of fluid flowing at low re ( ~10 ) is forced perpendicular to a series of obstacles of defined size and spacing ( see arrows ) . if particles are smaller than the lane width , they continually zigzag between the obstacles , returning to their original lane assignment after traversing several rows of obstacles ( zigzag mode ) . however , when the particles are larger than the lane width , they collide with the obstacles and displace only in one direction ( displacement mode not shown ) , allowing for separation to occur . ( b ) a series of channels of successively smaller width is microfabricated thus creating a cell sieve . mohamed et al ( 2004 ) have designed a device that uses a relatively simple size - based separation approach to isolate rare cells , such as metastatic cancer cells , from peripheral blood . the device contains four successively narrow arrays of parallel channels ranging from 20 m down to 2.5 m in thickness ( figure 1b ) . the device essentially achieves separation by acting like a sieve , trapping larger cells such as mononuclear cells and neuroblastoma cells further upstream in the larger channels , and allowing smaller cells such as erythrocytes to pass through . the authors experimented with several designs of varied channel width and height before identifying the critical dimensions required to trap the cells of interest . the channel width and height were tuned to ensure that the larger , rare cells ( in this case , neuroblastoma cells or mononuclear cells ) were always trapped while allowing the smaller , bulk cells ( in this case , erythrocytes ) to always pass through . thus , the approach can be applied to separation systems of mixed cell populations provided that the two cell types being separated are sufficiently different in size . to make the process more cost effective , a combination of soft lithography ( pdms , poly(dimethylsiloxane ) ) and polymers ( polyurethanes ) were used for fabrication . when cultured neuroblastoma cells were mixed with either plain medium or peripheral blood and passed through the device , the larger neuroblastoma cells were consistently retained in the channels 10 m wide and 20 m deep while erythrocytes traversed through the device completely . similarly , mononuclear cells in a sample of whole blood were trapped in channels 2.5 m wide and 5 m deep . while the technique is not without its limitations , including the issue of nonspecific cell adhesion to the channel walls , the technique is more effective in terms of cost and time than conventional cell sorting techniques such as fluorescence - activated cell sorting ( facs ) or magnetic - activated cell sorting ( macs ) . besides using a series of obstacles to achieve size - based cell separation , cells shevkoplyas et al ( 2005 ) have developed a biomimetic microfluidic device to facilitate separation of leukocytes from erythrocytes in whole blood samples . the device does not require a power source ( except for a small pressure gradient created by a water column to facilitate blood flow ) , and achieves efficient autoseparation in a single pass using low sample volumes ( ~l ) . it is biomimetic in that it contains a series of bifurcating , rectangular microchannels , emulating the natural structure of blood vessels in vivo . furthermore , it relies on the natural phenomenon of plasma skimming within a 2d channel to enrich the leukocyte population . when the erythrocytes and leukocytes interact and collide , the erythrocytes , which are smaller and more deformable than leukocytes , preferentially migrate toward the center of the channel , forcing the leukocytes to migrate to the outer wall of the microchannel . by diverting the leukocyte - rich plasma layer through several bifurcations , the authors achieved a 34-fold amplification in the leukocyte - to - erythrocyte ratio compared with the inlet concentration . the device can prove particularly useful in applications where high concentrations of leukocytes or their dna and rna are required for analysis . differential motility in laminar flow was used by cho et al ( 2003 ) to separate motile sperm cells from non - motile sperm cells . central to their design is the assumption that viable sperm cells are motile and will therefore migrate against a laminar fluid stream . in contrast , non - motile sperm simply follow the fluid stream lines along with other debris or round cells in the sample . the motile sperm will move across the fluid stream and exit via a different outlet at the bottom of the device , distributing themselves along the width of this outlet , while the non - motile sperm will exit at the outlet situated at the top of the device . the design does not require a power source since it utilizes a height difference between the inlet and outlet as well as surface tension of the fluid stream to create a gravity - driven pump , facilitating a constant fluid flow rate , regardless of the fluid reservoir volume . the authors demonstrated that motile sperm can be purified to levels of nearly 100% . additionally , the yields of motile sperm at the outlet compared with the inlet were significantly improved and comparable with other conventional separation techniques such as direct swim - up , swim - up from centrifuged pellet , and density gradient separation . cho et al have proposed several useful applications for the technique , such as home - based screening test for infertility , vasectomy , vasectomy reversal , and sperm toxicology tests . further improvements to the geometry and configuration of the chip , such as serial connections , may lead to even higher yields . these systems typically create a hydrodynamically focused stream of cell suspension which is interrogated using a laser beam and an array of photodetectors . fluorescent labels are attached to one or more cell types in a heterogeneous suspension and the cells are sorted individually based on ( a ) how they scatter the incident laser light and ( b ) the wavelength of light that they emit . a recent review by huh et al ( 2005 ) describes a number of microfluidic systems based on this principle . an important advantage of microfluidic facs systems is their low cost compared with conventional systems . in the context of medicine and point - of - care diagnostics furthermore , microscale facs systems have been shown to operate comparably to their macroscale analogs in terms of purity and throughput ( wolff et al 2003 ) . fu et al ( 1999 ) have described a device made of silicone elastomer and glass with embedded platinum electrodes to direct the cells by electro - osmotic flow . an external laser and photomultiplier tube ( pmt ) interfaced with a computer are used to excite and detect fluorescence . flow of the cell suspension is controlled by changing the electro - osmotic potentials in response to the pmt signal . the forward algorithm , cells that normally flow from the inlet to the waste reservoir are redirected to a collection channel if the fluorescence is above a preset threshold . with the reverse algorithm , cells flow at a high rate from the inlet to waste until the detection of fluorescence from a target cell induces the flow to reverse direction to allow a second detection step , followed by flow to the collection channel . forward algorithm is analogous to the mode of operation of most conventional facs systems , whereas the reverse is a novel algorithm that can not be implemented in conventional systems . a key advantage of the reverse algorithm is that it can be used to make more than one measurement on individual cells . this device was used to separate a mixed population of fluorescent and non - fluorescent escherichia coli cells . in a subsequent publication , the same group ( fu et al 2002 ) described a more advanced version of this device which consists of integrated microvalves and micropumps . the incorporation of these components on - chip allows for the elimination of some macroscopic components and better control of functions such as sample dispensing and recovery , and flushing . with the same sample of mixed cell populations ( fluorescent and non - fluorescent mixture of e. coli cells ) , this device allowed enrichments of up to 90-fold with a throughput of 10 cells / hour . a further step towards a fully integrated microfluidic facs system has been taken by krger et al ( 2002 ) . in their device , the laser and detectors are incorporated along with the microfluidic components in a single chip . the only macroscopic parts of this system are a laptop computer , charge - coupled device ( ccd ) camera , and syringe pumps . another example of an advanced microfluidic facs system with integrated waveguides and a cell culture chamber has been described by wolff et al ( 2003 ) . with this device , the authors were able to separate fluorescent latex beads from chicken red blood cells at high throughput ( 12 000 cells / second ) . this high rate was made possible by a high - speed hydrodynamic valve with a response time of 2.5 milliseconds , which was interfaced with the pmt . the authors also developed a second - generation device ( figure 2 ) designed for improved hydrodynamic focusing and on - chip cell culture . this device was used to separate normal yeast cells from yeast cells containing green fluorescent protein . the detected cells were directed to the culture chamber by the high - speed valve and allowed to grow and divide for several days with continuous flow of fresh medium . the incorporation of the culture chamber illustrates how microfluidic facs systems can reduce the risk of sample loss due to external handling or dead volumes , a feature that would be particularly relevant for the isolation of rare cells . cells flow through a channel into an observation region . upon detection of a target cell , a high - speed hydrodynamic valve switches fluid flow to send the cell into the holding / culturing chamber . source : wolff a , perch - nielsen ir , larsen ud , et al . 2003 . integrating advanced functionality in a microfabricated high - throughput fluorescent - activated cell sorter . abbreviations : pmt , photomultiplier tube ; facs , fluorescence - activated cell sorting . the imag cell separation system ( bd biosciences , san jose , ca , usa ) is one example of several available separation systems . when exposed to a mixed cell population , the magnetic beads attach to the surface of desired cells via antibody antigen interaction . the desired cell subpopulation can then be separated in the presence of a strong magnetic field either in separation columns or on integrated microchips . magnetic separation is applicable only to cells that can be separated by specific surface antigens . for cells that are commonly distinguished by intercellular proteins ( eg magnetic sorting is of particular interest since it can easily be miniaturized and utilized in applications where small sample volume is required . this is the main advantage of microscale magnetic separation over the well established and efficient macroscale analogs . the major challenge in the design of microscale magnetic separation systems , which is the focus of most studies , is achieving efficient separation in a continuous ( flow through ) system . while several examples of microscale magnetic separation systems have been described in recent years , macroscale systems remain more attractive in terms of purity ( > 95% ) and throughput ( ~ 10 cells / hour ) ( thiel et al 1998 ) . deng et al ( 2002 ) have developed a magnetic micro - filtration system for separation of magnetic beads from non - magnetic beads with up to 95% efficiency . in this set - up , nickel posts 10 m in diameter were positioned in a microfluidic channel . a heterogeneous suspension of beads on the order of 1 m was flowed into the channel in the presence of an external magnetic field that magnetized the nickel posts . as a result , the magnetic beads adhered to the posts while nonmagnetic beads were removed by the fluid flow . upon removal of the external magnet and while keeping flow velocity unchanged , the magnetic beads were removed from the microfluidic channel . a heterogeneous particle population can be separated using a combination of flow and magnetic field in the process called magnetophoresis depending on the particle size and magnetic properties , the particles were deflected into the magnetic field and carried into different outlet channels . although the two systems described were not tested with cells , they appear very promising for cell separation applications . the combination of magnetic microbeads and external magnetic field was utilized in a y - shaped microfluidic device by furdui and harrison ( 2004 ) to separate immunogenic t cells from whole blood with an efficiency of up to 40% ( figure 3a ) . in the first step , paramagnetic beads coated with protein - a / anti - human cd3 were flowed into the micro - device and immobilized using an external magnet . subsequently , whole blood was flowed over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , the t cells were flushed out of the microfluidic device and utilized for pcr analysis . while this purity appears low in comparison with standard methods , such as separation using antibody - coated erythrocytes in conjunction with ficoll - hypaque density gradient centrifugation ( > 90% efficiency ; wilson et al 1976 ) , the microfluidic approach has the advantage of being easy to integrate with downstream analysis systems . magnetic cell separation and sorting . ( a ) y - shaped device used to separate immunogenic t cells from whole blood in three steps . paramagnetic protein a / anti - cd3-coated paramagnetic beads are flowed into the microdevice and immobilized using an external magnet . subsequently , whole blood is introduced over the bed of magnetic beads , resulting in the capture of t cells . upon removal of the external magnet , ( b ) combination of fluid flow ( arrows ) and ferromagnetic strips used to separate leukocytes on planar surfaces . bright dots : time - lapse image of a tagged leukocyte moving along a ferromagnetic strip ( left ) . untagged red blood cells are moving along the direction of the fluid flow ( right ) . the matrix consists of two wire meshes superimposed at 180. source : lee h , purdon am , westervelt rm . magnetic cell separation can also be achieved on planar surfaces , as demonstrated by inglis et al ( 2004 ) . leukocytes were separated from red blood cells by flowing the blood over the surface coated with ferromagnetic strips . the cells were labeled with anti - cd45 conjugated superparamagnetic nanoparticles ( 20100 nm diameter ) such that each positive cell carried around 5000 particles . the strips were positioned at an angle of 9.6 relative to the direction of fluid flow , causing the deflection of labeled cells ( figure 3b ) . magnetic labeling can also potentially enable precise manipulation and positioning of cells at the desired location . to prove this principle , lee et al ( 2004 ) constructed a microelectromagnetic matrix by positioning two wire meshes on top of one another in a cross - pattern ( figure 3c ) . the electric current in each wire was independently controlled via a computer , enabling precise control of the magnetic field at each point in the grid . with this set - up , the authors were able to separate nonviable yeast cells from viable cells by moving the latter individually to a distant location within the grid in a controlled fashion . the solution containing the live / dead - stained , magnetic bead - bound yeast cells was introduced into the microfluidic channel , integrated with the wire mesh . one viable yeast cell and two nonviable cells were initially trapped by a single magnetic field peak . subsequently , the single peak was split into two smaller peaks ; one of the peaks held the nonviable cells and the other one held the viable cell . the magnetic peak that held the viable cell was then moved by adjusting the current in the wires , so that the viable cell was moved away from the nonviable cells . adhesion - based cell separation systems are akin to chromatography columns where a mixture is passed through a column packed with beads or other materials capable of binding to the undesired constituents of the feed . in cell separation , antibodies immobilized on surfaces are used for binding . an important advantage of this technique is that it can be used to separate cell populations that have the same size and/or density , such as subpopulations of human lymphocytes . another advantage of this approach is that there is no need for preprocessing incubation of the starting cell mixture with fluorescent or magnetic antibody tags . conventional macroscale adhesion based systems are described by the term cell affinity chromatography ( cac ) . these systems can generally provide high purity ( > 95% ) and high throughput ( 1010 cells / hour ) ( mandrusov et al 1995 ; putnam et al 2003 ) . a major limitation of these systems is their packed bed design , which maximizes the surface area to volume ratio but also results in long residence times ( in the order of 12 hours ) ( mandrusov et al 1995 ; putnam et al 2003 ; ujam et al 2003 ) . microfluidic cac systems provide high surface areas per unit volume but their small overall volume keeps residence times short ( order of minutes or less ) ( murthy et al 2004 ; sin et al 2005 ) . microfluidic cac systems designed to separate t and b lymphocytes from mixed suspensions have been described by murthy et al ( 2004 ) and sin et al ( 2005 ) . these investigations employed microfluidic chambers made of glass and pdms , whose interior surfaces were functionalized with antibodies capable of binding to surface antigens on the surface of the target cell type . both investigations used t and b lymphocyte cells lines , molt-3 and raji , to create the mixed suspensions , and anti - cd5 and anti - cd19 antibodies to capture the respective cell types . in both reports , the cells captured on the surface were extremely pure ( > 97% purity ) . nonspecific binding of the undesired cell type was overcome by attaching poly(ethylene glycol ) ( peg ) on the surface along with the antibody molecules ( murthy et al 2004 ) . in these approaches , while the number of cells that can be captured was limited by the relatively small surface area of each device , the authors proposed that throughput can be increased by designing a device with larger surface area or by running several devices in parallel . in the above two reports , the antibodies used for separation were specific to the desired cell type . anti - cd5 binds to the t cell line but not the b cell line ; and anti - cd19 binds to the b cell line but not to the t cell line . but what about situations where there is no unique cell surface receptor that distinguishes the target cell type from other cells in the mixed suspension ? chang et al ( 2005 ) have described an adhesion - based microfluidic separation technique that offers a way to address this challenge . this technique emulates the physiological process by which leukocytes from the blood stream are captured by blood vessel walls during an inflammatory response . blood vessel walls achieve this capture by presenting a variety of adhesion proteins ( such as selectins ) to the rapidly passing blood cells and selectively capturing only the cells that bear complementary ligands . the authors designed microfluidic devices out of silicon and glass where the capture surfaces were square and rectangular posts arranged in square and offset arrays , respectively . in this respect , their device is similar in design to that of huang et al ( 2004 ) and offers an interesting comparison even though the mode of separation is different ( adhesion - based in chang et al and size - based in huang et al ) . chang et al coated the post surfaces with e - selectin igg molecules and examined cell capture using hl-60 and u-937 myeloid cell lines , both of which express ligands for e - selectin . with the help of flow experiments as well as finite volume simulations , the authors observed that the movement of cells through their device occurs in three phases : ( 1 ) rolling , ( 2 ) transient capture on the trailing surface of the posts , followed by ( 3 ) detachment and subsequent flow . the authors postulate that each of these three phases is influenced by the strength of bonds between the cells and the selectin molecules on the posts . in the square array device , they then detached and always moved to the very next post downstream . in the offset array devices , however , the cells traveled arbitrary distances following the capture and release events , not always moving to the very next array . although this report does not include experiments with mixed cell suspensions containing both cell types , the authors did measure a significant difference in transit time between the two cell types in their square array device : 1.4 0.8 mm / second for hl-60 and 2.7 1.4 mm / second for u-937 . this difference could conceivably be utilized to separate cell populations that are indistinguishable in size and surface receptor expression . adhesion - based separation can also be performed in a static microfabricated system , as opposed to a fluidic system . revzin et al ( 2005 ) have described the fabrication of microwells made of peg for the isolation of lymphocyte subpopulations . these wells are functionalized with antibodies and are capable of capturing target cells bearing complementary antigens with high selectivity . cd5 + t lymphocytes ( molt-3 cell line ) adhered preferentially to microwells coated with anti - cd5 ( 95% of the available wells were occupied ) and showed only minimal adhesion to microwells coated with avidin and anti - cd19 ( well occupancies were 5% and 13% , respectively ) . with a microwell size of 15 m 15 m , the authors observed that each well contained a single lymphocyte ( figure 4a ) . the significance of this capability was demonstrated by the selective removal of individual t lymphocytes from an array of microwells by laser capture microdissection ( lcm , figure 4b ) . lcm is technique whereby individual cells from different array locations are made to adhere to an adhesive film placed on top of the substrate surface by a pulsed and focused laser beam . the adhesive film is mounted on a cap that is subsequently removed from the substrate surface . the microwell platform offers a simple and effective method of isolating cell subpopulations based on antigen expression . when combined with lcm extraction , this technology could potentially be employed to extract useful proteomic or genomic data from the selected cells . ( a ) poly(ethylene glycol ) ( peg ) microwells containing individual t and b lymphocytes . this type of capture allows subsequent extraction of individual cells by laser capture microdissection ( shown schematically in ( b ) ) . source : revzin a , sekine k , sin a , et al . 2005 . most electrophoretic separation processes employ a process known as dielectrophoresis ( dep ) , whereby particles with an induced electric polarization , such as dipoles , quadrupoles , and octopoles , can be trapped within a nonuniform electric field . negative dep forces cause polarized objects to be repelled away from an electrode , while positive dep forces attract objects toward an electrode . since dep depends on the existence of a gradient in an electric field , an alternating current ( ac ) voltage is usually employed . the response of a cell to dep - mediated forces is influenced by cell type , its density , and even its metabolic and physiologic state , making these important parameters for separation . there are essentially three variations of dep currently being employed for micro- and nanoscale devices : dep retention , dep migration , and dep - field flow fractionation ( dep - fff ) . dep retention ( or dep affinity ) is used to hold cells against a fluid flow , so that cells only weakly influenced by dep forces are carried along with the fluid , while cells held more strongly by dep forces can be eluted later . dep migration is similar to dep retention , but instead it utilizes dep to cause cells to migrate to different regions of an electrode . finally , dep - fff uses dep forces to differentially levitate cells against gravity in a fluid flow profile , such as a parabolic flow profile . this causes cells with different dielectric properties to travel at different velocities along the channel . like magnetic separation , the primary advantage of microscale electrophoretic separation systems is the ability to handle small sample volumes . voldman et al ( 2002 ) have developed a microfabricated separation system capable of trapping and sorting single cells on the basis of their response to certain stimuli . the authors describe it as a microfabrication - based dynamic array cytometer ( dac ) , since it allows cells to be physically arrayed into dep - based traps , and dynamically probed and sorted thereafter . an array of dep traps , each comprising four trapezoidally arranged electrodes of opposite polarity , was fabricated to create a non - uniform , quadrupole electric field ( figure 5a ) . cells became electrically polarized in the presence of the quadrupole field and were physically trapped within a potential energy well against a fluid flow ( figure 5b ) . a high frequency ( ~mhz ) ac field was chosen to minimize power dissipation and heat - induced damage to the cells , to prevent transmembrane polarization of cells , and also to minimize corrosion of the electrodes . the cells ( hl-60 cell line ) were fed into the chip via a reservoir , and then exposed to a stimulus ( calcein ) . they were then individually loaded into the traps and optically interrogated over time for their response to the stimulus ( calcein loading ) by optical microscopy . each of the traps was electrically addressable , allowing for manipulation and release of a single cell or multiple cells simultaneously . thus , based on their responses , the cells could be sorted by turning on or off each individual trap . the authors demonstrated this capability in calcein - labeled hl-60 cells as a proof - of - concept of the technique . a limitation that the authors point out in their dac system is that in some cases , more than one cell became confined in a trap . ( a ) a pseudo - colored sem image showing a dioelectrophoresis ( dep ) trap consisting of four trapezoidaly arranged gold electrodes . this configuration induces a dipole moment in the cell in the opposite direction to the electric field . the cell is repelled from the field and stably trapped at the quadrupole s field minimum . the application of higher flow rate results in the ejection of one cell ( dark grey arrow ) from the trap leaving the other cell behind ( grey arrow ) . huang et al ( 1999 ) used dep - fff to separate cultured human breast cancer mda-435 cells from cd34 + hematopoietic stem cells . the device consisted of three layers , including a patterned layer of eight interdigitated electrodes 50 m in width and spacing , a top glass layer , and a teflon spacer in between comprising the separation chamber . a syringe pump was used to create a constant flow through the channel , forming a parabolic velocity profile . since the heights attained in the fluid channel are both a function of the cell dielectric properties and the frequency of the applied voltage , the two cell populations were individually subjected to dep - fff over a range of frequencies to obtain information about their elution characteristics as a function of frequency . these initial experiments showed that mda-435 cells took much longer to elute and even became trapped near the electrodes above 20 khz , while the cd34 + cells eluted more quickly and completely over a broader range of frequencies . the first was a trap - and - release protocol , which utilized a fixed , 40 khz frequency to trap the mda-435 cells near the electrode , while the second was a frequency sweep between 15 and 35 khz , which allowed the mda-435 cells to be levitated slightly , allowing them to flow at a lower speed down the channel . mixtures of the two cell types were then introduced into the chamber via an injection valve and allowed to equilibrate to different heights in the fluid channel for 5 minutes under an ac voltage of 10 v peak - to - peak . the cells were then subjected to either of the frequency protocols described above for 7 minutes to allow the cd34 + cells to completely elute . the frequency was then switched to 5 khz to allow the mda-435 cells to completely elute . the results showed that in both protocols , cd34 + cells were eluted with purity greater than 99% between 3 and 5 minutes , while mda-435 cells were eluted with 96% purity between 9 and 12 minutes for the trap - and - release protocol and with 99% purity for the frequency - swept protocol . these levels of purity and throughput are comparable to those of macroscale fluorescence - based and magnetic separation systems and better than that of macroscale electrophoretic systems . in another study by members of the same group ( yang et al 1999 ) , the authors found that the elution time depended on both the frequency and voltage of the ac field . larger voltages and frequencies resulted in larger dep forces and , accordingly , longer elution times . there was no significant difference in elution time between the two cell types as a function of flow rate , indicating that the height obtained within the velocity field was mainly a function of the dep forces and not hydrodynamic lift forces created by flow within the channel . in contrast to the previous study , the authors found that mda-435 cells were levitated up to 15 m higher than erythrocytes and were eluted nearly twice as fast . at a frequency of 10 khz and a flow rate of 1 ml / minute , the authors obtained mda-435 cell fractions greater than 98% in purity between 10 and 12 minutes , and erythrocyte fractions greater than 99% purity between 18 and 26 minutes . more recently , huang et al ( 2003 ) also developed a dep - based device to separate several strains of bacteria from erythrocytes , simulating the removal of biological warfare agents from blood . the separation was achieved through dep migration , since the two cell types migrated to different parts of the microelectrode structure . the device was constructed by sequential lamination of five layers : a patterned polyimide layer consisting of interdigitated dep electrodes , two pressure - sensitive acrylic adhesive layers , one of which contained microfluidic channels , a poly - carbonate substrate layer at the bottom , and a glass cover plate at the top . cell suspension samples of 5 l were loaded into the device and an ac voltage of 10 v peak - to - peak at 10 khz was applied for 5 minutes . the cells were then washed , at first with the ac voltage on and then with the voltage off , to collect the eluted samples . the authors observed that the bacteria preferentially migrated and attached to the electrodes in the presence of a dep force . in contrast , blood cells accumulated in the recessed areas between the electrodes , where field strengths were at a minimum . when the three strains of bacteria were combined and mixed with blood , the dep device was able to simultaneously separate the multiple strains from blood cells . pcr amplification of bacterial strain - specific genes did not reveal bands prior to separation , but revealed strong bands after separation , indicating that these pcr products were previously masked due to the presence of blood components . in general , higher collection efficiencies were obtained for lower initial ratios of bacteria to erythrocytes , suggesting that the separation efficiency of the device is limited by the available surface area of its electrodes to about 1 10 bacteria per run . while the above sections encompass most of the available microscale separation techniques , a number of others approaches are under development . the strategy in this approach is to lyse the undesired cell populations in a mixed suspension while leaving behind the desired cells . sethu et al ( 2004 ) have developed a microfluidic device for the separation of leukocytes from erythrocytes by lysis of the latter . this separation is usually performed by macroscale lysis using osmotic lysing agents ( such as deionized water , sodium chloride buffer , ammonium chloride sodium bicarbonate buffer ) , by density centrifugation , or some combination of macroscale lysis and centrifugation . an important characteristic of macroscale lysis that the limiting step is not the actual cell lysis ( which occurs in 2030 seconds ) , but rather the diffusion of the lysing agent in the cell suspension , which can increase the time required for complete lysis to over 5 minutes . in the device designed by sethu et al , blood cells are introduced into a channel that is also fed from either side by channels carrying lysis buffer ( figure 6 ) . the result is that the blood cells flow in a narrow stream ( 1836 m wide ) at the middle of the channel with lysis buffer on either side . this arrangement significantly reduces the time required for diffusion of the lysis buffer into the cell suspension , resulting in complete lysis within 2840 seconds , depending on the ratio of blood to lysis buffer . the reduction in the total time required for this process also has the benefits of avoiding perturbation or damage to leukocytes resulting from prolonged contact with lysing agents or from centrifugation . ( a ) a stream of blood cells is induced to flow along the center of the main channel by two adjacent streams of lysis buffer . this narrowing , shown in ( b ) , minimizes the need for lysis buffer diffusion and allows contact with the flowing blood at nearly the single cell level . sohn et al ( 2000 ) found that the capacitance of cells can be related to their dna content . using a microfluidic device fabricated from pdms and glass with embedded electrodes , the capacitance was measured across a portion of the main channel with the cells flowing past one by one . comparison of capacitance values with measurements of dna content obtained using conventional techniques yielded a linear graph with different cell types lysing at different points . for example , this technique can distinguish between mammalian erythrocytes ( which contain no dna and therefore have zero capacitance ) and leukocytes ( whose dna content , and therefore capacitance , are nonzero ) . gawad et al ( 2001 ) have described a microfluidic device that can distinguish between erythrocytes and other cells types by measurements of spectral impedance done on individual cells passing through a channel . spectral impedance of an individual cell is a function of a number of variables , including cytoplasm conductance , cell size , and membrane capacitance . this device was fabricated with polyimide and glass , and contains integrated channels and electrodes . the broad spectrum of cell separation technologies described in this review illustrates the high level of interest and activity in this area . the described size- and density - based approaches offer a great potential for separation of cell subpopulations for which specific markers are not known or can not be used ( eg , to prevent cell activation ) . affinity - based approaches ( fluorescence- , magnetic- , adhesion - based , and electrophoretic ) can be employed for fast ( ~minutes ) and continuous separation with high specificity ( ~99% ) . for all of the approaches , the design of the devices is such that they can be operated in a massively parallel fashion to increase scale and throughput without compromising purity and efficacy . furthermore , micro - fluidic separation systems can be easily incorporated with devices that perform downstream analysis such as single - cell lysis ( irimia et al 2004 ) and proteomic and genomic analysis ( huang et al 2002 ; hashimoto et al 2005 ; parano et al 2005 ; situma et al 2005 ) . given the advanced level of design , fabrication , and measurement capabilities , we expect that the focus in the coming years will shift from proof - of - concept prototypes to devices that can be economically produced and easily operated in applications such as point - of - care clinical diagnostics , drug discovery , and chemical biological agent detection .

this review describes recent work in cell separation using micro- and nanoscale technologies . these devices offer several advantages over conventional , macroscale separation systems in terms of sample volumes , low cost , portability , and potential for integration with other analytical techniques . more importantly , and in the context of modern medicine , these technologies provide tools for point - of - care diagnostics , drug discovery , and chemical or biological agent detection . this review describes work in five broad categories of cell separation based on ( 1 ) size , ( 2 ) magnetic attraction , ( 3 ) fluorescence , ( 4 ) adhesion to surfaces , and ( 5 ) new emerging technologies . the examples in each category were selected to illustrate separation principles and technical solutions as well as challenges facing this rapidly emerging field .

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a definitive diagnosis of cirrhosis is made by means of histological findings after liver biopsy . however , taking into consideration the high incidence of virus hepatitis and liver cirrhosis , a significant number of patients undergo imaging examinations before the definitive diagnosis of cirrhosis has been established . several studies [ 1 - 11 ] showed that morphological signs that can be identified on routine clinical imaging examinations often accompany cirrhosis . therefore , knowing these signs as well as knowing the differences of their diagnostic performance in cirrhotic liver can be useful in clinical practice . different morphological signs have been described on different imaging techniques ( ultrasound , computer tomography , magnetic resonance imaging ) including the transverse diameter of segment 4 , caudate - to - right lobe ratio ( c / rl ) , the right posterior notch ( rpn ) , the right lobar atrophy , porta hepatis enlargement , and expanded gallbladder fossa ( egf ) . these morphological signs are the result of fibrosis accompanied by decreased blood flow within portal veins . the caudate - to - right lobe ratio is a quantitative measurement that demonstrated to have a high specificity and accuracy in diagnosing cirrhosis [ 5 , 9 ] . the size of the caudate lobe has been also correlated with the hepatic functional reserve in cirrhotic patients . it has been demonstrated that the caudate lobe was larger in patients with compensated cirrhosis than in patients with decompensated cirrhosis . however , the main disadvantage of this quantitative assessment is represented by the fact that is time consuming since several measurements of the caudate and right lobe must be calculated . therefore these measurements are rarely done in daily clinical work and have not gained widespread use . the expanded gallbladder fosa ( egf ) and the right posterior notch ( rpn ) are simple qualitative visual signs of cirrhosis [ 1 , 2 ] . both findings showed a high specificity and a high positive predictor value for the mr diagnosis of cirrhosis [ 1 , 2 ] . despite of their low sensitivity , these signs can be routinely used in clinical practice [ 1 , 2 ] . to our knowledge , a comparison between the c / rl , egf , and rpn in cirrhotic liver has not been studied . the purpose of this study was to evaluate and to compare the diagnosis performance of these signs in diagnosing cirrhosis . our institutional medical database was cross - referenced to identify all consecutive patients who underwent mr imaging for suspected liver lesions or for evaluation of the severity of liver cirrhosis during a 15-month period . one hundred twenty - five cirrhotic patients and 165 non - cirrhotic patients underwent mr imaging . the patients with hepatocellular carcinoma larger than 2 cm and the patients who undergone hepatic resection or therapeutic interventional procedures ( e.g. ablation , chemoembolization ) were not included in this study . finally , 95 cirrhotic patients were included in the cirrhosis group ( table 1 ) . hepatitis c virus infection hepatitis b virus infection hepatitis d virus infection in the control group were included patients who fulfilled the following criteria : no history of chronic liver diseases and no previous hepatic or biliary surgery . the patients with focal liver lesions greater than 2 cm were also not included in this study since the presence of those lesions might modify the liver shape and consequently might influence the interpretation criteria . the control group consisted of 57 subjects ( 26 men , 31 women , age range 18 - 83 ; mean age 51 ) . institutional review board approval with waived informed patient consent was obtained for this retrospective study . all mr examinations were performed using a 1.0-t mr system ( gyroscan intera , philips medical systems , netherlands ) . for optimal signal reception , the imaging protocol included a transverse respiratory - triggered , non - enhanced t2-weighted turbo spin - echo ( tse ) sequence [ repetition time ( tr)/echo time ( te)= 1600ms/100ms , flip angle 90 , matrix size 256 * 512 , field - of - view 37 - 42 * 50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] and a breath - hold , transverse non - enhanced t1-weighted fast - field echo ( ffe ) sequence in- and out - of - phase [ tr / te=15ms/6.9ms for in - phase acquisition and tr / te=15ms/3.45ms for out - of - phase acquisition ; flip angle 25 , 1 nsa , matrix size 256x256 , field of view 37 - 42x50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] were acquired . after acquisition of the non - enhanced sequences , gadobutrol ( gadovist , bayer schering pharma ag , berlin , germany ) was injected intravenously at a dose of 0.1mmol per kilogram of bodyweight with a flow rate of 2ml / sec followed by a 20ml saline flush at the same flow rate using a power injector ( spectris , medrad , indianola , pa , usa ) . dynamic gradient - recalled mr imaging was timed to capture the arterial , portal venous and equilibrium phase and was performed with a fast - field echo ( ffe ) sequence [ tr / t 5.1ms/1.69ms , flip angle 25 , matrix size 208 * 256 , field of view 37 - 43 * 44 - 50 cm , section thickness 4.2 mm ] . two radiologists ( with 6 and 4 years of experience in abdominal mri , respectively ) independently assessed data sets in two different reading sessions ( 1 , the evaluation of the presence or absence of rpn ; and 2 , the presence or absence of egf ) . to reduce any bias , reading sessions were separated by 4-week intervals and data sets were analyzed in random order from both cirrhotic patients and control subjects . the evaluation of both readers was made by using the non - enhanced t1 weighted images and dynamic gradient - recalled mr images . both readers were blinded to all clinical patient data . in the first reading session , the readers were asked to define the presence or absence of rpn and in the second reading session , the readers were asked to define the presence or absence of egf . in both assessments it was used a 2-point grading scale : 0- absence of the sign ; 1-presence of the sign . the rpn was defined as a sharp indentation in the right posterior surface of the liver ( fig 1 ) and the egf is represented by the enlargement of the pericholecystic space ( fig 2 ) . space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . the medial segment of the lateral lobe should not be seen on the same axial image . 56-year - old man with liver cirrhosis child a. axial enhanced mr image ( 15/3.45 , 25 flip angle ) . the c / rl is high ( 1.37 ) and the rpn is easily identified ( arrow ) . note the fact that the sharp notch that defines rpn and represents the landmark of the caudate lobe is situated on the line through the bifurcation of the right portal vein . space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . the c / rl ( the width of the caudate lobe / the width of the right lobe ) was calculated by one radiologist ( 6 years experience in abdominal mri ) . the reader chose images for measurement on the basis of the clarity of the portal veins on enhanced dynamic gradient - recalled mr images . the measurements were made by using the bifurcation of the right portal vein as the landmark of the caudate lobe ( fig 3 ) . axial contrast - enhanced ffe mr image ( 5.1/1.69 , 25 flip angle ) obtained during the portovenous phase at the level of right portal vein bifurcation shows the method of calculating c / rl . line 1 , line 2 , and line 3 are parallel to the midsagittal plane . line 2 is drawn through the right lateral wall of the bifurcation of the right portal vein and line 3 is drawn through the most lateral margin of the right lobe . line 4 is perpendicular to line 1 and is situated at midway between the posterior wall of the main portal vein and the anterior wall of the inferior vena cava . descriptive results regarding the presence or absence of rpn and egf were reported in relative numbers . a kappa value of 0 indicated poor agreement , a value of 0.01 - 0.20 slight agreement , a value of 0.21 - 0.40 fair agreement , a value of 0.41 - 0.60 moderate agreement , a value of 0.61 - 0.80 good agreement , and a value of 0.81 - 1.00 implied an excellent agreement . the qui - square test was used to evaluate the statistical difference of the diagnostic value of the signs between cirrhotic group and control group . the diagnosis accuracy of the c / rl sign was calculated by using the roc curve . the sensitivity and specificity of the c / rl sign for diagnosis of cirrhosis were calculated having a cut - off point of 0.50 . the relative risk ( odds ratio ) of c / rl the statistical significance of any difference between the three signs in diagnosis of cirrhosis was calculated using friedman and wilcoxon test and the statistical significance of any difference of each sign between different child - pugh classes of cirrhosis was calculated using kruskal - wallis and student 's t - test . all statistical analyses were performed by using commercially available software ( spss 13.0 , chicago , il ) . our institutional medical database was cross - referenced to identify all consecutive patients who underwent mr imaging for suspected liver lesions or for evaluation of the severity of liver cirrhosis during a 15-month period . one hundred twenty - five cirrhotic patients and 165 non - cirrhotic patients underwent mr imaging . the patients with hepatocellular carcinoma larger than 2 cm and the patients who undergone hepatic resection or therapeutic interventional procedures ( e.g. ablation , chemoembolization ) were not included in this study . finally , 95 cirrhotic patients were included in the cirrhosis group ( table 1 ) . hepatitis c virus infection hepatitis b virus infection hepatitis d virus infection in the control group were included patients who fulfilled the following criteria : no history of chronic liver diseases and no previous hepatic or biliary surgery . the patients with focal liver lesions greater than 2 cm were also not included in this study since the presence of those lesions might modify the liver shape and consequently might influence the interpretation criteria . the control group consisted of 57 subjects ( 26 men , 31 women , age range 18 - 83 ; mean age 51 ) . institutional review board approval with waived informed patient consent was obtained for this retrospective study . all mr examinations were performed using a 1.0-t mr system ( gyroscan intera , philips medical systems , netherlands ) . for optimal signal reception , the imaging protocol included a transverse respiratory - triggered , non - enhanced t2-weighted turbo spin - echo ( tse ) sequence [ repetition time ( tr)/echo time ( te)= 1600ms/100ms , flip angle 90 , matrix size 256 * 512 , field - of - view 37 - 42 * 50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] and a breath - hold , transverse non - enhanced t1-weighted fast - field echo ( ffe ) sequence in- and out - of - phase [ tr / te=15ms/6.9ms for in - phase acquisition and tr / te=15ms/3.45ms for out - of - phase acquisition ; flip angle 25 , 1 nsa , matrix size 256x256 , field of view 37 - 42x50 - 71 cm , section thickness 7 mm and intersection gap 1.0 mm ] were acquired . after acquisition of the non - enhanced sequences , gadobutrol ( gadovist , bayer schering pharma ag , berlin , germany ) was injected intravenously at a dose of 0.1mmol per kilogram of bodyweight with a flow rate of 2ml / sec followed by a 20ml saline flush at the same flow rate using a power injector ( spectris , medrad , indianola , pa , usa ) . dynamic gradient - recalled mr imaging was timed to capture the arterial , portal venous and equilibrium phase and was performed with a fast - field echo ( ffe ) sequence [ tr / t 5.1ms/1.69ms , flip angle 25 , matrix size 208 * 256 , field of view 37 - 43 * 44 - 50 cm , section thickness 4.2 mm ] . two radiologists ( with 6 and 4 years of experience in abdominal mri , respectively ) independently assessed data sets in two different reading sessions ( 1 , the evaluation of the presence or absence of rpn ; and 2 , the presence or absence of egf ) . to reduce any bias , reading sessions were separated by 4-week intervals and data sets were analyzed in random order from both cirrhotic patients and control subjects . the evaluation of both readers was made by using the non - enhanced t1 weighted images and dynamic gradient - recalled mr images . both readers were blinded to all clinical patient data . in the first reading session , the readers were asked to define the presence or absence of rpn and in the second reading session , the readers were asked to define the presence or absence of egf . in both assessments it was used a 2-point grading scale : 0- absence of the sign ; 1-presence of the sign . the rpn was defined as a sharp indentation in the right posterior surface of the liver ( fig 1 ) and the egf is represented by the enlargement of the pericholecystic space ( fig 2 ) . space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . the medial segment of the lateral lobe should not be seen on the same axial image . 56-year - old man with liver cirrhosis child a. axial enhanced mr image ( 15/3.45 , 25 flip angle ) . the c / rl is high ( 1.37 ) and the rpn is easily identified ( arrow ) . note the fact that the sharp notch that defines rpn and represents the landmark of the caudate lobe is situated on the line through the bifurcation of the right portal vein . space is bounded laterally by the edge of the right hepatic lobe , medially by the edge of the lateral segment of the left lobe . the c / rl ( the width of the caudate lobe / the width of the right lobe ) was calculated by one radiologist ( 6 years experience in abdominal mri ) . the reader chose images for measurement on the basis of the clarity of the portal veins on enhanced dynamic gradient - recalled mr images . the measurements were made by using the bifurcation of the right portal vein as the landmark of the caudate lobe ( fig 3 ) . axial contrast - enhanced ffe mr image ( 5.1/1.69 , 25 flip angle ) obtained during the portovenous phase at the level of right portal vein bifurcation shows the method of calculating c / rl . line 1 , line 2 , and line 3 are parallel to the midsagittal plane . line 2 is drawn through the right lateral wall of the bifurcation of the right portal vein and line 3 is drawn through the most lateral margin of the right lobe . line 4 is perpendicular to line 1 and is situated at midway between the posterior wall of the main portal vein and the anterior wall of the inferior vena cava . descriptive results regarding the presence or absence of rpn and egf were reported in relative numbers . a kappa value of 0 indicated poor agreement , a value of 0.01 - 0.20 slight agreement , a value of 0.21 - 0.40 fair agreement , a value of 0.41 - 0.60 moderate agreement , a value of 0.61 - 0.80 good agreement , and a value of 0.81 - 1.00 implied an excellent agreement . the qui - square test was used to evaluate the statistical difference of the diagnostic value of the signs between cirrhotic group and control group . the diagnosis accuracy of the c / rl sign was calculated by using the roc curve . the sensitivity and specificity of the c / rl sign for diagnosis of cirrhosis were calculated having a cut - off point of 0.50 . the relative risk ( odds ratio ) of c / rl , egf and rpn for the diagnosis of cirrhosis was also calculated . the statistical significance of any difference between the three signs in diagnosis of cirrhosis was calculated using friedman and wilcoxon test and the statistical significance of any difference of each sign between different child - pugh classes of cirrhosis was calculated using kruskal - wallis and student 's t - test . all statistical analyses were performed by using commercially available software ( spss 13.0 , chicago , il ) . the interobserver agreement between the 2 readers was excellent ( 0.81 ; 95% ci : 0.92 , 1.0 ) in the evaluation of the presence or absence of rpn and egf . there was a significant statistical difference of the diagnostic value of c / rl , rpn , and egf between cirrhotic patients and control group ( p<0.001 ) . table 2 summarizes the sensitivities , specificities , accuracies , and the relative risk of c / rl , rpn , and egf for cirrhosis with regard to the diagnosis of cirrhosis . there was no statistical significant difference between c / rl and rpn ( p=0,382 ) for the diagnosis of cirrhosis ( fig 1 ) . there were statistical significant differences between c / rl and egf ( p=0,000 ) and between rpn and egf ( p=0,010 ) for the diagnosis of cirrhosis ( fig 5 ) . there was a statistical significance difference between c / rl and egf and between rpn and egf for child a class of cirrhosis ( table 3 ) . summary of the diagnostic performance of the c / rl , the rpn , and the egf and the relative risk of all three morphological signs in the diagnosing cirrhosis . 42-year - old woman with liver cirrhosis child b. axial unenhanced mr image ( 15/3.45 , 25 flip angle ) shows the right posterior hepatic notch ( large arrow ) . on the same image the pericholecystic space is bounded medially by the edge of the medial segment of the left lobe ( small arrow ) and not by the lateral segment . the statistical differences between c / rl , rpn , and egf in different classes of cirrhosis . cross - sectional imaging has been used for the diagnosis of cirrhosis based on the quantitative evaluation of lobar changes of the liver as well as on qualitative findings such as the right posterior hepatic notch and the expanded gallbladder fossa . in cirrhotic liver there is an atrophy of the right lobe concomitant with a hypertrophy of the caudate lobe due to alterations in portal blood flow and hepatic fibrosis . regardless the compensated or uncompensated type of cirrhosis the caudate lobe is larger in these patients compared with the noncirrhotic patients . harbin et al have shown that on ultrasonography or ct examinations the cirrhotic livers can be differentiated from noncirrhotic livers by using c / rl with a sensitivity of 84% , a specificity of 100% , and an accuracy of 94% . in their study , the c / rl was calculated by using the main portal vein bifurcation as the landmark of the caudate lobe . however , awaya et al have proposed a modified c / rl for the quantitative evaluation of cirrhotic liver . based on the fact that the caudate lobe receives blood supply from the posterior segmental branch in 51% of the cases and the intrahepatic course of the caudate branches is short and less influenced by fibrosis , the authors have considered that the landmark of the caudate lobe should be the bifurcation of the right portal vein . comparing the roc curve of the c / rl described by harbin with awaya s modified ratio for diagnosing cirrhosis there was a significant higher az value for modified c / rl ( 0.797 ; p=0.040 ) than for harbin s ratio ( az=0.737 ) ( 9 ) . in our study we used the c / rl modified by awaya and we obtained an az value of 0.848 for the diagnosis of cirrhosis . the different az values may be the result of the fact that the patients included in awaya s study were reported having only mild or moderate cirrhosis in 65% of cases . when we calculated the sensitivity , the specificity , and the accuracy of c / rl having a cut - off point of 0.50 we obtained a sensitivity of 72% , specificity of 87% , and accuracy of 78% . our results are different from those reported by harbin . since the az value of the c / rl used in our study has shown higher values comparing with harbin s results , we would have been expect that the sensitivity , the specificity , and the accuracy would have been higher . however , in their study the authors have used different landmarks for c / rl and different criteria for including cirrhotic patients and control subjects . harbin did not specify the underlying cause of cirrhosis and patients with diffuse liver diseases were also included in the control group . the evaluation of the c / rl requires performing contrast - enhanced cross sectional imaging in order to identify the portal vein bifurcations . therefore , the method gained limited use in clinical practice for assessment of cirrhotic patients . the right posterior hepatic notch and the expanded gallbladder fossa are two simple qualitative visual findings for cirrhosis [ 1 , 2 ] . ito et al described the rpn as a sharp indentation on the posteroinferior liver surface different from the renal impression . the presence of the rpn in cirrhosis is the result of the atrophy of the right lobe and hypertrophy of the caudate lobe . thus , both signs , c / rl and rpn , are dependent on the same lobar changes of the liver . this may be the reason why the diagnostic sensitivity , specificity , and accuracy together with odds ratio of each sign are similar in our study . when comparing the signs , we did not find any statistical significance difference between them for the diagnosis of cirrhosis . moreover , there was no difference between these signs in the diagnosis of different classes of cirrhosis . the sensitivity , specificity , and accuracy of rpn were reported by ito to be 72% , 98% , and 82% , respectively . our results show lower values ( sensitivity 67% , specificity 87% , and accuracy 75% ) . the rpn is a morphological sign more frequently seen in patients with alcoholic cirrhosis compared with patients with viral cirrhosis since the volume index of the caudate lobe is significant greater in alcoholic cirrhosis than in viral cirrhosis . in our study population there was included only a small percentage of patients with alcoholic cirrhosis ( 5% ) . in comparison , in the study of ito , 13% of the patients included in their study were with alcoholic cirrhosis . this difference in the patient population together with the fact that the rpn is statistically more frequent in the alcoholic type of cirrhosis might explain the differences in the diagnosis performances between the two studies . in contrast to c / rl and rpn , the expanded gallbladder fossa is a sign that reflects different segmental liver changes in a cirrhotic liver . although , the hypertrophy of the caudate lobe and atrophy of the right lobe contribute to the presence of egf , another two segmental changes have an important role in the visualization of the sign : the atrophy of the medial segment of the left hepatic lobe and the cephalocaudal enlargement of the lateral segment of the left hepatic lobe . the atrophy of the segment 4 in cirrhotic liver has been demonstrated by lafortune who showed that regardless the cause or the severity of cirrhosis , the mean diameter of the segment 4 measured on ultrasonography is significant decreased in cirrhosis compared with normal liver . torres et al demonstrated on a volumetric evaluation of the cirrhotic liver a decrease in volume by 10.9% of the medial segment of the left lobe when compared with normals . on the other hand , the enlargement of the lateral segment of the left hepatic lobe is seen only in compensated viral induced cirrhosis whereas in decompensated cirrhosis there is an atrophy of this segment . it has been also demonstrated that the left lobe does not increase in volume with the progression of the cirrhosis in contrast to the right lobe which shows a significant decrease in volume . there is also a marked variability in the volumetric measurements of the lateral segment of the left lobe in normal livers . these variations of the left lobe volume can be responsible for the low sensitivity ( 49% ) and low odds ratio ( or=10.18 ) of egf . the low sensitivity of this sign in our study has been previously reported [ 1 , 2 ] . however , the specificity of egf in this study is high ( 91% ) and comes to confirm the results of other authors . when comparing egf with c / rl and rpn , there was a statistical significance difference between the last two signs and egf in diagnosing cirrhosis ( p<0.010 ) . these results suggest that the egf is a useful sign in evaluation of cirrhosis when is used in conjunction with other morphological findings reported in previous studies [ 2 , 5 , 9 , 10 , 19 ] . our study results demonstrated a significant statistical difference between c / rl and egf ( p=0.002 ) and between rpn and egf ( p=0.037 ) for child a class of cirrhosis . no differences were found between signs for child b and child c classes of cirrhosis ( p>0.05 ) . these data might express that in compensated and less severe clinical cirrhosis the morphological changes are mainly represented by the hypertrophy of caudate lobe and atrophy of the right lobe . we did not evaluate the diagnosis performance of combined c / rl , rpn , and egf . however , we tried to assess the individual role of each sign in the diagnosis of cirrhosis . consideration of two findings or of all three signs of cirrhosis would improve the diagnosis accuracy . another limitation is represented by the fact that we did not correlate the morphological signs with the underlying cause of cirrhosis . further studies are needed to determine if these signs might help to differentiate the cirrhosis induced by different causes . a potential criticism of this study may also be the exclusion of the patients with chronic liver diseases including fibrosis . a relevant evaluation would involve an assessment of the morphological findings in cirrhotic patients in comparison with the findings in patients with chronic liver diseases . finally , the c / rl , the rpn , and the egf can not be only calculated and visualized on mr liver examinations . . however , mr examinations is at this moment extensively used in the evaluation of cirrhosis since this method offer a more accurate and complete evaluation of the cirrhotic liver . in conclusion , the caudate to right lobe ratio and the right posterior hepatic notch have similar performance regarding the diagnosis of cirrhosis . the expanded gall - bladder fossa is a sign with low sensitivity and high specificity and represents an important additional finding of cirrhosis .

background & aims the purpose of the study is to evaluate the accuracy of the c / rl , rpn , and egf in diagnosing cirrhosis . methods the study population included 95 cirrhotic patients in the cirrhosis group ( 56 men , 39 women , age range 14 - 76;mean age 52.3 ) and 57 subjects in the control group ( 26 men , 31 women , age range 18 - 83;mean age 51 ) . all mr examinations were performed by using the same protocol . two radiologists independently assessed data sets in two different reading sessions . the sensitivity , specificity , and accuracy and the relative risk of the signs in diagnosing cirrhosis were calculated . the diagnosis accuracy of the c / rl sign was calculated using the roc curve . the statistical significance of any difference of each sign between different classes of cirrhosis was also calculated . results the interobserver agreement between the readers was excellent ( 0.81;95% ci:0.92 , 1.0 ) . there was a significant statistical difference of the diagnostic value of c / rl , rpn , and egf between cirrhotic patients and control group ( p<0.001 ) . the sensitivity , specificity , and accuracy of c / rl were 72% , 87% , and 78% ; 67% , 87% , and 75% for rpn ; and 49% , 91% , and 65% for egf . c / rl ( or=18.95 ) and rpn ( or=14.74 ) showed a higher risk for cirrhosis compared to egf ( or=14.74 ) . there was a statistical significance difference between c / rl and egf ( p=0.002 ) and between rpn and egf for child a class of cirrhosis ( p-0.037 ) . conclusion the c / rl and rpn have similar performance regarding the diagnosis of cirrhosis having a higher diagnostic performance compared to egf in cirrhosis .

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participants from this study were included in a prospective longitudinal study designed to evaluate optic nerve structure and visual function in glaucoma ( the diagnostic innovations in glaucoma study ) conducted at the hamilton glaucoma center at the department of ophthalmology , university of california , san diego ( ucsd ) . the institutional review board approved the study methodology , which adhered to the tenets of the declaration of helsinki and to the health insurance portability and accountability act . at each visit during follow - up , patients underwent a comprehensive ophthalmologic examination including review of medical history , best - corrected visual acuity , slit - lamp biomicroscopy , intraocular pressure , gonioscopy , dilated ophthalmoscopic examination , stereoscopic optic disc photograph ( kowa nonmyd wx3d ; kowa optimed , inc . , torrance , ca , usa ) , sd - oct testing ( spectralis ; heidelberg engineering , dossenheim , germany ) , and sap using the swedish interactive threshold algorithm ( sita standard 24 - 2 ; carl zeiss meditec , inc . , subjects were excluded if they had any ocular or systemic disease that could affect the optic nerve or the visual field . all patients had a diagnosis of glaucoma at baseline , based on the presence of repeatable visual field defects on sap and/or glaucomatous optic neuropathy on optic disc stereophotographs . repeatable visual field defects on sap were defined as at least three consecutive abnormal sap results with pattern standard deviation ( psd ) with p < 0.05 , and/or glaucoma hemifield test results outside normal limits . glaucomatous optic neuropathy was evaluated by masked assessment of stereophotographs and defined based on the presence of neuroretinal rim thinning , excavation , notching , or characteristic rnfl defects . healthy subjects were recruited from the general population through advertisements or from the staff and employees at the ucsd . healthy subjects had intraocular pressures less than 22 mm hg with no history of increased intraocular pressure and at least two reliable normal visual fields in both eyes , which were defined as a pattern sd within 95% confidence limits and a glaucoma hemifield test result within normal limits . for inclusion in the analysis , each glaucoma patient or healthy subject was required to have at least five visits ( with five sap tests and five oct tests ) over a follow - up duration of at least 2 years . the spectralis sd - oct ( software version 5.4.7.0 ) all images were reviewed by the ucsd imaging data evaluation and analysis center to ensure that the scan was centered , the signal strength was more than 15 db , that there were no artifacts , and that there were no rnfl segmentation algorithm errors . the oct parameter used in the study was the global rnfl thickness corresponding to the average of all rnfl thickness measurements acquired from a 3.45-mm circle centered on the optic disc consisting of 1536 a - scan points . visual fields were performed using sap sita 24 - 2 and evaluated by the ucsd visual field assessment center ( visfact ) . visual fields were excluded if they had more than 33% fixation losses or more than 15% false - positive errors . visual fields were excluded in the presence of the following artifacts : eyelid , rim artifacts , fatigue effects , inappropriate fixation , or evidence that the visual field results were caused by a disease other than glaucoma or inattention . visual fields exhibiting a learning effect ( i.e. , initial tests showing consistent improvement on visual field indices ) were also excluded . the mean sensitivity ( ms ) in decibels of each eye was calculated by converting each threshold point into a linear scale ( apostilbs ) and then averaging all 52 threshold points . global parameters of structural and functional damage were selected for evaluation in this study to provide a fair comparison between the different tests . as comparable localized parameters would be difficult to establish for sap and oct , we restrained our analyses to global structural loss as measured by the oct rnfl average thickness and global functional loss as measured by sap ms . we used ms instead of mean deviation ( md ) , as md is an age - corrected parameter , whereas global rnfl thickness and ms are not . a challenge in evaluating the relative ability of different tests in detecting glaucoma progression resides in equating their specificities , so that sensitivities can be meaningfully compared . when evaluating progression based on slopes of change , the specificity can be set by the type i error . a statistically significant slope at p < 0.05 implies that in only 5% of the cases the slope would be declared statistically significantly different from zero in cases where no change truly existed . however , such approach may be insufficient to establish specificity due to possible changes over time that can occur from aging , instead of glaucoma progression . therefore , to take into account possible age effects , we established confidence limits for the rates of change of oct average rnfl thickness and sap ms using longitudinal data available from healthy eyes . slopes of change were obtained using linear mixed models and cutoffs corresponding to the fifth percentile of rates of change in the healthy group were obtained for each parameter . it is expected that 95% of healthy eyes will have rates of change slower ( i.e. , more positive ) than the cutoff value . after cutoffs were obtained from the healthy group , individual slopes were calculated for each glaucoma eye and for each test using ordinary least squares regression . progression was then determined to have occurred if the slope was statistically significant at p < 0.05 and also faster ( i.e. , more negative ) than the fifth percentile cutoff calculated from the healthy group . this approach equated the specificities of the tests by requiring statistically significant slopes at the same type i error and also by taking into account age - related changes by establishing confidence limits from the healthy group . for each glaucoma eye , slopes of change for each test were calculated using progressively cumulative visits over time , with a minimum of five visits . for example , for an eye that had a total of 10 visits during follow - up , slopes of change were calculated from data including only the first 5 visits , as well as from the data including the first 6 visits , first 7 visits , and so on , up to 10 visits . therefore , for an eye with 10 visits during follow - up , six slopes were calculated for each test . progression ( yes / no ) was then determined based on the criteria set on the previous paragraph for each one of these slopes . after progression ( yes / no ) was determined at each visit , a logistic regression model was used to evaluate the relative odds of progressing by oct and sap in the population . for the logistic regression , eyes that progressed exclusively by oct were assigned the value 1 , whereas eyes that progressed exclusively by sap were assigned the value 0 . as we were interested in evaluating the factors associated with progressing by one method versus the other , eyes that progressed by both methods or by none were excluded . as each eye could have had multiple slopes for each test depending on the number of visits during follow - up , each eye had a combination of 1s and 0s during follow - up , depending on whether progression was detected solely by oct or by sap at each point during follow - up . a generalized estimating equation ( gee ) model was used to deal with correlated observations from each eye . the gee logistic model evaluated the effect of the number of tests during follow - up and disease severity at baseline on the relative odds of progressing by oct versus sap . baseline disease severity was classified according to sap md in early ( > 6 db ) , moderate ( < 6 and > 12 statistical analyses were performed using commercially available software stata , version 13 ( statacorp lp , college station , tx , usa ) . the level ( type i error ) the spectralis sd - oct ( software version 5.4.7.0 ) was used to measure peripapillary rnfl thickness in the present study . all images were reviewed by the ucsd imaging data evaluation and analysis center to ensure that the scan was centered , the signal strength was more than 15 db , that there were no artifacts , and that there were no rnfl segmentation algorithm errors . the oct parameter used in the study was the global rnfl thickness corresponding to the average of all rnfl thickness measurements acquired from a 3.45-mm circle centered on the optic disc consisting of 1536 a - scan points . visual fields were performed using sap sita 24 - 2 and evaluated by the ucsd visual field assessment center ( visfact ) . visual fields were excluded if they had more than 33% fixation losses or more than 15% false - positive errors . visual fields were excluded in the presence of the following artifacts : eyelid , rim artifacts , fatigue effects , inappropriate fixation , or evidence that the visual field results were caused by a disease other than glaucoma or inattention . visual fields exhibiting a learning effect ( i.e. , initial tests showing consistent improvement on visual field indices ) were also excluded . the mean sensitivity ( ms ) in decibels of each eye was calculated by converting each threshold point into a linear scale ( apostilbs ) and then averaging all 52 threshold points . global parameters of structural and functional damage were selected for evaluation in this study to provide a fair comparison between the different tests . as comparable localized parameters would be difficult to establish for sap and oct , we restrained our analyses to global structural loss as measured by the oct rnfl average thickness and global functional loss as measured by sap ms . we used ms instead of mean deviation ( md ) , as md is an age - corrected parameter , whereas global rnfl thickness and ms are not . a challenge in evaluating the relative ability of different tests in detecting glaucoma progression resides in equating their specificities , so that sensitivities can be meaningfully compared . when evaluating progression based on slopes of change , the specificity can be set by the type i error . a statistically significant slope at p < 0.05 implies that in only 5% of the cases the slope would be declared statistically significantly different from zero in cases where no change truly existed . however , such approach may be insufficient to establish specificity due to possible changes over time that can occur from aging , instead of glaucoma progression . therefore , to take into account possible age effects , we established confidence limits for the rates of change of oct average rnfl thickness and sap ms using longitudinal data available from healthy eyes . slopes of change were obtained using linear mixed models and cutoffs corresponding to the fifth percentile of rates of change in the healthy group were obtained for each parameter . it is expected that 95% of healthy eyes will have rates of change slower ( i.e. , more positive ) than the cutoff value . after cutoffs were obtained from the healthy group , individual slopes were calculated for each glaucoma eye and for each test using ordinary least squares regression . progression was then determined to have occurred if the slope was statistically significant at p < 0.05 and also faster ( i.e. , more negative ) than the fifth percentile cutoff calculated from the healthy group . this approach equated the specificities of the tests by requiring statistically significant slopes at the same type i error and also by taking into account age - related changes by establishing confidence limits from the healthy group . for each glaucoma eye , slopes of change for each test were calculated using progressively cumulative visits over time , with a minimum of five visits . for example , for an eye that had a total of 10 visits during follow - up , slopes of change were calculated from data including only the first 5 visits , as well as from the data including the first 6 visits , first 7 visits , and so on , up to 10 visits . therefore , for an eye with 10 visits during follow - up , six slopes were calculated for each test . progression ( yes / no ) was then determined based on the criteria set on the previous paragraph for each one of these slopes . after progression ( yes / no ) was determined at each visit , a logistic regression model was used to evaluate the relative odds of progressing by oct and sap in the population . for the logistic regression , eyes that progressed exclusively by oct were assigned the value 1 , whereas eyes that progressed exclusively by sap were assigned the value 0 . as we were interested in evaluating the factors associated with progressing by one method versus the other , could have had multiple slopes for each test depending on the number of visits during follow - up , each eye had a combination of 1s and 0s during follow - up , depending on whether progression was detected solely by oct or by sap at each point during follow - up . a generalized estimating equation ( gee ) model was used to deal with correlated observations from each eye . the gee logistic model evaluated the effect of the number of tests during follow - up and disease severity at baseline on the relative odds of progressing by oct versus sap . baseline disease severity was classified according to sap md in early ( > 6 db ) , moderate ( < 6 and > 12 db ) , and advanced ( < 12 db ) . statistical analyses were performed using commercially available software stata , version 13 ( statacorp lp , college station , tx , usa ) . the level ( type i error ) the study included 462 eyes of 305 glaucoma patients and 62 eyes of 49 healthy controls . table 1 shows the baseline clinical and demographic characteristics of the subjects included in the study . glaucoma patients had a median of 8 visits ( range , 518 ) during an average follow - up of 3.6 0.9 years . for healthy subjects , the median number of visits was 7 ( range , 510 ) , over an average follow - up period of 3.8 0.9 years . mean age at baseline was 70.0 8.2 years in the glaucoma group and 69.1 8.5 years in the healthy group ( p = 0.470 ) . average sap md was 4.8 5.9 and 0.0 1.2 db , and global rnfl thickness at baseline was 78.4 17.6 and 94.9 11.0 m , in glaucoma and healthy subjects , respectively . baseline clinical and demographic characteristics of healthy subjects and glaucoma patients included in the study as the number of visits for glaucoma eyes ranged from 5 to 18 visits , the number of slopes ranged from 1 to 14 for each eye . there were in total 1910 slopes for all the glaucomatous eyes included in the study . the fifth percentile cutoff values for rates of change estimated from the healthy group were 0.35 db / y for sap ms and 1.48 m / y for global rnfl thickness . progression was determined for each eye and each test if the slope was faster than these cutoff values and also if the slope was statistically significant at p < 0.05 . from the 1910 slopes , progression was detected solely by oct in 363 slopes ( 19.0% ) , solely by sap in 183 slopes ( 9.5% ) , and by both oct and sap in 80 slopes ( 4.1% ) . progression was not detected in 1284 slopes ( 67.2% ) . as we were interested in factors explaining the relative odds of progressing by one test versus the other , subsequent analyses were conducted with the 546 slopes that showed progression exclusively by oct or exclusively by sap . table 2 shows results of progression by oct versus sap according to the number of visits and baseline disease severity . the raw data suggested that oct was able to detect more eyes as progressing in comparison to sap , except for eyes with moderate and severe disease and when a large number of tests were available ( fig . total number of visits with progression detected by sap and sd - oct , according to baseline disease severity and the number of visits during follow - up estimated probabilities of detecting progression by sd - oct versus sap , according to different levels of disease severity at baseline and number of tests acquired during follow - up . this was confirmed by the results of the gee logistic model , as shown in table 3 . each 1 db higher md was associated with a 5% increase in the odds of detecting progression by oct versus sap ( odds ratio [ or ] = 1.05 per 1 db ; 95% confidence interval [ ci ] : 1.011.09 ; p = 0.005 ) . that is , eyes with less severe disease at baseline had higher chance of being detected as progressing by oct but not by sap , whereas an increase in disease severity at baseline increased the chance that the eye would be detected as progressing by sap but not oct . a higher number of visits during follow - up was associated with a decrease in the relative odds of progressing by oct versus sap ( or = 0.86 per visit ; 95% ci : 0.810.93 ; p although an increase in the number of visits would be expected to increase the chance of detecting progression by both oct and sap , it is important to keep in mind that the model evaluated the odds of progressing by one test versus the other . the results show that for eyes with few visits during follow - up , oct had a higher chance of detecting progression compared with sap . importantly , there was also a significant interaction between baseline disease severity and number of visits in explaining the relative odds of progressing by oct versus sap . for eyes with early disease and relatively small number of visits , the odds of detecting progression by oct were in general much larger for oct compared with sap ( fig . conversely , for eyes with advanced disease and relatively large number of visits available during follow - up , the odds of detecting progression by sap were greater than by oct . figures 2 and 3 show cases of eyes included in the study illustrating differences in the ability to detect progression by oct and sap . logistic gee model evaluating factors explaining the relative odds of progressing by oct versus sap example of an eye of a glaucoma patient with glaucomatous optic neuropathy but normal visual field at baseline ( mild disease ) . significant progression was seen on sd - oct rnfl thickness measurements over time , with a rate of change of 2.28 m / y ( p = 0.001 ) . progression was not seen on sap mean sensitivity measurements over time , with a slope of 0.25 db / y ( p = 0.188 ) . example of an eye of a glaucoma patient with moderate disease at baseline ( baseline md deviation of 10.96 db ) . significant progression was seen on sap with a slope of change in mean sensitivity of 0.51 db / y ( p = 0.009 ) . however , no significant change was seen on sd - oct measurements , with a slope of change of 0.02 m / y ( p = 0.961 ) . the present study demonstrated that the relative odds of identifying progression by structural or functional tests varies according to the number of tests performed during the follow - up and also according to the disease severity at baseline . these findings may have significant implications for the evaluation of glaucoma progression in clinical practice by allowing clinicians to tailor the frequency of sap and oct tests during follow - up , taking into account the relative odds of detecting progression at different levels of disease severity . several studies have previously shown substantial disagreement when evaluating detection of glaucoma progression by structural and functional tests . progression was detected simultaneously by sap and sd - oct in only 80 ( 13% ) of them . in the vast majority of cases , progression was either shown by sd - oct or by sap , but not by both . therefore , we built a model evaluating the relative odds of progression by sd - oct versus sap in an attempt to understand factors that could explain why some eyes are detected as changing by one test but not by the other . the model showed that in eyes with less severe disease at baseline the odds of detecting progression by sd - oct were generally higher than the odds of detecting change by sap . each 1 db higher baseline md was associated with a 5% increase in the odds of progression being detected by sd - oct versus sap . this can be clearly seen on figure 1 . when slopes were calculated from eight visits , the probability of detecting progression by sd - oct when the baseline md was 1 db , for example , was 70% compared to only 30% for sap . equal probabilities ( i.e. , 50% each ) for detecting progression for sap and sd - oct would occur for a baseline md of 10 db . for eyes with baseline md worse than 10 db , the probability of detecting progression by sap would be larger than by sd - oct considering slopes calculated from eight visits . for example , for a baseline md of 20 db , the estimated probability of detecting progression by sap was 70% versus only 30% for sd - oct . these results are in agreement with previous suggestions in the literature that structural assessment by sd - oct would perform better for detection of disease progression in early stages of damage , whereas sap would perform better for more advanced cases . however , to the best of our knowledge , no previous study has provided a quantification of the impact of disease severity on the relative odds of detecting change by these two methods . the relationship between ability to detect change and disease severity may be explained by a number of factors . previous studies investigating the structure and function relationship in glaucoma have shown that when measurements are expressed in their original scales ( i.e. , db for sap and m for rnfl ) , a nonlinear relationship is seen . from this nonlinear relationship , it can be extracted that for eyes at relatively early stages of damage , substantial change in rnfl measurements might be seen when only relatively small changes in visual function are seen in decibels . in contrast , for eyes at advanced stages of disease , even relatively small neural losses will produce large changes in the decibel scale . it is important to note that the nonlinear relationship between structure and function seems to be mostly the result of the logarithmic scaling of visual field data , as transformation of functional data to a linear scale ( or structural data to a decibel scale ) seems to produce a closely linear relationship between functional and structural measurements . however , such post hoc transformations will not influence the ability of a test to detect change as long as the original measurements are still acquired in a decibel scale ( i.e. , the staircase procedures for threshold acquisition still acquired on decibel steps ) . another factor influencing the relative ability of structural and functional tests in detecting progression is the presence of floor effects . for sd - oct average rnfl thickness measurements , a floor effect seems to occur when measurements are close to 50 m , although there are variations according to specific instruments . when the floor is reached , even further neural losses will go relatively undetected by rnfl thickness measurements . the presence of such floor has been shown by several previous investigations and seems to be related to the presence of nonneural or glial tissue , as well as to the dynamic range of the instrument . this would seem to contradict our findings of a relatively superior ability of sap in detecting progression as disease severity gets worse . however , it is important to note that such relationship between disease severity and sap variability is valid for measurements expressed in the decibel scale . even though variability in decibels increases with worsening visual field damage , due to the logarithmic scaling , a 1-db change in later stages of the disease corresponds to a much smaller change in terms of neural loss or structural damage than a 1-db change in early disease . therefore , even relatively small losses of neural tissue in later stages of the disease may produce relatively large changes in visual sensitivity as measured in decibels , overcoming at least in part the limitation of increased variability and making detection of change in decibels relatively easier . as expected , the number of tests ( visits ) acquired during follow - up also significantly influenced the ability to detect change . it is expected that a greater number of visits will make detection of change easier for both structural and functional tests . however , we also found a significant interaction between baseline disease severity and number of tests in explaining the relative odds of detecting change by sd - oct versus sap . this is illustrated by figure 1 and also by the analysis of the raw data presented in table 2 . when only a small number of tests were available during follow - up ( less than eight ) , detection of progression was more common for sd - oct than sap for all stages of the disease , even in eyes with moderate and advanced damage . conversely , when a large number of tests were available ( > 12 tests ) , the odds of detecting change were higher for sap compared with sd - oct for eyes with moderate and advanced disease at baseline . our findings may be important in providing clinicians with information that can be used to tailor the frequency and type of test according to the stage of disease . however , such test customization strategy might be difficult to implement in clinical practice , as there are no clear cutoffs of when to perform one test versus the other . additionally , even though under certain circumstances the relative odds of detecting change may be higher for test a compared with test b , this does not necessarily imply that all cases of progression will be detected by test a. therefore , algorithms that provide a combination of structural and functional data allowing effective monitoring throughout the disease continuum might offer a better solution to this issue . this was done to allow equating the specificities of the tests , an essential requirement for comparing their abilities to detect change . as there is no current gold standard for detecting change in glaucoma , our comparison was based on the relative proportion of glaucoma eyes that were detected as having statistically significant slopes of progression considering rates of change that would have only a 5% chance of occurring in a healthy population . this was done to provide not only a statistically but also a clinically relevant criteria . future studies should , however , investigate the relative ability to detect change by using other methods such as event - based detection of localized change or other techniques . as another limitation of our study although this assumption is likely to correspond to how these tests are currently used in clinical practice for relatively short - term management decisions , it is likely that the true course of the disease is not linear in the long run . recent investigations have also suggested nonlinear models for investigation of rates of visual field change that might provide an improvement over current simple linear models . our results show that the ability to detect glaucoma progression by sap and sd - oct is significantly influenced by the stage of disease . our results may provide useful information for guiding clinicians on the relative utility of these tests for detecting change throughout the disease continuum . furthermore , they may also serve to guide the construction of indices or strategies combining structural and functional information for detection of change over time .

purposethe purpose of this study was to evaluate the effect of disease severity and number of tests acquired during follow - up on the relative odds of identifying progression by structural or functional tests in glaucoma.methodsthis was an observational cohort study involving 462 eyes of 305 patients with glaucoma and 62 eyes of 49 healthy subjects . glaucoma patients and healthy subjects were followed for an average of 3.6 0.9 and 3.8 0.9 years , with a median ( interquantile range ) of 8 ( 69 ) and 7 ( 68 ) visits , respectively . at each visit , subjects underwent visual field assessment with standard automated perimetry ( sap ) and retinal nerve fiber layer ( rnfl ) evaluation by spectral - domain optical coherence tomography ( sd - oct ) . slopes of change in sap mean sensitivity and oct rnfl thickness over time were estimated by linear regression using progressively cumulative visits over time . cutoff values for age - related expected rates of change for each test were obtained from the healthy group . progression by sd - oct and/or sap was determined if the slope of change was statistically significant and also lower ( faster ) than the fifth percentile cutoff calculated from the healthy group . a generalized estimating equation logistic regression model was used to evaluate the relative odds of progressing by oct versus sap in glaucoma eyes.resultseyes with less severe disease at baseline had a higher chance of being detected as progressing by sd - oct but not by sap , whereas an increase in disease severity at baseline increased the chance that the eye would be detected as progressing by sap but not sd - oct . each 1 db higher md was associated with a 5% increase in the odds of detecting progression by sd - oct versus sap ( odds ratio = 1.05 per 1 db ; 95% confidence interval : 1.011.09 ; p = 0.005).conclusionsthe ability to detect glaucoma progression by sap versus sd - oct is significantly influenced by the stage of disease . our results may provide useful information for guiding clinicians on the relative utility of these tests for detecting change throughout the disease continuum .

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as has been the case in most other industries , the promise of new technologies and innovations has been cited as the way to address the healthcare industry s current challenges ; in particular , to circumvent the so - called iron triangle , simultaneously improving access , cost , and quality of care . at the same time , research based institutions across the globe are experiencing significant stress from more competition for less funding . from fy 2003 to 2015 , for example , the nih lost 22% of its capacity to fund research due to budget cuts , sequestration , and inflationary losses . this resulted in fewer grants and discoveries , along with talented scientists and investigators leaving research and/or the us . as such , it is more important than ever to increase the efficiency and effectiveness with which investments in fundamental research and development translate into products , services , and procedures that improve the health and wellbeing of people around the world . examples abound of the power of technology and innovation to enable disruptive step changes in performance while simultaneously slashing costs . moore s law in semiconductors , which projects a doubling of cpu capacity every 18 to 24 months , typifies the exponential power of technology . innovator and futurist ray kurzweil extended moore s law to show that when a specific technology platform approaches some kind of physical limit , a new one emerges to extend the exponential growth , bypassing perceived barriers . eroom s law typifies the experience in the pharma industry : rather than show an improvement , the trend for new drug approvals by the us food and drug administration per inflation - adjusted us dollars spent on r&d is a decrease of 50% over 9 years a negative rather than positive exponential growth . while no generally accepted law exists for healthcare as a whole , labor productivity in healthcare , for example , is in decline , with technology not improving productivity as it has in other industries . successful innovation in healthcare requires navigating a long and challenging journey between an unmet need or discovery generated from basic research to a viable commercial product or service . while there are many potential stumbling points along the way , the so - called valley of death that exists between academic research and commercialization of a new product is often cited as a key obstacle for health innovations . the valley of death has been shown to occur most frequently in the presence of non - economic investments , such as government expenditures on early stage , basic research without attention to the likelihood of commercially motivated investment at later stages if successful . technologies that emerge from academia which do not have a promising commercialization pathway and clearly articulated value proposition are simply not considered by financially - motivated investors and , as a result , languish in the lab . financial investors are not typically impressed with the great technical results that investigators focus on developing and are rewarded for publishing they expect them . they understand the reality is that the vast majority of new ventures fail not because of flawed science ( technical risk ) , but because the market does not perceive a need or will not pay for the product or service ( market risk ) . many academic investigators and research teams are simply not prepared or motivated to think beyond their work in the lab to anticipate or address commercial issues such as market risk . some are even discouraged from considering commercial issues while conducting research due to lingering concerns about aligning too closely with industry . a commonly held misconception by many investigators is that once they are successful publishing results in a high impact journal , the commercial value will be so self - evident that companies will flock to get commercial rights . as a result , many leave commercialization related issues to be addressed only after a technology is shown to work ( i.e. proof - of - concept demonstrated ) , expecting them to be transferred to a company that will bring a product or service into practice . as a result , academic innovators pursuing translational research still often approach it with the mindset of doing science . they do not develop an appreciation for which of the many paths available to them is most likely to lead to patient impact and hence financial return for investors . today s challenging funding situation , with historically low pay - lines , creates even more pressure for investigators , particularly new ones , to focus on novelty and quickly move on to new areas once publishable results have been generated . this often leaves institutional technology transfer offices ( ttos ) with the very challenging task of seeking licensees to technologies that have been shown to work , but with no clear commercial value and without much , if any , technical support . simultaneously , financially driven investors are becoming less willing to take risks , particularly risks that they can not control or may only pay - off in the longer term . they are looking to invest in opportunities for which they are well positioned to manage the risks , such as implementation related issues . they do not want to take risks in areas for which they have no control , such as market acceptance , reimbursement and regulatory acceptance . as a result of these increasing pressures from both sides , the only teams surviving the commercialization journey through today s more treacherous valley of death are ones with the most robust preparation , demonstrably de - risking both the technology and the market to succeed in attracting investors . universities and academic medical centers are facing increasing competition to not only win scarcer research funding , but also to attract the best investigators and students . to compete successfully at both , they need to do more in preparing and supporting those investigators interested in translating research into practice : excellence in science is still required , but is no longer sufficient . investigators that wish to expand beyond basic research need new skills and resources that can help them focus on creating and developing technologies that have a better chance of surviving the commercialization journey long enough to engage investors and get products to the market . one approach being taken at some institutions is to add a business development function to an institution s tto , expanding its role to include while this approach can be very effective in advancing some projects , it does not address the fundamental issue of improving the commercial readiness or attractiveness of technologies emerging from labs . in addition , balancing the roles of guardian and promoter of ip in a single cost center with increasingly constrained budgets can add an obstacle to commercialization which discourages all but the few that are selected for support . another approach taken by institutions , including stanford biodesign and the coulter foundation as well as by funders , including the nih and the nsf , are creating programs to assist academic investigators advance technologies toward commercialization while they are conducting their research . they are providing educational opportunities to help investigators understand , anticipate and address commercialization challenges . efforts range from didactic educational programs to accelerators which provide hands - on skills development and bridge / product development funding as well as support in the form of mentoring and pitch competitions , the goal being to help teams achieve a commercial exit : the point at which financially motivated investors such as angels , vcs and strategic players invest . figure 1 schematically represents a range of these approaches taken by organizations to improve the effectiveness of the translational research they support . figure 1.the gap filled by hcps in the portfolio approaches used by academic programs to improve the impact of translational research they support . the gap filled by hcps in the portfolio the horizontal axis represents the typical focus of the effort . at one end of the spectrum the focus is on building the skills of individuals , such as university based degree programs . the other end of the spectrum is focused on helping advance projects , such as accelerator programs . the vertical axis represents the typical intensity of the efforts . at one end of the spectrum are activities that take a few hours and cost very little to deliver , such as webinars . at the other end are programs that take years and cost in the hundreds of thousands of dollars , such as university based degree programs . an emerging option for academic investigators is the development of healthcare commercialization programs ( hcps ) . these programs attempt to strike a balance by cost - effectively advancing a project into commercialization , while simultaneously building the skills of team members so that they can learn while contributing to an entrepreneurial activity . two recent examples are a modified version of the i - corps program , adapted in collaboration with the nih and another being the coulter foundation c3i program . in 2013 , the national heart , lung and blood institute ( nhlbi ) took a fresh approach to improving the effectiveness of the translation of academic based research into implementation . it established three centers to accelerate innovations from the bench to the clinic : the nih centers for accelerated innovations ( ncais ) . the ncais are to help advance projects into patient care by providing product development funds and project facilitation , while also providing skills development to empower academic investigators with a greater understanding of the commercial path of their technologies . the ncais were provided additional funds by the nsf in the spring and summer of 2015 to explore how the i - corps program could help advance ncai supported projects into practice . the university of california ncai applied its pre - existing i - corps program and approach for teams from across the university of california network , with a focus on teams from the san francisco area . the boston ncai , b - bic ( through its collaborator the consortia to improve medicine with innovation and technology : cimit ) and cleveland s ncai ( the nih center for accelerated innovation at cleveland clinic ; ncai - cc ) took a different approach . they choose to blend several methodologies into a program intended to address the unique challenges facing healthcare innovations . they each built on the fundamentals of the i - corps program while integrating commercialization experiences gained from introducing therapeutic and healthtech products . cimit built on its 18 + year history and experiences facilitating more than 600 projects with its unique approach to facilitating teams as well as its work with the coulter foundation and academic based programs at its member institutions . cleveland clinic incorporated specific workshops focused on therapeutics , including regulatory and intellectual property with support of the nhlbi staff . a brief overview of each and some results are outlined below : cimit s focus is on healthtech innovations , the overlap of medicine and engineering , including devices , diagnostics , e / digital health and big data . its initial pilot program had eight weekly sessions and was comprised of a cohort of 9 teams . each team had a clinical lead , technical lead and entrepreneurial lead , which were supported by an executive from the cimit accelerator team . the overall results were very promising , with four teams successfully attracting commercial funding within 6 months of completing the course , receiving more than $ 11 m in commercial investment . three are now pursuing licensing opportunities instead of trying to create a newco , and two projects are being rethought after learning of the lack of a market need . programmatically , these quick kills are viewed as being a success along with the teams that are able to advance to commercialization as it saves time and resources so that teams can work on more impactful problems . the teams all reported significant progress in a compressed period of time , and similar results an example of a self - reported team assessment is:our experience at the cimit program and all the support from the faculty has been instrumental to our progress . less than a year ago , i was an md - phd student with no business experience and no intentions of commercializing technology . in fact , before the course , we did nt even know who our customer was or what our product was , let alone our business model . cimit taught us to make hypotheses about our business and test them rigorously by speaking to all the stakeholders in the ecosystem . not only did we convince ourselves of who our customer was and what we should be building , all the data we gathered also convinced investors . our experience at the cimit program and all the support from the faculty has been instrumental to our progress . less than a year ago , i was an md - phd student with no business experience and no intentions of commercializing technology . in fact , before the course , we did nt even know who our customer was or what our product was , let alone our business model . cimit taught us to make hypotheses about our business and test them rigorously by speaking to all the stakeholders in the ecosystem . not only did we convince ourselves of who our customer was and what we should be building , all the data we gathered also convinced investors . the ncai - cc hcp program was seven weekly sessions , specifically designed for therapeutics . it provided specialization workshops on regulatory and intellectual property consideration with nhlbi staff and focused on the development of a target product profile , an accepted tool for pharmaceutical development projects . the cohort included five teams , each with a principal investigator , entrepreneurial lead and a faculty member , one of the ncai product development directors . the teams all began at different baselines but advanced their understanding of their commercialization process . they defined the types and quality of data that would be required to attract a pharmaceutical company s interest in commercial development . one team identified a path that includes formation of a new company and application for sbir support . examples of the self - reported assessments include:taught to think differently about our technology with a business / practical perspective never learned in graduate school or as a postdochelped prepare me for industry i became better suited to write business reports such as a strategic plan focused for upper managementit was not until we talked to clinicians , patients and regulatory experts that we were able to capture a holistic view of how our technology fit in the therapeutic ecosystem . taught to think differently about our technology with a business / practical perspective never learned in graduate school or as a postdoc i became better suited to write business reports such as a strategic plan focused for upper management it was not until we talked to clinicians , patients and regulatory experts that we were able to capture a holistic view of how our technology fit in the therapeutic ecosystem . the cimit and ncai - cc faculty coordinated activities in developing , delivering and critiquing their programs . despite the differences between the programs due to the different clinical foci , the faculty developed consensus while there is strong agreement that there is no single right way to run an hcp , the following points are intended to help guide others wishing to develop an hcp . the lessons reinforce the applicability of some of the fundamentals of the i - corps program as well as ways that an hcp should differ from i - corps to address the unique needs and challenges of healthcare innovators and innovations . the fundamental premise of the i - corps program was found to apply very well to healthcare : get team members out of the lab to talk with stakeholders , customers in particular , and apply the scientific approach of developing , testing and validating hypotheses about the business . there are many facets of the i - corps program that were found to apply ; some key examples include : a weekly goal of around 10 exploratory interviews with a range of stakeholders needs to be stressed , and may be the most challenging as well as valuable thing for participants to learn . most successful investigators are good advocates and teachers , re - learning to listen to feedback is often a challenge , particularly when based on partial information.the flipped - classroom structure with off - line assignments and weekly two - hour interactive session focused on practical applications of the materials and on teams presenting their work for robust feedback in a mix of physical and web - based meetings is time - efficient and allows participants to continue with their day jobs.programs should run at least eight to ten weeks to allow time for a sufficient number of interviews along with the potential of at least one pivot - a change in direction based on validated feedback from the market . a weekly goal of around 10 exploratory interviews with a range of stakeholders needs to be stressed , and may be the most challenging as well as valuable thing for participants to learn . most successful investigators are good advocates and teachers , re - learning to listen to feedback is often a challenge , particularly when based on partial information . the flipped - classroom structure with off - line assignments and weekly two - hour interactive session focused on practical applications of the materials and on teams presenting their work for robust feedback in a mix of physical and web - based meetings is time - efficient and allows participants to continue with their day jobs . programs should run at least eight to ten weeks to allow time for a sufficient number of interviews along with the potential of at least one pivot - a change in direction based on validated feedback from the market . it is not surprising that an effective hcp would differ from the i - corps program since it was developed with a different focus . i - corps was designed to support principal investigator led academic teams with sbir awards in a broad range of industries . while many commercialization challenges transcend industries , there are some unique challenges facing healthcare innovators that require simultaneous attention in an hcp . some key examples include : 1.buying dynamics : the user of the products / services ( user ) and those responsible for making a buying decision ( economic buyer ) in healthcare are usually not the same people , can be very hard to identify and may change from one institution to the next . in addition , users and economic buyers frequently have competing or misaligned objectives . this is in contrast , for example , to selling to consumers who make the purchasing decision and use the products.2.reimbursement complexity : reimbursement for products / services in healthcare is a complex and highly regulated process . this is in contrast to most industries in which the price a company can charge is established by a free market.3.regulatory pathway : while regulations exist in every industry , the impact that early and what may appear to be subtle decisions can have a major impact on the level of testing and validation that a medical product or service must undergo before being approved for sale or use . the decision can mean the difference of years before introduction and 10 s of millions of dollars of investment and therefore commercial viability . the option to start with a minimally viable product ( mvp ) and iterate with customer experiences to improve it is not the same as it is in industries with products that have less potential for harm , and are therefore less regulated.4.culture : healthcare professionals , be they doctors , nurses , therapists , technicians , etc . must invest many years and a great deal of money into education in order to practice . the option for them to leave a position to focus on a start - up comes at a very high professional and personal risk and therefore is often not practical . however , their constant engagement in a development effort and focus on addressing important unmet clinical needs is critical to success . their engagement must be managed within their other time constraints , which are generally only increasing , and done in a way consistent with their expectations and culture.5.funding requirements : while there is a wide range of funding required to develop a new product across industries , healthcare innovations are typically at the higher cost end . while the low - range may be $ 100k for an app and a few $ million for a consumer device , it now costs about $ 2 billion to bring a new drug to the market . in 2010 , medical devices typically cost between $ 31 million for those that require a 510(k ) and $ 94 million for higher - risk devices requiring the pma regulatory pathway ( excluding reimbursement and sales / marketing activities ) . as such , more resources , both quantity and magnitude investors with smaller dollars are therefore less inclined to invest due to the potentially negative impact that later stage investments can have on the value of their investment . buying dynamics : the user of the products / services ( user ) and those responsible for making a buying decision ( economic buyer ) in healthcare are usually not the same people , can be very hard to identify and may change from one institution to the next . this is in contrast , for example , to selling to consumers who make the purchasing decision and use the products . reimbursement complexity : reimbursement for products / services in healthcare is a complex and highly regulated process . this is in contrast to most industries in which the price a company can charge is established by a free market . regulatory pathway : while regulations exist in every industry , the impact that early and what may appear to be subtle decisions can have a major impact on the level of testing and validation that a medical product or service must undergo before being approved for sale or use . the decision can mean the difference of years before introduction and 10 s of millions of dollars of investment and therefore commercial viability . the option to start with a minimally viable product ( mvp ) and iterate with customer experiences to improve it is not the same as it is in industries with products that have less potential for harm , and are therefore less regulated . culture : healthcare professionals , be they doctors , nurses , therapists , technicians , etc . must invest many years and a great deal of money into education in order to practice . the option for them to leave a position to focus on a start - up comes at a very high professional and personal risk and therefore is often not practical . however , their constant engagement in a development effort and focus on addressing important unmet clinical needs is critical to success . their engagement must be managed within their other time constraints , which are generally only increasing , and done in a way consistent with their expectations and culture . funding requirements : while there is a wide range of funding required to develop a new product across industries , healthcare innovations are typically at the higher cost end . while the low - range may be $ 100k for an app and a few $ million for a consumer device , it now costs about $ 2 billion to bring a new drug to the market . in 2010 , medical devices typically cost between $ 31 million for those that require a 510(k ) and $ 94 million for higher - risk devices requiring the pma regulatory pathway ( excluding reimbursement and sales / marketing activities ) . as such , more resources , both quantity and magnitude investors with smaller dollars are therefore less inclined to invest due to the potentially negative impact that later stage investments can have on the value of their investment . below are some key distinguishing features and attributes the authors suggest to build on the i - corps fundamentals to address these healthcare specific issues in constructing and conducting an effective hcp : 1.team composition : teams need to include the perspectives and proactive engagement from the clinical as well as the technical and business environments . while all team members need to share work and responsibilities , the person with the business / entrepreneurial focus needs to take the lead in coordinating the team s efforts as well as organize the outreach and implementation of stakeholder interviews . clinicians are critical team members , and being time efficient is crucial to enable their participation . this approach differs from i - corp that has fixed team member roles and only includes a technical and entrepreneurial lead along with an industry mentor supplied by the team . successful implementation requires that each team member understands and is prepared to put in the time required , a total of about 40 hours / team / week . senior faculty members should be discouraged from taking a team member slot if they intend to just oversee the work.2.executive faculty : given the inter - related complexities associated with healthcare innovations and need to balance multiple trade - off s simultaneously to effectively de - risk projects , the authors urge that the hcp faculty presenting materials be healthcare industry experts and use healthcare examples . in particular , rather than having team - supplied mentors , the hcp should be responsible for supplying qualified , experienced industry veterans to provide teams with consistent feedback and guidance . to be cost effective , the same individuals can deliver most of the program s customized content as well as support teams . it is important that in addition to the work they each do with a specific team that they work as group in providing regular synthesized feedback to teams . while increasing the cost , having experienced executives with a diversity of backgrounds ( technical , regulatory , operational , marketing , manufacturing , etc . ) working together helps teams understand trade - offs and make better decisions to efficiently and cost effectively de - risk projects . a key to successful implementation is finding and recruiting these individuals , which is a challenge . the authors suggest tapping into existing entrepreneurial networks locally or nationally to get access to people with the diversity of skills needed.3.content : the content used in hcps should be designed to help teams address the specific challenges facing healthcare innovations . it should cover the complex buying dynamics , reimbursement complexities , practice workflow implications , regulatory consequences and constraints in addition to traditional entrepreneurial topics . the author s experience is that there is enough difference between pharma / biotech and healthtech , both in terms of content and the expertise of faculty and industry experts , to justify separate programs . for example , the coulter foundation and stanford biodesign have well developed , publically available healthtech content for reference , so there is little need to create new content . we suggest curating a repository using the participants feedback that can be accessed by team members as a reference library , not only during but also after the program.4.style : like i - corps , teams need frank , objective feedback . but unlike the rough treatment that is part of the i - corps pedagogy , faculty must also develop strong rapport with the team members . they should be seen as being part of the team and not as outside assessors . keeping objective metrics ( like the number of interviews ) visible keeps pressure on teams and engenders some inter - team competition . team - team interactions are encouraged and generate some of the key learning opportunities by seeing the challenges and mistakes of other teams , as well as successful strategies they employ to overcome them.5.ecosystem leverage : in addition to the above ways an hcp should be conducted , the context in which an hcp is used can help address the funding challenges . the primary goal of the program is for each team to establish if they can deliver against a validated value proposition with a convincing pitch that articulates the narrative of a viable commercialization pathway or conclude that a quick kill it is critical that teams start their journey into the valley of death with a convincing pitch , but even better if they have some resources as they start their journey . so , rather than conduct an hcp as a stand - alone activity at the end of a funding cycle ( such as the i - corps program after sbir funding ) , the authors strongly encourage hcps to be conducted as an integral part of an ecosystem or program , like the ncai program , while its other resources are available to assist teams advance . this approach can significantly improve funding efficiency by applying the benefits of an hcp early , for example as a this helps focus available resources on the teams with the highest likelihood of translational success and focus teams on tasks that are most needed to advance projects to commercially relevant , as well as technically and clinically important milestones . team composition : teams need to include the perspectives and proactive engagement from the clinical as well as the technical and business environments . while all team members need to share work and responsibilities , the person with the business / entrepreneurial focus needs to take the lead in coordinating the team s efforts as well as organize the outreach and implementation of stakeholder interviews . clinicians are critical team members , and being time efficient is crucial to enable their participation . this approach differs from i - corp that has fixed team member roles and only includes a technical and entrepreneurial lead along with an industry mentor supplied by the team . successful implementation requires that each team member understands and is prepared to put in the time required , a total of about 40 hours / team / week . senior faculty members should be discouraged from taking a team member slot if they intend to just oversee the work . executive faculty : given the inter - related complexities associated with healthcare innovations and need to balance multiple trade - off s simultaneously to effectively de - risk projects , the authors urge that the hcp faculty presenting materials be healthcare industry experts and use healthcare examples . in particular , rather than having team - supplied mentors , the hcp should be responsible for supplying qualified , experienced industry veterans to provide teams with consistent feedback and guidance . to be cost effective , the same individuals can deliver most of the program s customized content as well as support teams . it is important that in addition to the work they each do with a specific team that they work as group in providing regular synthesized feedback to teams . while increasing the cost , having experienced executives with a diversity of backgrounds ( technical , regulatory , operational , marketing , manufacturing , etc . ) working together helps teams understand trade - offs and make better decisions to efficiently and cost effectively de - risk projects . a key to successful implementation is finding and recruiting these individuals , which is a challenge . the authors suggest tapping into existing entrepreneurial networks locally or nationally to get access to people with the diversity of skills needed . content : the content used in hcps should be designed to help teams address the specific challenges facing healthcare innovations . it should cover the complex buying dynamics , reimbursement complexities , practice workflow implications , regulatory consequences and constraints in addition to traditional entrepreneurial topics . the author s experience is that there is enough difference between pharma / biotech and healthtech , both in terms of content and the expertise of faculty and industry experts , to justify separate programs . for example , the coulter foundation and stanford biodesign have well developed , publically available healthtech content for reference , so there is little need to create new content . we suggest curating a repository using the participants feedback that can be accessed by team members as a reference library , not only during but also after the program . unlike the rough treatment that is part of the i - corps pedagogy , faculty must also develop strong rapport with the team members . they should be seen as being part of the team and not as outside assessors . keeping objective metrics ( like the number of interviews ) visible keeps pressure on teams and engenders some inter - team competition . team - team interactions are encouraged and generate some of the key learning opportunities by seeing the challenges and mistakes of other teams , as well as successful strategies they employ to overcome them . ecosystem leverage : in addition to the above ways an hcp should be conducted , the context in which an hcp is used can help address the funding challenges . the primary goal of the program is for each team to establish if they can deliver against a validated value proposition with a convincing pitch that articulates the narrative of a viable commercialization pathway or conclude that a quick kill it is critical that teams start their journey into the valley of death with a convincing pitch , but even better if they have some resources as they start their journey . so , rather than conduct an hcp as a stand - alone activity at the end of a funding cycle ( such as the i - corps program after sbir funding ) , the authors strongly encourage hcps to be conducted as an integral part of an ecosystem or program , like the ncai program , while its other resources are available to assist teams advance . this approach can significantly improve funding efficiency by applying the benefits of an hcp early , for example as a phase 0 award . this helps focus available resources on the teams with the highest likelihood of translational success and focus teams on tasks that are most needed to advance projects to commercially relevant , as well as technically and clinically important milestones . hcps offer institutions and funders an additional option to improve the efficiency with which the healthcare projects they support reach practice and improve people s health . it offers a cost effective way for them to increase the likelihood of selecting and advancing projects to commercialization and patient impact while also helping build innovation capacity . they teach academic based investigators important skills in a learn - by - doing mode to enable them to contribute to entrepreneurial activities without attempting to turn them into entrepreneurs . while no single approach for hcps is being advocated , the lessons learned synthesized from a diversity of programs can act as a guide for those interested in establishing a hcp to maximize the impact of their available resources .

academic investigators are generating a plethora of insights and technologies that have the potential to significantly improve patient care . however , to address the imperative to improve the quality , cost and access to care with ever more constrained funding , the efficiency and the consistency with which they are translated into cost effective products and/or services need to improve . healthcare commercialization programs ( hcps ) are described and proposed as an option that institutions can add to their portfolio to improve translational research . in helping teams translate specific healthcare innovations into practice , hcps expand the skillset of investigators and enhance an institution s innovation capacity . lessons learned are shared from configuring and delivering hcps , which build on the fundamentals of the national science foundation s innovation corps program , to address the unique challenges in supporting healthcare innovations and innovators .

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bladder cancer is the second most common cancer in the urogenital tract . in about 85% of cases at the time of diagnosis of cancer is limited only to the urinary bladder in the remaining 15% of the observed expansion beyond the urinary bladder . the first confirmed carcinogenic factors were the aromatic amines : o - toluidine , 2-naftylamine , 4-aminobiphenyl , and many other chemicals contained in tobacco smoke . in recent years currently there are about 1.1 billion smokers . in poland , there are about 9.5 million people smoking , including 43% of men and 22% of omen . passive smoking is also becoming a widespread social phenomenon , mainly at home ( 47% men & 52% women ) and work ( 55% men & 27% women ) . in poland , smoking is considered a shameful habit so patients asked about their smoking history do not readily admit to active smoking or they say they quit smoking just before evaluation . most patients know that smoking is harmful since they are ashamed to admit to active smoking ( embarrassed for their ignorance ) . they do not realize that it is possible to assess the reliability of the collected information about the history of smoking . we do not consider smoking danger warnings placed on cigarette packs and media - advocated smoking cessation as helpful to the no - smoking cause . the international agency for research on cancer predicts that by 2020 the number of deaths related to smoking will rise to 10 million per year , of which 70% will be in developing countries . at present approximately 20% of cancer deaths it is , however , satisfying that recently administrative rulings managed to limit the number of public places where smoking is permitted . research was carried out , which allegedly confirmed the positive results of this reduced smoking . the aim of this study was to assess the reliability of a subjects smoking history , and assess compliance with the recommendations of smoking cessation on the basis of a questionnaire and the measurement of cotinine , a marker of exposure . the study is a part of the long - term study conducted in our department funded by the national research committee , grant no . the evaluation of patients treated in the department of general , oncologic and pediatric urology of collegium medicum nicolaus copernicus university in bydgoszcz in the period from june 2009 to december 2010 who had confirmed primary or recurrent bladder cancer by histopathological examination was undertaken . every patient who agreed to participate in the study signed an informed consent form and completed a questionnaire on smoking history and tobacco smoke exposure . it was asked whether the patient smokes , if so , how many cigarettes per a day and for how many years . smokers were asked when they stopped smoking , how many cigarettes were smoked per day , and for how long . a model questionnaire on the history of smoking : 1 . non - smoker and non - exposed to the tobacco smokenever smokedstopped smoking < 1 year ago2 - 5 years ago>5 years ago 2 . smokersthe number of cigarettes smoked , packages ( 1 pack = 20 pieces)since i smoke ? 3 . exposed to tobacco smoke ' passive smokingplace of exposure to tobacco smokehomeworkanother placethe residence time in tobacco smoke ( hours)number of smokers 1 . exposed to tobacco smoke ' passive smoking each patient had their urine cotinine levels determined immediately after admission to the hospital to reduce the number of false - negative results arising from the discontinuation of smoking during hospitalization . cotinine ( name of the synthetic ( s)-1- methyl-5-(3-pyridinyl)-2-pirolydyna ) is an objective indicator of exposure to tobacco smoke and is found in the urine , even several hours after smoking ; with a half - life of 20 - 40 hours . in the body , it can be detected at very low concentrations in the limit 0.57 nmol / l = 0.1 ng / ml ( 6 ) . the analysis of cotinine , the main metabolite of nicotine , were determined by high performance liquid chromatography ( hplc / dad ) . application to the markings cotinine hplc / dad allowed the side to ensure adequate sensitivity of determination ( ng / l ) also identify the compound , determined by analysis of mass spectra . downloaded from those covered in the study sample of urine ( about 50 ml ) for analysis were stored at minus 20 c. cotinine extraction from urine was carried out in alkaline ( ph = 11.1 mol naoh ) . after evaporation of organic solvent in a nitrogen atmosphere the dry residue was dissolved in methanol and analyzed by hplc / dad with the following conditions : liquid chromatograph f - my agilent technologies ( binary pump & autosampler ) column eclipse xdb c8 zorba ( 150x4 , 6 mm , 5 l ) at 25 c , injection volume 20 ml . the mobile phase was methanol / ammonium buffer ph = 3.5 with a flow of 0.8 ml / min . detection of cotinine level in urine was performed in 96 patients , of which 35 patients ( 36% ) obtained results indicating active smoking ( cotinine levels in the urine of > 200 ng / ml , values range from 257 to 7929 ng / ml ) and in 4 patients ( 4% ) had cotinine levels equivalent to passive smoking ( level between 0 - 200 ng / ml , range of values from 40 to 187 ng / ml ) while in the remaining 57 patients ( 59% ) had no cotinine in urine ( table 1 ) . subjects groups on basis cotinine level we analyzed the questionnaire completed by patients and compared it with test results of cotinine in the urine . among the 96 patients,18 admitted to smoking ( at about 19% ) , in fact , in 35 ( 36% ) patients were confirmed by cotinine levels indicating active smoking , in 4 patients , exposure to tobacco smoke at home . among smokers , the average smoking time is approximately 33 years ( range from 20 to 45 years ) . the number of cigarettes smoked was on average 15.6 pieces per day ( range of values from 15 to 20 pieces a day ) within 18 months of observation in 3 patients in the first test was confirmed in the urine cotinine ( values from 2709 to 4760 ng/ ml ) in a subsequent study found no cotinine in 2 patients , with a group of non - smokers in another examination confirmed that indicates the level of active smoking ( values from 839 to 1110 ng/ ml ) was not confirmed in the questionnaire . it is widely know from numerous publications that bladder cancer and smoking are strongly linked ; however , no significant correlation between the type of tobacco smoking , nicotine content of the smoked cigarettes , and higher risk of bladder cancer was confirmed . all cigarette smoking , both strong ( with a higher nicotine content ) and weak ( with reduced nicotine content ) , with or without a filter , has the same negative effect on our body without clinically important differences . there are no further reports assessing how smoking affects the course of bladder cancer , the number of recurrences , and the progression of the disease . no such analysis is probably due to the limited reliability of the smoking questionnaire and due to expensive and time - consuming methods of monitoring cotinine in urine in all patients , which can only be afforded by a few university centers . the destructiveness of tobacco should always be presented to all smoking patients in an attempt to persuade the smokers to stop smoking . this study found that not all patients admit to smoking , and not all follow the recommendations to quit smoking . in the present study patients did not know that the information contained in it would be verified objectively before filling in the questionnaire . therefore , 18% of patients admitted to active smoking and 4% of patients were unaware of the adverse effects of tobacco smoke exposure . three smoking patients quit smoking by the 18-month follow - up while two from the group of non - smokers had confirmed active smoking in subsequent examinations , although the questionnaire strongly denied that those patients smoke . using a marker of exposure to tobacco smoke each patient was determined in urine cotinine levels immediately after admission to hospital to reduce the number of false - negative results resulting from smoking cessation during hospitalization , despite the fact that the period of persistence of cotinine in the urine is so long that it enables detection , even after several hours . detection of cotinine in urine proved to be a convenient and easy way that was acceptable to the patient and chosen by the authors of the study to eliminate the stress factor associated with the collection of a blood sample . application to the markings cotinine hplc / dad allows side to ensure adequate sensitivity of determination ( ng / l ) also denoted the identification of compound . statement and determine the levels of cotinine in the urine of patients who do not smoke tobacco will allow the realization of risks arising from the presence in tobacco smoke to those who reside there but involuntarily and unknowingly risk individuals [ 11 , 12 ] . in our study , people exposed to tobacco smoke , passive smoking was not aware that being in smoky areas are directly exposed to the harmful effects of tobacco smoke . satisfactory is the fact that in recent years through administrative rulings have managed to limit the number of public places where smoking is permitted . still , smoking in enclosed areas , especially in households causes harmful substances that remain after combustion of nicotine on the surfaces of various objects for many hours and adversely affect people who are in such areas , in particular , children . on the question how smoking affects of bladder cancer evaluation will answer the study conducted in our clinic during the long observation of patients with bladder cancer . detection of cotinine level allows for credible assessment of medical history related to tobacco smoking.questions and answers indicated that the credibility of the questionnaire is limited to the disclosure and does not accurately relate smoking by the patients.assessment of active and passive smoking should be carried out based on evaluation of the questionnaire and measurement of the nicotine metabolite in the urine.monitoring of cotinine level allows for an objective assessment and to effectively convey the recommendations for quitting tobacco smoking . detection of cotinine level allows for credible assessment of medical history related to tobacco smoking . questions and answers indicated that the credibility of the questionnaire is limited to the disclosure and does not accurately relate smoking by the patients . assessment of active and passive smoking should be carried out based on evaluation of the questionnaire and measurement of the nicotine metabolite in the urine . monitoring of cotinine level allows for an objective assessment and to effectively convey the recommendations for quitting tobacco smoking .

an analysis of the reliability of a questionnaire on smoking in 96 patients with transitional cell carcinoma ( tcc ) of the bladder . the credibility of the questionnaire was evaluated based on the detection of cotinine , an objective marker of tobacco smoke exposure , in urine . it was confirmed that approximately 18% of smokers did not admit to smoking , did not comply with recommendations to stop smoking , and about 4% of non - smokers were exposed to tobacco smoke unknowingly .

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anal incontinence is a bothersome ailment associated with many health complaints and discomfort in daily life : hygienic problems , limitations in occupational and social life , sexual dysfunction , reduced quality of life , and altered self - esteem . prevalence and severity of anal incontinence are measured by patient self - reporting and no objective assessment methods exist . obstetric anal sphincter injury ( oasis ) is one of the main causes for female ai reported in nonpregnant women . additionally , multiple vaginal deliveries can increase the risk of ai regardless of anal sphincter injury [ 2 , 3 ] . age , obesity and medical conditions such as diabetic neuropathy and gastrointestinal disorders also increase the risk of anal incontinence [ 2 , 4 , 5 ] . prevalence of anal incontinence among women differs largely ( 228% ) in previous studies and differs between different study populations [ 46 ] . postpartum studies show a high prevalence of ai in women having suffered from oasis , 3859% [ 68 ] . women attending gynecological outpatient clinics have higher prevalence of ai ( 1628% ) compared with the general female population ( 4.4% ) [ 2 , 5 ] . women with pelvic floor disorders have higher prevalence of ai than women without pelvic floor disorders . community - based studies show differences in prevalence of ai between age groups , with increasing prevalence by increasing age [ 4 , 5 , 9 ] . most frequent ai is found among nursing home residents ( 5060% ) , among the oldest women with frequent additional complaints and comorbidity . few previous studies have assessed the prevalence of anal incontinence in a female population of fertile age , and few studies have included nulliparous women [ 1114 ] . the aim of this study was therefore to assess the prevalence and risk factors for anal incontinence in an unselected female population across parity groups in second trimester of pregnancy . this study is part of a comprehensive perineum research study , which was approved by the regional committee for medical research ethics in south - eastern norway ( ref . this study was conducted as a survey of pregnant women attending free of charge routine ultrasound examination at second trimester , from september 2009 to august 2010 , in a large university hospital in oslo , norway . the pregnant women attending the ultrasound screening in our hospital represent a nonselected population from the entire oslo area . all pregnant women in norway are offered a free of charge second trimester routine ultrasound examination in gestational week 1820 , and 98% attend . in our hospital , this routine ultrasound is performed by specially educated midwives at the fetal medicine unit . the hospital receives admission notes from the local general practitioners when the woman is pregnant in the first trimester . the invitation to participate in our study , the questionnaire , and the informed consent were included as a part of the invitation to the routine ultrasound appointment . midwives performing the routine ultrasound examination reminded the women of the study and collected the questionnaire and the signed informed consent . from the 7256 women who were posted a questionnaire , 973 women were not found in our postpartum labor ward database : they did not achieve 18 weeks pregnancy ( pregnancy loss ) , they did not deliver in our hospital , or they had moved out of oslo area or norway , resulting in 6283 women eligible for study participation . four women returned two questionnaires ( twice during the same pregnancy ) , and one woman returned the questionnaire shortly after the index delivery . thus , five filled - out forms were excluded from the analyses and the study group consisted of 2846 ( of 6283 invited ) women , resulting in a response rate of 45% . the questionnaire consisted of 105 questions concerning anal and urinary incontinence , general health condition , drug use , and worries concerning pregnancy and delivery . the major part of the questions was chosen from validated questionnaires such as due and ottesen , st . mark 's , nugg , hunt , and cambridge worry scale ( cws ) [ 19 , 20 ] . additionally , we collected demographic data , obstetrical history , educational level , household income , and country of origin of the participant . anal incontinence was identified by self - reported leakage of gas , loose , or solid stools , lack of ability to defer defecation for 15 minutes ( fecal urgency ) , use of pads or plugs , and alteration of lifestyle described in st . mark 's score from 0 to 2 were analyzed as a control group ( no or infrequent ai ) . urinary incontinence was defined as self - reported symptoms of stress or urge urinary incontinence . thus , women with cesarean delivery only ( never having delivered vaginally before ) were categorized as vaginal primiparous . continuous data were categorized and the independent variables are presented as frequencies . univariate analysis was performed to identify significant risk factors for anal incontinence . the results from this regression analysis are presented as adjusted odds ratios ( aors ) for ai with 95% ci . for each model the assumptions underlying a valid logistic regression analysis were checked and found to be adequately met . mark 's score 3 or more ) in the entire study group was 8.4% ( 238/2846 ) . most of the women ( 80.3% ) reported complete anal continence ( 2268/2846 ) with st . mark 's score 0 , and 11.3% ( 322/2846 ) women reported infrequent ai with st . mark 's score 1 or 2 . inability to control flatus was the most frequent complaint , reported by 18.0% ( 513/2846 ) . of these , fecal incontinence was reported by 6.0% ( 171/2846 ) and fecal urgency by 3.2% ( 90/2846 ) of the women . urinary incontinence ( ui ) was significantly associated with reported anal incontinence among all parity groups and 32.4% of the women with ai also reported ui ( p < 0.001 ) . prevalence of ui was threefold among parous women compared to nulliparous women and increased slightly with increasing vaginal parity ( p < 0.001 ) ( data not shown ) . the majority of the 2846 women had answered the questionnaire when they were in the second trimester ( 84% ) , 12.2% in the first trimester , and 1.2% in the third trimester . mean height was 168 cm and mean weight was 66.7 kg . mean bmi was 23.6 , range 16.042.4 . smoking was infrequent ; only 2% ( 57/2851 ) women reported that they smoked during this pregnancy ( table 1 ) . there was a significant difference in prevalence of ai among women with different obstetric histories . mark 's score of 3 or above ) increased with increasing vaginal parity ( data not shown ) . of the nulliparous women , 7.8% ( 139/1792 ) reported anal incontinence . in the univariate analysis , non - western background , low household income , being unmarried or single , low educational level , age 35 or more , answering the questionnaire in the first trimester ( as opposed to second trimester ) , dermatological disease , ulcerative stomach disease , hypertension , rheumatoid arthritis , and muscle - skeletal complaints were significantly associated with anal incontinence among the nulliparous women ( tables 1 and 2 ) . in the multivariate analysis , low educational level , dermatological disease , and rheumatoid arthritis remained as significant factors for ai ( table 2 ) . after excluding the 140 women with previous cesarean delivery only , the subgroup of vaginal parous women consisted of 914 women . overall anal incontinence among parous women was 9.8% ( 90/914 ) . in the group of women with one previous vaginal delivery , 8.5% ( 61/714 ) reported ai , whereas the group of women with more than one previous vaginal delivery , as many as 14.5% ( 29/200 ) , reported ai ( p = 0.004 ) . of the parous women , 15.9% had previously delivered at least one macrosomic ( > 4000 g ) infant ( 145/914 ) , 156 reported previous delivery with vacuum extraction , and 24 women reported a previous forceps delivery . an obstetric history with instrumental delivery or a macrosomic infant previous delivery with oasis was reported by 41 women ( 4.5% ) and was strongly associated with ai . in the univariate analysis , previous delivery with oasis , non - western background , low household income , being unmarried or single , low educational level , age 35 or more , bmi 25 or more , dermatological disease , and use of pain killers were significantly associated with anal incontinence among the parous women ( tables 1 and 2 ) . in the multivariate analysis , previous delivery complicated with oasis and dermatological disease remained as significant risk factors for ai ( table 2 ) . the risk of ai was threefold among women with previous oasis compared to women without ( 24.4% and 8.1% , resp . ) . the higher risk of ai associated with previous oasis remained threefold in the more severe forms of ai , if defining ai as self - reported st . mark 's score of 5 or above ( 12.2% and 3.8% ) or if 7 or above ( 7.3% and 2.3% ) instead of 3 or above ( table 3 ) . the subgroup of parous women with previous cesarean only ( n = 140 ) and no vaginal deliveries was also analyzed separately . in this subgroup the prevalence of ai was the lowest ( 6.4% , 9/140 ) compared with all other parity groups but was too small to further analysis of risk factors . when the analyses were repeated with this subgroup of women added to the subgroup of nulliparous women , the conclusions remained unaltered ( data not shown ) . women who reported having a dermatological disease reported also more anal incontinence than women without this disease ( table 2 ) . there was a significant association between dermatological disease and several other complaints : allergy , migraine and headache , constipation , and psychological problems . women who reported dermatological problems also more frequently reported use of vitamins , allergy medication , and stomach and bowel regulators . these women also reported more worries about the cambridge worry scale than the women without dermatological problems . this population - based study showed that previous obstetric anal sphincter injury was the strongest risk factor for self - reported ai among pregnant parous women . among the nulliparous women , a low educational level and comorbidity the group of women with previous deliveries with cesarean section only had the lowest prevalence of ai , indicating that pregnancy per se may not represent a major risk factor for ai . these findings support the notion that the process of vaginal delivery may be more damaging to the anal continence mechanisms than pregnancy per se . we found a surprisingly high frequency of self - reported ai among nulliparous women ( 7.8% ) . low socioeconomic status ( low income , low education ) is a well - known reason for lower health status and increased morbidity , and previous studies show that self - rated health predicts morbidity well [ 2123 ] ; therefore , there is now a reason to doubt the correctness of the self - reported ai . socioeconomic differences have been found in occurrence of almost all conditions and illnesses [ 22 , 23 ] . this might explain part of the results for the group of nulliparous pregnant women in our study , where low educational level remained as significant risk factor for ai in the multivariate analysis . a previous oasis was the strongest risk factor for self - reported anal incontinence in all analyses in parous women , with and without adjusting for other factors , and in all categories of severity of anal incontinence . in our study , women with previous oasis reported a lower prevalence of ai than women in previous studies on nonpregnant women [ 68 , 24 ] . all the participants in our study were pregnant , and the low prevalence of ai among women with previous oasis might indicate that fewer women with severe complaints of ai embark on a new pregnancy . the risk of ai increased with increasing number of vaginal deliveries , a result similar to previous studies . interestingly , previous delivery with a macrosomic infant ( > 4000 g ) was not associated with self - reported ai during pregnancy in our study , and was not a previous delivery with vacuum extraction or forceps . this is in contrast to some previous studies , where previous forceps delivery and macrosomy are reported as risk factors for ai [ 8 , 26 , 27 ] . in our study of pregnant women , maternal age was not a significant risk factor for ai in the subgroup of parous women , probably because our study group was young , the oldest participant was 45 years old , and age related increased risk of anal incontinence is probably more important in older age groups [ 2 , 5 , 27 ] . women with overweight ( bmi 2529.9 ) and obesity ( bmi > 30 ) were more likely to suffer from anal incontinence than women with normal bmi ( < 25 ) in our study , but in the multivariate analysis this effect disappeared , due to the strong effect of previous oasis . the large effect of oasis exceeded all other factors ( except dermatological illness ) . many previous studies of ai describe only the frequency of the different components of ai . we chose to describe the prevalence of ai as a score , to be able to perform multivariable analyses of the assessed variables in our study . mark 's score 3 as cutoff for ai was to be able to compare the results from this study to our previous study and also to our future study , a followup of the participating women after delivery . as a limit of 3 for defining ai may be questioned for clinical relevance , we repeated all statistical analyses with different cutoffs ( 4 , 5 , and 7 ) for st . the main conclusions remained unaltered , oasis was the most important predictor for ai ( for all these cutoffs for st . mark 's score ) among parous women , and low socioeconomic status and comorbidity were the most important indicators for ai among nulliparous women . variables with a p value over 0.05 were also included in the primary analyses to ensure that no risk factors were missed among our registered variables , but no such factors were revealed . similarly to our previous study of nonpregnant women , urinary incontinence was reported by 19.2% of the participants . prevalence of urinary incontinence was threefold among women who reported ai compared to women without ai , in both nulliparous and parous subgroups of women ( p < 0.001 ) . this might indicate that some women are in higher overall risk for incontinence , possibly associated with tissue type , or the pathophysiological mechanism may be the same for both diseases . strength of this study is that the pregnant population was unselected and consisted of all parity groups , including nulliparous women . the majority of studies on female anal incontinence have assessed nonpregnant women 624 months after delivery . another strength of this study is that we also assessed comorbidity and medication use in addition to obstetrical history . to our knowledge , no previous studies have assessed anal incontinence and comorbidity among pregnant women . among nulliparous women further research is needed to explore whether this is a consistent finding across population groups and to explore which mechanisms could underlie such an association . a weakness in our study is that the response rate among the invited women was less than 50% , and this can cause self - selection bias among the study participants . similar selection bias was observed in the norwegian moba study , where higher educated women more likely agreed to participate . however , the effect of such selection bias was found low in the moba study , and we have no reason to believe that our response rate of 45% negatively affected our study either . low prevalence of comorbidity and medication use may indicate that the participants did not have lower health status than nonparticipants or the general population in oslo . bias of women with a previous oasis and complaints of ai having been more eager to participate in the study is unlikely , since the prevalence of ai in the subgroup of women with previous oasis was lower ( 24.4% ) than that in the previously reported studies ( from norway ) [ 6 , 7 ] . we compared the study population 's basic clinical data with an anonymous electronic database covering all patients delivering at the same time period as the participants in this study . the distribution of nulliparous women in our study population was higher than in the overall delivery population in our hospital ( 63% and 52% , resp . ) . we did not find any differences in mean age between responders and nonresponders , but the distribution of women with non - western background was higher among the nonresponders ( data not shown ) , as expected , as the questionnaire and patient information were in norwegian . all data in this study was based on self - reporting from the participants , and thus information of their obstetric history can include errors . it is likely that women remember correctly the number of previous deliveries , delivery mode , and infant birth weight , but not all women are aware of having suffered from oasis when they leave the hospital after delivery . lacking information of oasis might strengthen our conclusions of oasis being a strong risk factor for ai : if women unaware of previous oasis reported ai and were analyzed as having no previous oasis , the risk of ai after oasis is even higher than calculated in this study . on the other hand , if more women who were unaware of having oasis reported no ai , our results would show too strong effect of oasis as a risk factor to ai . we found an association between self - reported dermatological disease and self - reported ai for all parity groups , which has not been reported before . we can only speculate reasons for this association ; women that have ai may also be more sensitive to dermatological bother than others , perhaps associated with the fecal incontinence with affection of perianal skin . possibly , there could be a common tissue specific risk for both ai and dermatological conditions . women who reported dermatological problems also reported more worries ; another explanation could be that these women were in general more sensitive to different symptoms and signs . in a further study more detailed questions about the type of dermatological disease would be of interest when assessing comorbidity in relation to symptoms of ai . we have performed a large number of analyses due to a large number of detailed information about obstetric history and maternal characteristics . the study population is relatively young , and thus , frequency of comorbidity and medication use was very low among the participants , which can give results by chance . this did not alter the conclusions ; low educational level among nulliparous and oasis among parous women were the most important factors associated to ai . we conclude that among parous women , previous oasis is the most important risk factor for anal incontinence and other obstetrical events only had a minor effect on development of ai . as oasis is a modifiable risk factor , and frequency may rapidly be altered after introduction of obstetrical perineal support programs [ 3033 ] , prevention of obstetrical sphincter injury is likely the most important factor for reducing bothersome anal incontinence in fertile women .

the aim of this study was to assess the prevalence and risk factors of anal incontinence in an unselected pregnant population at second trimester . a survey of pregnant women attending a routine ultrasound examination was conducted in a university hospital in oslo , norway . a questionnaire consisting of 105 items concerning anal incontinence ( including st . mark 's score ) , urinary incontinence , medication use , and comorbidity was posted to women when invited to the ultrasound examination . results . prevalence of self - reported anal incontinence ( st . mark 's score 3 ) was the lowest in the group of women with a previous cesarean section only ( 6.4% ) and the highest among women with a previous delivery complicated by obstetric anal sphincter injury ( 24.4% ) . among nulliparous women the prevalence of anal incontinence was 7.7% and was associated to low educational level and comorbidity . prevalence of anal incontinence increased with increasing parity . urinary incontinence was associated with anal incontinence in all parity groups . conclusions . anal incontinence was most frequent among women with a history of obstetric anal sphincter injury . other obstetrical events had a minor effect on prevalence of anal incontinence among parous women . prevention of obstetrical sphincter injury is likely the most important factor for reducing bothersome anal incontinence among fertile women .

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the incidence of hypotension in non - obstetric patients ranges between 5% and 66% , depending on the definition of hypotension limit , timing of measurements , and differences between data acquisition methods and patient characteristics . previous cohort studies reported that hypotension due to spinal anesthesia was approximately twice as common in hypertensive patients [ 13 ] . it has been shown that intraoperative hypotension is associated with myocardial ischemia and increased stroke risk . in addition , because hypertension is an independent risk factor for coronary heart disease , congestive heart failure , and cerebrovascular diseases , it is considered that the negative effects of hypotension will occur more easily in this patient group . the number of hypertensive patients in the adult population in turkey is estimated to be approximately 15 million ( 31.8% ) , and almost one - third is unaware of their condition . although hypertension is a problem frequently encountered by anesthesiologists , and despite advances in the definition and management of hypertension , there are only a few randomized controlled studies that have investigated the hemodynamic effects of spinal anesthesia in these patients . in this study we aimed to compare normotensive patients and hypertensive patients under control by medical treatment , with respect to the hemodynamic effects of spinal anesthesia performed with hyperbaric bupivacaine . the study was approved by the ethics committee of haseki training and research hospital , and all patients gave written and verbal consent prior to the study . sixty patients ages 1865 with asa scores i ii and who were scheduled for elective surgeries of lower abdomen , lower limbs , and perineum under spinal anesthesia were included into the study . blood pressures and heart rates were measured under standard conditions and the basal values were recorded . patients with a history of hypertension and who regularly used antihypertensive medications constituted group h ( n=30 ) , and patients with basal blood pressure values below 140/90 mm hg and no history of use of drugs effective on the cardiovascular system constituted group n ( n=30 ) . hypertension was inadequately controlled but blood pressure was below 180/100 mm hg in all patients of group h. antihypertensive drugs were given on the morning of surgery . patients in whom lumbar puncture failed in the first attempt , who had blood loss necessitating transfusion , or had any contraindications against spinal anesthesia were excluded from the study . an intravenous catheter was placed in the premedication room , then the ecg , non - invasive blood pressure ( nibp ) and peripheral oxygen saturation ( spo2 ) were monitored ( drger infinity delta , drger medical systems , inc . the patients were premedicated with 11.5 mg midazolam and received 10 ml / kg isotonic nacl according to the ideal body mass calculated with formula ( [ height100 ( height150)/4 ] for male and [ height100 ( height150)/4]0.9 for female ) in 20 minutes , then they were taken into the operating room . spinal anesthesia injected with a 22-gauge quincke spinal needle from the l34 intervertebral space with the patient in sitting position . as the local anesthetic , 3.5 ml of 0.5% hyperbaric bupivacaine ( marcaine 0.5% , spinal heavy 0.5% ( 5 mg / ml ) astrazeneca ) was applied to the subarachnoid space for 15 seconds . the patients were then immediately brought into supine position with the head elevated to 30 degrees . sensory block was evaluated with the pinprick test , and motor block was evaluated with the modified bromage scale ( 0=no motor block , 1=inability to raise extended leg , but able to move knees and feet , 2=inability to raise extended leg and move knee , but able to move feet , 3=inability to flex ankle joint ) . the systolic ( sbp ) , diastolic ( dbp ) , and mean ( mbp ) blood pressures , and heart rate ( hr ) were measured and recorded by an assistant at the following time points : basal ( the average of 3 consecutive measurements in the premedication room ) , after fluid loading , and 1 , 3,5 , 10 , 20 , 30 , 40 , 50 , and 60 minutes after spinal anesthesia . a decrease of mbp of more than 30% from the baseline was accepted as hypotension and was treated with intravenous ephedrine 5 mg . a decrease in hr to below 50 beats / minute was accepted as bradycardia and was treated with intravenous atropine 0.5 mg . patients who received ephedrine and atropine , and the time of treatment were recorded . during surgery , all patients inspired 100% o2 at a flow rate of 2 l / minute using a face mask , and isotonic nacl was infused at 8 ml / kg / hour . data were evaluated with the spss 17.0 statistics software for windows ( spss inc . , categoric variables between the groups ( sex , asa , the frequency of hypotension and bradycardia ) were compared with the chi - square test . numeric variables ( sex , body weight , height , and hemodynamic parameters ) were evaluated with the kolmogorov - smirnov test with respect to normal distribution . repeated measurements analysis of variance ( rmanova ) was used for intragroup comparison of the hemodynamic data , and bonferroni test was used for post hoc multiple comparison . the data are shown as mean s.d . and p<0.05 was accepted as statistically significant . there was no significant difference between the groups with respect to age , sex , weight , height , maximal height of sensory block , incidences of hypotension and bradycardia , or the amount of fluids infused ( p>0.05 ) . however , in group h the number of patients with asa ii physiologic scores was statistically higher compared to group n ( p<0.001 ) ( table 1 ) . antihypertensive medications used by the patients are also shown in table 1 . in the hypertensive patient group , the sbp , dbp , and mbp values were significantly higher than the normotensive patient group at all measurement times ( p<0.05 ) . comparison within the groups did not reveal any significant differences in either group compared to the basal values ( p>0.05 ; figures 13 ) . we also found that hr values did not show a significant change compared to the basal measurements ( p>0.05 ; figure 4 ) . six patients in group h and 7 patients in group n received atropine due to bradycardia . we observed that bradycardia resolved with a single dose of 0.5 mg intravenous atropine . in patients who developed hypotension , a single dose of 5 mg ephedrine treatment was sufficient ( table 1 ) . in this study we showed that there was no difference between the incidence of hypotension in hypertensive versus normotensive patients who underwent spinal anesthesia with hyperbaric bupivacaine . however , the number of patients who developed hypotension was greater in the hypertensive group ( 6/30 ) compared to the normotensive group ( 1/30 ) . in contrast to our results , racle et al . reported that hypotension due to spinal anesthesia with 0.5% isobaric bupivacaine was more frequent in hypertensive patients than in normotensives . in their study , the incidence rate of hypotension was 33% ( 10 out of 30 patients ) in the hypertensive group and 10% ( 3 out of 30 patients ) in the normotensive group , which were higher than our results . on the other hand , 2 other studies showed , as in our study , that the incidence rates of hypotension in hypertensive and normotensive patient groups were not different . however , the rates of hypotension were 55.5% and 67% in hypertensive patients , and 43.8% and 73% in normotensive patients , which were higher than our findings . the patients in the studies of racle and nishikawa were 1 or 2 decades older than ours ( mean age was above 75 ) . hypotension due to spinal anesthesia is caused by decreased systemic vascular resistance and venous return to the heart , caused by sympathetic system blockage . there is an increase in the basal sympathetic system activity and plasma norepinephrine levels associated with aging . therefore , sympathetic blockage after spinal anesthesia may cause a greater decrease in the systemic vascular resistance in elderly patients . hypertensive patients also have increased sympathetic activity and norepinephrine levels , as well as decreased parasympathetic activity . persistent sympathetic stimulation , independent of hypertension itself , causes loss of elasticity in the arterial wall and induces structural changes that in turn result in increases in peripheral vascular resistance . it has been shown that there is a close relationship between basal sympathetic activity and decrements in blood pressure that occur after sympathetic blockage . the reduction in blood pressure after spinal anesthesia is correlated with the degree of preoperative blood pressure . fukuda and nishikawa found that when preoperative blood pressure was normalized with antihypertensive treatment , there was no difference between the incidence of hypotension following spinal anesthesia in hypertensive versus normotensive patients . our results support these findings the reason for this is the improvement in vascular structural changes and the achievement of a decrease in basal sympathetic activity with the use of effective antihypertensive treatment . prehydration under the guidance of heart rate variability , an indicator of sympathovagal balance , was seen to decrease the risk of hypotension after spinal anesthesia . venous capacitance is decreased in hypertensive patients ; therefore it is thought that a sympathetic block after spinal anesthesia will cause a further decrease in the central volume and venous return to the heart . the fact that antihypertensive treatment provides an improvement in venous capacitance as much as peripheral vascular resistance explains why we did not find any significant decrease in the incidence of hypotension between normotensive and hypotensive patients . in this study , although we showed that there was no difference between the 2 groups with respect to the frequency of hypotension , the sbp , dbp , and mbp were higher in hypertensive patients compared to normotensive patients at all measurement times this is related to the higher basal blood pressure values in the hypertensive group compared to the normotensive group . our study did not measure the basal sympathetic activities of the patients , which is this study s most significant limiting factor . although an improvement in this increase and blood pressure regulation is achieved with appropriate antihypertensive treatment , lack of measurement of the basal sympathetic activities of the patients was the most significant limiting factor . in conclusion , we found that there was no significant difference between normotensive and hypertensive patients in the incidence of hypotension caused by spinal anesthesia with 0.5% hyperbaric bupivacaine .

backgroundhypotension is the most common problem with spinal anesthesia . this prospective study aimed to compare normotensive and hypertensive patients with respect to the hemodynamic effects of spinal anesthesia performed with hyperbaric bupivacaine.material/methodssixty patients who were scheduled to undergo various elective operations under spinal anesthesia were included into the study . the patients were separated into 2 groups : hypertensive patients constituted group h ( n=30 ) and normotensive patients constituted group n ( n=30 ) . after fluid loading , spinal anesthesia was performed with 3.5 ml 0.5% hyperbaric bupivacaine . demographic characteristics and incidence of hypotension and bradycardia were compared . systolic ( sbp ) , diastolic ( dbp ) , and mean blood pressures ( mbp ) and heart rate ( hr ) were also compared before and after spinal anesthesia.resultsthere was no significant difference between the groups with respect to demographic characteristics , maximal height of sensory block , incidences of hypotension and bradycardia , and the amount of fluids infused ( p>0.05 ) . in the hypertensive patient group , the sbp , dbp , and mbp values were significantly higher than in the normotensive patient group at all measurement times ( p<0.05 ) . comparison within the groups did not reveal any significant differences in either group compared to the basal values ( p>0.05 ) . there were no significant differences in hr between or within groups ( p>0.05).conclusionsthere was no significant difference between normotensive and hypertensive patients in the incidences of hypotension caused by spinal anesthesia with 0.5% hyperbaric bupivacaine .

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chronic pain and depression are among the most common health problems reported by patients attending primary care [ 13 ] . diagnosing pain and its coexistence with depression and getting an overview of the pain patient 's situation can be challenging and can have great consequences for the treatment outcome . numerous studies have found pain associated with a significant limitation in daily activities , physical activities and with poor self - rated health [ 3 , 4 ] . pain is also associated with increased prevalence of depressive disorders [ 5 , 6 ] . studies from primary care have demonstrated that 2240% of all contacts to primary care are due to pain . persistent pain is often comorbid with depression both in general pain conditions [ 4 , 8 ] and in disease - specific conditions . though health care providers are advised to pay special attention to pain symptoms in patients with depression , studies have demonstrated that primary care physicians tend to associate pain with depression to a significant lesser extent than other somatic symptoms , although pain is the most prevalent somatic symptom in patients with depression . this tendency to underestimate the intensity of burden has also been proven for the symptom pain with significant differences between the patient 's and the healthcare provider 's grading of the intensity of the pain [ 11 , 12 ] . patients with depression seek care more often for somatic symptoms than for psychological symptoms , why care providers must have an increased awareness for this coexistence . there is evidence that optimizing antidepressant therapy and pain self - management in primary care patients may result in improvement of both depression and pain . coexistence of depression and pain has also shown to be a major risk factor for opioid misuse both by using opioids for stress and sleep problems instead of for pain alone , and by using more opioids than prescribed . it is therefore important that healthcare provider and the patient have an ongoing awareness and understanding of this connection between depression and pain and preferably share understanding of the current state of the condition . dolotest ( figure 1 ) has been developed to provide such a shared understanding of the patients situation . it is a validated , pain- and health - related quality of life ( hrqol ) assessment tool integrating measurement of the intensity of pain with assessment of the intensity of the impact of the pain including mood . the aim of this study was to determine to which degree dolotest is useful as a screening tool for depression in primary care for adult ( all persons 18 years or older ) seeking primary care , and to find the sensitivity and specificity for dolotest used for screening for depression . the study is a diagnostic study among the population of patients visiting primary care physician no matter the reason for the visit . laegehuset noerretorv in grindsted , denmark in the period from october 1st 2008 to december 31st , 2008 . all patients being 18 years old or older visiting the clinic and not complying with the exclusion criteria were asked to fill in the questionnaires : dolotest and major depression inventory the study included a study population of 715 patients equal to a response rate of 68,5% . when arriving at the clinic , a clinic nurse screened for inclusion / exclusion criteria . patients who could enter the study were handed the questionnaires together with a letter of introduction and the standardized dolotest patient introduction . patients were also asked about age and gender and whether their visit was due to a pain problem or not . patients were excluded if they meet exclusion criteria : cognitive dysfunction , visual impairment , and patients whose physical condition made filling the test impossible or prior participation in the study . dolotest is a validated pain- and health related quality of life ( hrqol ) assessment tool integrating measurement of the intensity of pain with assessment of the intensity of the impact of the pain on eight 100 mm visual analogue scales ( vas ) . the design of dolotest provides a presentation of the test result as a visual profile ( dolotest - profile ) , with no problems toward the centre of each vas - line and worst possible toward the periphery . the larger the dolotest - profile the worse the hrqol , which is immediately understandable for both the patient and the healthcare provider . the dolotest - profile enables the patient to take part in the evaluation of the test result and improve communication about the situation including problems related to depression like mood , sleep problems , tiredness functional problems , and allows the patient and the healthcare provider to evaluate response to treatment together . the test results are also available as a dolotest - score , the sum of all scored domains , ranging from 0 to 800 . mdi is a validated self - assessment questionnaire developed to cover the diagnostic criteria in who 's icd-10 classification of major depressive disorders . mdi was chosen because it follows the icd-10 criteria and is easy to use as a self - assessment questionnaire . the mdi can be used both as a measuring instrument and as a diagnostic instrument with algorithms leading to the icd-10 categories of depression . when used as a diagnostic instrument like in this study , the mdi items are dichotomized to indicate the presence or absence of each of the symptoms . data comes from 715 consecutive patients coming to primary care , no matter the reason for their visit . statistical analysis was made using mann - whitney test to compare groups and chi - square test to compare frequencies using analyse - it software v. 2.12 . the study was according to danish law ethical approved by the danish data protection agency , copenhagen , denmark , and all patients participated voluntary and were provided with both oral and written information . a total of 1044 patients were asked to participate . of those , 302 ( 31,5% ) were not included either because they did not want to participate or due to the exclusion criteria . of the 715 patients , 458 ( 64.1% ) were women and 257 ( 35.9% ) men . there were no statistically significant difference between the gender distribution in the study group and the excluded group ( p = 0.59 ) . the average age in the study population was 45.7 years and in the excluded group 55.4 years ( p < 0.0001 ) . table 1 shows that 246 patients ( 34.4% ) stated their visit was due to a pain problem ( study group ) and 469 patients ( 65.6% ) came for other reasons than pain ( control group ) . the study and control groups were sociodemographic identical with no statistical significant difference between the gender and age distributions , 33.6% of the woman and 35.8% of the men came due to pain ( p = 0.56 ) . table 2 shows that in all domains there are statistically significant difference ( p < 0.0001 ) between the study group and for the control group . low spirits and pain were the only domains in study group with statistically significant differences between men and women , women having the highest score . in the total study population , the women in average scored statistically significantly higher than men in all domains except for pain and problems with more strenuous activity . in the control group , women scored statistically significantly higher in al domains except pain , where the women also scored higher than men , however not statistically significant . the dolotest - score was statistically significantly higher for women in the whole study population and also for the control group . in the study population the women 's dolotest - score had a tendency to be higher than the men 's . table 1 shows the prevalence of patients with depression for the study and control groups . 115 ( 16.1% ) of all the patients met criteria for depression using the mdi - scale . table 2 presents the prevalence of depression together with the average mdi - rating score for the whole study population as well as specified for the study group and for the control group . in the whole study population , 32 men ( 12.8% ) and 83 women ( 18.1% ) met the criteria for depression using the mdi scale , and the corresponding prevalence for control group was 11 men ( 6.7% ) and 38 women ( 12.5% ) with statistically significant difference between genders in both groups ( p = 0.05 ) . in the study population , 21 men ( 22.8% ) and 45 women ( 29.2% ) met criteria for depression ( p = 0.27 ) . the prevalence of patients with depression in study group was statistically significantly higher than in the control group ( p = 0.0002 ) for men and p < 0.0001 for women . for patients meeting criteria for depression and patients not meeting the depression criteria , there was a difference for all dolotest domains ( p < 0.0001 ) ( table 3 ) . the domains were scored equally by men and women meeting criteria for depression due to the mdi scale ; however , for patients not meeting criteria for depression , women scored statistically significantly higher than men on five of the domains ( problems with light physical activity , reduced energy and strength , low spirits , reduced social life , and sleeping problems ) ( table 3 ) . the numbers in table 3 are visualized by presented as average dolotest profiles in figure 1 . a cut - off value for the domain to what extent do you experience low spirits for depression was found using roc curve analysis . it is desirable to find a cut - off with both a high sensitivity and a high specificity , and from a clinical point of view defining a cutoff with few false positive and high predictive value of positive test is important to help making an accurate clinical decision . selecting a cutoff point of 65 ( figure 2 ) will provide a sensitivity of 0.78 ( 95% ci : 0.700.85 ) and a specificity of 0.95 ( 95% ci : 0.930.96 ) the predictive value of positive test being 0.74 for detecting that a patient meet criteria for depression . dolotest has in this study proved useful as a screening tool for patients ' experienced pain and the impact of pain on the patient 's hrqol as well as a screening tool for patients meeting criteria for depression and thereby for the coexistence of pain and depression . a cut - off value of 65 on the low spirits domain provides both high sensitivity ( 0.78 ) and high specificity ( 0.95 ) and a predicted value on positive test at 0.74 . this study is the first to present health related quality of life ( hrqol ) data as dolotest profiles and dolotest scores for all patients seeking a primary care both for patients whose visit are due to a pain problem and for patients whose visit has other reasons . furthermore , the study contributes to the growing knowledge about coexistence of pain and depression , finding the prevalence of depression to be statistically significantly higher for both genders in the group visiting primary care physician because of a pain problem than for other problems combined . pain symptoms have been found to be associated with reduced psychological and physical health as well as with limitations in daily activities and with social isolation [ 19 , 20 ] and are found to be a major reason for seeking health care [ 19 , 20 ] . in this study , 34.4% of the patients stated that their visit to physician was due to pain , which is in accordance with findings in other studies . our study shows that 16.1% of all patients met criteria for depression , and 26.8% whose visit was due to pain , met depression criteria . these findings are in good correlation with the literature , though this study captures the pain intensity during the last week , for example , not necessarily persistent pain . the findings in this study are clinically important since the visual dolotest profiles can facilitate both awareness and often otherwise difficult communication about depression and pain , a coexistence leading to increased risk of reduced function , reduced work capacity , increased risk of opioid misuse , reduced hrqol , and other related problems . it has been shown that suffering from both pain and depression at the same time besides the low hrqol is associated with dramatically increased health care costs [ 22 , 23 ] compared to depression alone , and that treating depression alone does not give sufficient improvement on somatic symptoms like pain [ 21 , 24 ] . based on this knowledge , health care providers are encouraged to be aware of pain symptoms in patients with depression as well as depressive symptoms in patients with pain [ 21 , 25 ] . furthermore , it is stated that targeted screening for the cooccurrence of pain and depression is warranted . the respect trial found that effective treatment of either depression or pain is dependent on the treatment of the other when they are co - existing . the study concludes it makes no longer sense to treat one condition without considering the other . it is therefore of high importance to provide healthcare professionals with tools to screen and identify these patients at risk for suboptimal treatment if not found . in other studies using visual analogue scale ( vas ) as screening tool for depression , it has shown to be a reliable method and vas has thereby showed to be a valuable tool for assessment of mood . a study looking at using one question from the subjective health complaints ( shc ) inventory found this useful with sensitivity 79% , specificity 81% and predictive value of positive test to be 67% . in the present study , dolotest has proved as a tool screening both pain and depression , since the dolotest domain low spirits is associated with depression . a cut off value of 65 has been found optimal for the dolotest domain low spirits with a sensitivity of 78% and specificity of 95% to detect persons meeting criteria for depression , and with a predicted value on positive test at 0.74 . pain is not included in neither the icd-10 nor the dsm - iv criteria for depression , and it is also noteworthy that pain is not included in the most used tools for depression diagnostics like hospital anxiety and depression scale ( hads ) and beck depression inventory ( bdi ) or in the major depression inventory ( mdi ) used in the present study , which increase the risk for not finding the coexistence . both persistent pain and somatization have been found to be frequently associated with depression , yet few studies on somatization are controlled for depression . data is regarded representative for denmark as the present study was conducted in a clinic serving both urban and rural areas . in denmark , all citizens have free and equal access to primary care , as the primary care is financed through the danish taxation system . as expected the age distribution was statistically significantly higher in the excluded group due to the exclusion criteria cognitive and visual impairment . the scoring on both the dolotest and mdi was made by the patients themselves and not controlled . same physician made all diagnoses of meeting criteria for depression based on the mdi scorings ; however , a depression diagnosis was not provided through interview or other further examination . dolotest used as a screening tool for depression criteria had a sensitivity of 78% and specificity of 95% , and a predicted value on positive test at 0.74 . patients meeting criteria for depression had statistically higher pain score and reduced health related quality of life than patients not meeting criteria for depression . dolotest has in this study proved an easy - to - use screening tool in clinical settings for pain and the impact of pain as well as a screening tool for patient meeting criteria for depression . this ease the process of identifying patients with the important coexistence enabling easy identification of this large group of patients needing individualized treatment for more than the pain alone in order to get best possible health related quality of life . treating both pain and depression when co - existing is very important since it otherwise is found to be associated with risk of reduced function , reduced work capacity , increased risk of opioid misuse , and increased healthcare costs .

background . coexistence of pain and depression has significant impact on the patient 's quality of life and treatment outcome . dolotest is a pain and hrqol assessment tool developed to provide shared understanding between the clinician and the patient of the condition by a visual profile . aim . to find the sensitivity and specificity of dolotest as a screening tool for depression for patients in primary care . methods . all patients coming to a primary care clinic were asked to fill in a dolotest and a major depression inventory . results . 715 ( 68.5% ) of 1044 patients entered the study . 34.4% came due to pain . 16.1% met depression criteria , and 26.8% of patients coming due to pain met criteria for depression . 65.6% of the men and 54.2% of the women meeting the criteria for depression came due to pain . depressed patients had statistically significant higher scores on all dolotest domains . selecting the cutoff value for the domain low spirits to be 65 ( 0100 ) for depression gave a sensitivity of 78% ( 7085% ) and a specificity of 95% ( 9396% ) for meeting depression criteria . conclusion . dolotest can with a high sensitivity and specificity identify persons meeting criteria for depression and is an easy - to - use screening tool to identify patients with the coexistence of pain and depression .

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human papillomavirus ( hpv ) is a non - enveloped deoxyribonucleic acid ( dna ) virus belonging to the family papillomaviridae . this family includes more than 130 genotypes,1 many of which infect the mucosal areas of the human upper digestive tract and the anogenital region through sexual contact,2,3 leading to increased risk of development of cancer.1 these genotypes are grouped into high - risk and low - risk according to the degree of risk of development of cancer after infection . infection with the high - risk serotypes of hpv can lead to cervical cancer and are associated with other mucosal anogenital , and head and neck cancers.4,5 infection with the low - risk serotypes is known to cause benign or low - grade cervical tissue changes and genital warts ( condyloma acuminata ) on the cervix , vagina , vulva , and anus in women and on the penis , scrotum , and anus in men.6,7 genital hpv infection is one of the most common sexually transmitted infections in sexually active adolescents and young women.8,9 it has been estimated that at least 50% of sexually active adults have had a genital hpv infection2,9 and that globally 75% of individuals ( males and females ) will experience an hpv infection at least once in their lifetime , with the highest rates of infection occurring in those under the age of 25 years.10 in a recent meta - analysis , a global hpv prevalence of 11.7% was reported . the hpv prevalence in north america and europe was estimated at 11.5% and 14.2% , respectively , while the prevalence in africa was estimated at 21.1% , with sub - saharan africa topping the list at 24%.11,12 in nigeria , the prevalence of hpv is high in all female age groups , and highest in women aged 1523 years.12,13 studies have indicated that high - risk hpv genital infections in young females are transient and have little long - term significance.2,14,15 however , when the infection persists , as in 5%10% of infected women , there is a high risk of developing a precancerous lesion of the cervix , which can progress to invasive cervical cancer 1520 years later.4,1622 persistent infection following acquisition of a high - risk hpv is generally defined by continued detection of cervical dna of the same hpv type.23 cervical cancer is an important health problem worldwide , being the second most common cancer among women , and ranking first in many developing countries.9,17 half a million women develop cervical cancer annually and more than half die from the disease.9 in 2008 , more than 270,000 women died of cervical cancer worldwide , with nearly 85% of these deaths occurring in developing countries.13,24 cervical cancer is the second most common cancer in women aged 1544 years in nigeria and the incidence rate is 27/100,000.13,24 current estimates indicate that every year 14,089 women are diagnosed with cervical cancer and 8,240 die from the disease in nigeria.13 a prevalence of 26.3% for hpv in the general population has been reported in southern nigeria.25 the incidence of hpv in women with cervical cancer is reported to be 24.8%,12,24 while hpv prevalence in the general population ( among women with normal cytology ) is 23.7%.13 risk factors associated with hpv infection include heterosexuality , promiscuity , smoking,26,27 high parity , early sexual debut,3 infection with other sexually transmitted diseases,26 prolonged use of contraceptives , dietary factors , and genetic disorders such as whim ( warts , hypogammaglobulinemia , immunodeficiency , myelokathexis ) syndrome.28,29 infection with hpv , diagnosed by detection of antibodies to hpv in the serum or detection of hpv dna , is the primary risk factor contributing to development of cervical intraepithelial neoplasia and invasive cervix carcinoma . detection of anti - hpv has been shown to reflect the overall hpv infection rate in a population more effectively than detection of hpv dna.3 this study was conducted in an area where the seroprevalence of hpv has not as yet been reported . the study population comprised 350 women of reproductive age ( 1549 years ) attending the reproductive health clinic at ahmadu bello university teaching hospital , shika , zaria , nigeria . this clinic offers counseling and contraceptive services to delayers , spacers , and limiters . post abortion care , endometrial biopsy , papanicolaou smear , cervical punch biopsy , and colposcopy are other services offered . after obtaining ethical approval from the hospital s ethics committee , the women were recruited and counseled , and those who gave their consent were enrolled in the study . women who were pregnant , those who refused to give consent , and those not of reproductive age were not enrolled . a 3 ml blood sample was collected by venipuncture from each of the 350 women with the assistance of a laboratory scientist under the supervision of a physician . the blood samples were transported immediately to the laboratory in the department of microbiology , faculty of science , ahmadu bello university , zaria , which is located 5 km from the hospital . the blood samples were allowed to clot for 30 minutes and then centrifuged at 1,000 g for 10 minutes to separate the serum . the serum samples were stored at 20c until analysis . an enzyme - linked immunosorbent assay ( wkea med supplies corporation , changchun , people s republic of china ) was used to screen for hpv immunoglobulin g ( igg ) antibodies in the serum samples . all reagents were brought to room temperature and mixed thoroughly by gentle swirling before use . the cutoff value was calculated using the manufacturer s specifications . the average optical density value of the negative control wells plus 0.15 was taken as the cutoff value . a negative hpv result was interpreted as any sample with an optical density value less than the calculated cutoff value , and samples with an optical density greater than the calculated cutoff value were reported as positive for igg to hpv . the results and data from the questionnaires were analyzed , reduced to percentages , and presented as tables and figures . the statistical analysis was done using statistical package for the social sciences version 17 software ( spss inc . , associations between variables were identified by pearson s chi - square analysis and relationships between hpv and risk factors were identified by spearman s rank correlation . the study population comprised 350 women of reproductive age ( 1549 years ) attending the reproductive health clinic at ahmadu bello university teaching hospital , shika , zaria , nigeria . this clinic offers counseling and contraceptive services to delayers , spacers , and limiters . post abortion care , endometrial biopsy , papanicolaou smear , cervical punch biopsy , and colposcopy are other services offered . after obtaining ethical approval from the hospital s ethics committee , the women were recruited and counseled , and those who gave their consent were enrolled in the study . women who were pregnant , those who refused to give consent , and those not of reproductive age were not enrolled . a 3 ml blood sample was collected by venipuncture from each of the 350 women with the assistance of a laboratory scientist under the supervision of a physician . the blood samples were transported immediately to the laboratory in the department of microbiology , faculty of science , ahmadu bello university , zaria , which is located 5 km from the hospital . the blood samples were allowed to clot for 30 minutes and then centrifuged at 1,000 g for 10 minutes to separate the serum . an enzyme - linked immunosorbent assay ( wkea med supplies corporation , changchun , people s republic of china ) was used to screen for hpv immunoglobulin g ( igg ) antibodies in the serum samples . all reagents were brought to room temperature and mixed thoroughly by gentle swirling before use . the cutoff value was calculated using the manufacturer s specifications . the average optical density value of the negative control wells plus 0.15 was taken as the cutoff value . a negative hpv result was interpreted as any sample with an optical density value less than the calculated cutoff value , and samples with an optical density greater than the calculated cutoff value were reported as positive for igg to hpv . the results and data from the questionnaires were analyzed , reduced to percentages , and presented as tables and figures . the statistical analysis was done using statistical package for the social sciences version 17 software ( spss inc . , associations between variables were identified by pearson s chi - square analysis and relationships between hpv and risk factors were identified by spearman s rank correlation . the sociodemographic data for the women obtained from the questionnaires were analyzed and the results showed that women aged 4549 years were most frequently seen ( 32.6% , 114/350 ) in the clinic while those aged 1519 years were the least often seen ( 4.3% , 15/350 , table 1 ) . further analysis showed that 38.0% ( 133/350 ) of the women were from tribes other than the three major nigerian ethnic groups . further , 39.7% ( 139/350 ) were civil servants , 65.1% ( 228/350 ) were married , 56% ( 199/350 ) were in monogamous marriages , and 4.3% ( 15/350 ) admitted to smoking ( table 2 ) . the reproductive characteristics and sexual behavior of the women are shown in tables 3 and 4 , respectively . the data show that 54.9% ( 192/350 ) of the women had been married for less than 25 years and 56.3% ( 197/350 ) had had less than seven successful pregnancies ( parity ) , ie , reaching at least 28 weeks gestation . in total , 69.1% ( 242/350 ) of the women had children , with 57.1% ( 200/350 ) having 16 children . further analysis of the data showed that 60.3% ( 211/350 ) of the women had never used any form of contraception , and a sizeable proportion ( 3.1% , 11/350 ) had used other forms or non - modern contraception ( table 3 ) . the most frequent age of sexual debut was 19 years ( 26.9% , 94/350 ) while 8.9% ( 31/350 ) had their first child aged 1518 years ( table 4 ) . human papillomavirus igg antibodies were detected in serum samples from 42.9% ( 150/350 ) of women . antibodies to hpv initially increased with age , decreased in women aged 3039 years , and then increased again from age 40 years , peaking in women aged 4549 years ( 50.0% , 57/114 ) . women aged 3539 years had the lowest prevalence ( 31.7% , 13/41 , table 1 ) . the hpv infection rate did not differ significantly according to patient age ( =6.192 , df = 6 , p=0.402 ) . women belonging to tribes other than the three major nigerian ethnic groups had the highest hpv prevalence ( 44.0% , 66/119 ) , while igbo women had the lowest prevalence ( 4.7% , 7/25 ) . there was a marginal association between hpv infection rate and tribal origin ( =7.595 , df =3 , p=0.05 ) . hpv antibodies were detected significantly ( =3.938 , df = 1 , p=0.03 ) more often in christian women ( 47.1% , 99/203 ) than in muslim women ( 36.4% , 51/140 ) . retired women had the highest prevalence ( 75.0% , 9/12 ) and unemployed women had the lowest prevalence ( 35.1% , 20/57 ) ; however , this finding was not statistically significant ( =7.820 , df = 4 , p=0.098 ) . the seroprevalence of hpv was highest among widows ( 53.1% , 17/32 ) and lowest among divorcees ( 35.7% , 5/14 ) , but the difference was not statistically significant ( =1.739 , df =3 , p=0.773 ) . the seroprevalence of hpv igg antibodies was similar between monogamous ( 43.2% , 86/199 ) and polygamous women ( 42.7% , 32/75 ; =0.007 , df = 1 , p=0.935 ; odds ratio 1.023 ; 95% confidence interval 0.5981.749 ) . hpv infection was significantly associated with educational status ( =6.594 , df = 4 , p=0.043 ) . antibodies were detected with the highest prevalence among women with secondary education ( 49.2% , 29/59 ) while those with quranic education had the lowest prevalence ( 21.7% , 5/23 , figure 1 ) . antibodies to hpv were detected with a slightly higher frequency ( 43.1% , 140/325 ) in women who were not smoking compared with those who admitted to smoking ( 40.0% , 10/25 ; =0.090 , df=1 , p=0.765 ) . analysis of the results according to years of marriage showed that the highest frequency of antibodies to hpv was among women who had been married for 1620 years ( 55.3% , 21/38 ) and the lowest frequency in those who had been married for 2630 years ( 28.6% , 6/21 ; =8.734 , df = 7 , p=0.262 ) . no statistically significant relationship was found between seroprevalence of hpv and parity ( =3.700 , df = 4 , p=0.448 ) , but the prevalence was highest amongst women who had 79 pregnancies ( 46.7% , 21/45 ) and lowest in those of parity > 9 ( 25.0% , 3/12 ) . women with children had a higher prevalence of antibodies to hpv ( 44.6% , 108/242 ) than those without children ( 38.9% , 42/108 ) . although the difference observed was not statistically significant ( =1.004 , df = 1 , p=0.316 ) , women with children were 1.3 times more likely to be infected than women without children ( odds ratio 1.267 ; 95% confidence interval 0.7982.011 ) . women who had 79 children had the highest seroprevalence ( 46.9% , 15/32 ) while those with more than nine children had the lowest seroprevalence ( 16% , 1/6 ; =2.851 , df = 4 , p=0.583 ) . the seroprevalence of igg antibodies to hpv was not significantly associated with use of contraceptives ( =1.169 , df = 4 , p=0.883 ) ; women who were using other types of contraception ( eg , not modern or local contraceptives ) had the highest prevalence ( 54.5% , 6/11 ) while those using oral contraception had the lowest prevalence ( 39.4% , 13/33 ) . antibodies to hpv were detected most frequently among women who had their sexual debut aged 2023 years ( 51.1% , 46/90 ) and least often in women who had their sexual debut aged 2426 years ( 37.9% , 25/66 ) . however , this difference was not statistically significant ( =3.114 , df = 5 , p=0.682 ) . the frequency of detection of igg antibodies to hpv according to age at birth of first child was highest among women older than 30 years ( 68.8% , 11/16 ) and lowest among women aged 1518 years ( 35.5% , 11/31 ; =8.163 , df = 5 , p=0.147 ) . the prevalence of hpv was also investigated according to vaginal symptoms reported by the women during the study . women with normal vaginal discharge had the highest frequency of hpv antibodies ( 45.1% , 110/244 ) while women with abnormal discharge ( 37.7% , 40/106 ) had the lowest frequency ( =1.628 ; df = 1 ; p=0.202 ) . further , women with genital rash at the time of the study had the highest seroprevalence ( 61.9% , 13/21 ) , with the lowest seroprevalence seen in women with vaginal itching ( 33.3% , 26/78 ) . the difference in prevalence observed according to vaginal symptoms was almost statistically significant ( =7.461 , df =3 , p=0.059 ) . half of the women ( 50.0% , 9/18 ) with a family history of cervical cancer had igg antibodies to hpv compared with those without a family history of cervical cancer ( 42.5% , 141/332 ; =0.395 , df = 1 , p=0.348 ) . antibodies to hpv tended ( =0.806 ; df = 1 ; p=0.274 ) to be detected with a higher prevalence in women without a history or signs of whim as a genetic disorder ( 43.3% , 146/337 ) than in those with such a history ( 30.8% , 4/13 , table 5 ) . the sociodemographic data for the women obtained from the questionnaires were analyzed and the results showed that women aged 4549 years were most frequently seen ( 32.6% , 114/350 ) in the clinic while those aged 1519 years were the least often seen ( 4.3% , 15/350 , table 1 ) . further analysis showed that 38.0% ( 133/350 ) of the women were from tribes other than the three major nigerian ethnic groups . further , 39.7% ( 139/350 ) were civil servants , 65.1% ( 228/350 ) were married , 56% ( 199/350 ) were in monogamous marriages , and 4.3% ( 15/350 ) admitted to smoking ( table 2 ) . the reproductive characteristics and sexual behavior of the women are shown in tables 3 and 4 , respectively . the data show that 54.9% ( 192/350 ) of the women had been married for less than 25 years and 56.3% ( 197/350 ) had had less than seven successful pregnancies ( parity ) , ie , reaching at least 28 weeks gestation . in total , 69.1% ( 242/350 ) of the women had children , with 57.1% ( 200/350 ) having 16 children . further analysis of the data showed that 60.3% ( 211/350 ) of the women had never used any form of contraception , and a sizeable proportion ( 3.1% , 11/350 ) had used other forms or non - modern contraception ( table 3 ) . the most frequent age of sexual debut was 19 years ( 26.9% , 94/350 ) while 8.9% ( 31/350 ) had their first child aged 1518 years ( table 4 ) . human papillomavirus igg antibodies were detected in serum samples from 42.9% ( 150/350 ) of women . antibodies to hpv initially increased with age , decreased in women aged 3039 years , and then increased again from age 40 years , peaking in women aged 4549 years ( 50.0% , 57/114 ) . women aged 3539 years had the lowest prevalence ( 31.7% , 13/41 , table 1 ) . the hpv infection rate did not differ significantly according to patient age ( =6.192 , df = 6 , p=0.402 ) . women belonging to tribes other than the three major nigerian ethnic groups had the highest hpv prevalence ( 44.0% , 66/119 ) , while igbo women had the lowest prevalence ( 4.7% , 7/25 ) . there was a marginal association between hpv infection rate and tribal origin ( =7.595 , df =3 , p=0.05 ) . hpv antibodies were detected significantly ( =3.938 , df = 1 , p=0.03 ) more often in christian women ( 47.1% , 99/203 ) than in muslim women ( 36.4% , 51/140 ) . retired women had the highest prevalence ( 75.0% , 9/12 ) and unemployed women had the lowest prevalence ( 35.1% , 20/57 ) ; however , this finding was not statistically significant ( =7.820 , df = 4 , p=0.098 ) . the seroprevalence of hpv was highest among widows ( 53.1% , 17/32 ) and lowest among divorcees ( 35.7% , 5/14 ) , but the difference was not statistically significant ( =1.739 , df =3 , p=0.773 ) . the seroprevalence of hpv igg antibodies was similar between monogamous ( 43.2% , 86/199 ) and polygamous women ( 42.7% , 32/75 ; =0.007 , df = 1 , p=0.935 ; odds ratio 1.023 ; 95% confidence interval 0.5981.749 ) . hpv infection was significantly associated with educational status ( =6.594 , df = 4 , p=0.043 ) . antibodies were detected with the highest prevalence among women with secondary education ( 49.2% , 29/59 ) while those with quranic education had the lowest prevalence ( 21.7% , 5/23 , figure 1 ) . antibodies to hpv were detected with a slightly higher frequency ( 43.1% , 140/325 ) in women who were not smoking compared with those who admitted to smoking ( 40.0% , 10/25 ; =0.090 , df=1 , p=0.765 ) . analysis of the results according to years of marriage showed that the highest frequency of antibodies to hpv was among women who had been married for 1620 years ( 55.3% , 21/38 ) and the lowest frequency in those who had been married for 2630 years ( 28.6% , 6/21 ; =8.734 , df = 7 , p=0.262 ) . no statistically significant relationship was found between seroprevalence of hpv and parity ( =3.700 , df = 4 , p=0.448 ) , but the prevalence was highest amongst women who had 79 pregnancies ( 46.7% , 21/45 ) and lowest in those of parity > 9 ( 25.0% , 3/12 ) . women with children had a higher prevalence of antibodies to hpv ( 44.6% , 108/242 ) than those without children ( 38.9% , 42/108 ) . although the difference observed was not statistically significant ( =1.004 , df = 1 , p=0.316 ) , women with children were 1.3 times more likely to be infected than women without children ( odds ratio 1.267 ; 95% confidence interval 0.7982.011 ) . women who had 79 children had the highest seroprevalence ( 46.9% , 15/32 ) while those with more than nine children had the lowest seroprevalence ( 16% , 1/6 ; =2.851 , df = 4 , p=0.583 ) . the seroprevalence of igg antibodies to hpv was not significantly associated with use of contraceptives ( =1.169 , df = 4 , p=0.883 ) ; women who were using other types of contraception ( eg , not modern or local contraceptives ) had the highest prevalence ( 54.5% , 6/11 ) while those using oral contraception had the lowest prevalence ( 39.4% , 13/33 ) . antibodies to hpv were detected most frequently among women who had their sexual debut aged 2023 years ( 51.1% , 46/90 ) and least often in women who had their sexual debut aged 2426 years ( 37.9% , 25/66 ) . however , this difference was not statistically significant ( =3.114 , df = 5 , p=0.682 ) . the frequency of detection of igg antibodies to hpv according to age at birth of first child was highest among women older than 30 years ( 68.8% , 11/16 ) and lowest among women aged 1518 years ( 35.5% , 11/31 ; =8.163 , df = 5 , p=0.147 ) . the prevalence of hpv was also investigated according to vaginal symptoms reported by the women during the study . women with normal vaginal discharge had the highest frequency of hpv antibodies ( 45.1% , 110/244 ) while women with abnormal discharge ( 37.7% , 40/106 ) had the lowest frequency ( =1.628 ; df = 1 ; p=0.202 ) . further , women with genital rash at the time of the study had the highest seroprevalence ( 61.9% , 13/21 ) , with the lowest seroprevalence seen in women with vaginal itching ( 33.3% , 26/78 ) . the difference in prevalence observed according to vaginal symptoms was almost statistically significant ( =7.461 , df =3 , p=0.059 ) . half of the women ( 50.0% , 9/18 ) with a family history of cervical cancer had igg antibodies to hpv compared with those without a family history of cervical cancer ( 42.5% , 141/332 ; =0.395 , df = 1 , p=0.348 ) . antibodies to hpv tended ( =0.806 ; df = 1 ; p=0.274 ) to be detected with a higher prevalence in women without a history or signs of whim as a genetic disorder ( 43.3% , 146/337 ) than in those with such a history ( 30.8% , 4/13 , table 5 ) . in this study , hpv igg antibodies were detected in serum samples from 150 of 350 women studied , giving a seroprevalence of 42.9% . similar to the 40% reported in southern mozambique.31 the higher seroprevalence in the present study compared with that in a previous report from nigeria may be due to a difference in the study population . another reason could be the high sensitivity of the enzyme - linked immunosorbent assay that permits detection of the hpv antibody in samples with low hpv antibody which would probably otherwise have been scored as negative for hpv . it has been shown that detection of antibodies to hpv can evaluate the overall hpv infection rate in a population more effectively than hpv dna.3 in view of this , and because ahmadu bello university teaching hospital is a referral center where patients from zaria and its environs are seen , and 40% of the women attending the clinic there have been infected with hpv , this percentage represents the actual seroprevalence of hpv in the state . this high seroprevalence could be due to the lifestyle of the local population in the study area , where women are constantly exposed to the virus , by means of early sexual debut , early marriage , multiple sexual partners due to polygamy , and high divorce rates . acquisition of hpv infection has been shown to be strongly related to sexual behavior , and the prevalence of hpv increases with increasing number of sexual partners and early sexual debut.3,30 about one third of the women attending the reproductive health clinic were aged 4549 years , also the group in which hpv was most prevalent . the detection of hpv in older women in this study is consistent with a previous report,32 and could be due to the sexual mode of transmission of the virus which allows reinfection and persistence of the virus for years.33,34 these women might have acquired the virus at an earlier age , considering that the virus has been shown to persist in a significant percentage of women.20,22 antibodies to hpv were also detected in younger women in this study , probably because of early acquisition of infection as a result of early indulgence in sexual activity and early marriage . women in the study area often marry as young as age 15 years , and it has been reported that the age of sexual debut in nigeria is 910 years.35 however , detection of antibodies to hpv , which signifies infection , does not mean eventual development of cervical cancer . this is because most hpv infections in younger women are transient or asymptomatic , often spontaneously regress,36 and have little long - term significance . moreover , the incidence of cervical cancer in women younger than 30 years is very low , and 70% of cases of hpv infection resolve in one year and 90% in 2 years.22,37 however , persistent infection with one or more high - risk types of hpv is an important etiologic factor in the development of cervical intraepithelial neoplasia and progression to cervical cancer.20,22,3840 in addition , virologic , environmental , immunologic , and genetic factors have also been implicated in the development of cervical cancer.41 the prevalence of hpv according to occupational status of the woman was not statistically significant in this study . this means that all women , regardless of occupation were at similar risk of being infected . single and married women in this study had a similar seroprevalence of hpv , indicating a similar rate of sexual activity . the seroprevalence of hpv was higher in widows than in other studies , which reported a higher prevalence among married women.31,39 yet other studies have reported the highest prevalence among single women.33,41 the prevalence of antibodies to hpv was similar between women who married into monogamous homes and those who married into polygamous homes . this study presents a paradoxical picture that is in contrast with the widely held belief that sexual activity in individuals with multiple partners increases the risk of hpv infection . it has been shown that educational level is a socioeconomic factor with an effect on risk of hpv infection . more educated women had the highest infection rate in our study , with the highest prevalence seen in women with secondary school education . this result is consistent with previous research,32,39 but in contrast with a report by marrazzo et al42 who showed that the hpv infection rate decreased with increasing level of education . this observation could be due to early indulgence in sexual activity and lack of awareness of its consequences . however , in our study , hpv was not significantly associated with smoking and was detected with a higher prevalence among nonsmokers . this observation contrasts that of schlecht et al.39 an earlier study reported that daily cigarette smoking had a deleterious effect and contributed to development of low - grade squamous intraepithelial lesions.43 several epidemiologic studies have identified a role of cigarette smoking in invasive cervical cancer.26,27,44 nicotine and other cigarette metabolites have been found in cervical mucus.45 it is also suspected that the relationship between cigarette smoking and low - grade squamous intraepithelial lesions reflects a link between smoking and immune dysregulation,46 and it has been suggested that smoking may induce an impaired antibody response in young women infected with hpv16/18.27 studies have shown that use of oral contraceptives is a risk factor for acquiring hpv , and prolonged use of oral contraceptives is associated with development of squamous intraepithelial lesions.29,47 in this study , even though , hpv antibodies were detected with the highest prevalence among women who used non - modern contraceptives compared with those using modern contraceptives , the difference in prevalence was however , not statistically significant . this result contrasts with previous reports.33,39 other studies have recorded a higher prevalence of hpv infection in women who do not use any form of contraception.32,42 however , a recent study conducted in kano , nigeria , did not find a significant association between use of oral contraceptives and hpv infection.30 number of years of marriage had no significant effect on infection with hpv in this study , implying that hpv can be acquired at any time during marriage . women with high parity had the least prevalence , as previously reported.39 low parity was recently reported to be a significant risk factor for acquisition of hpv in northern nigeria.30 in contrast , okolo et al,25 in southern nigeria , observed that the prevalence of hpv increases with increasing parity . this increase in prevalence of hpv infection with increasing parity has been attributed to increased sexual activity.3,34 in our study , women who had children had a higher prevalence of antibodies to hpv than women with no children . although this difference in prevalence did not reach statistical significance , women who had children were 1.3 times more likely to be infected with hpv than those who did not . this observation is consistent with a report by firnhaber et al.48 it was also observed that antibodies to hpv were detected more often in women who had had multiple pregnancies than in women who had never been pregnant . a similar observation was made by trottier et al,34 who also agreed with the view that parity is a risk factor for acquisition of hpv infection . according to the american cancer society,49 women who have had three or more full - term pregnancies have an increased risk of developing cervical cancer due to having unprotected intercourse to become pregnant , further , studies have pointed to hormonal changes during pregnancy as a possible factor making women more susceptible to hpv infection or development of cancer.49 women in our study who had their sexual debut aged 2023 years had the highest prevalence of hpv antibodies . this agrees with previous reports of an increased hpv among women who indulge in sexual activity at an early age.25,30,32,41 acquisition of hpv infection is strongly associated with sexual behavior . the prevalence of hpv increases with number of sexual partners and earlier sexual debut.3 antibodies to hpv were detected more often in women who had had their first child at age older than 30 years . in 2012 , the american cancer society reported that women who were younger than 17 years when they had their first pregnancy were almost twice as likely to develop cervical cancer later in life than women who did not become pregnant until they were aged 25 years or older.49 abnormal discharge was found in 37.7% of our women , although this finding was not statistically significant . abnormal discharge is the commonest sign observed when there is infection in the reproductive organs.50 discharge is always seen when neoplasia is present and in the advanced stages of hpv infection.51 the majority of the women attending our reproductive health clinic had normal vaginal discharge . abnormal vaginal symptoms included itching , rash , and ulcer , with vaginal itching being the most common ( p=0.059 ) . half of the women with a family history of cervical cancer had antibodies to hpv . cervical cancer may run in some families . if cervical cancer is known in a family to be genetic , the chances of developing the disease are 23 times higher than if no one in the family has it.49 some researchers suspect that in some cases this familial tendency is caused by an inherited condition that makes some women less able to fight off hpv infection than others.49 four of the 13 women who had a history of or some signs of whim as a genetic disorder had antibodies to hpv , but the antibodies were not detected with a statistically significance difference . it has however been reported that the main clinical problem of patients with whim , is unusual susceptibility to infection with hpv51,52 and the most common signs are repeated bacterial infections , neutropenia and extensive infection with hpv leading to dermal and genital warts.52 hpv igg antibodies were detected in 42.9% of the women enrolled in this study , indicating that the women had been infected and that the virus is circulating with a high prevalence in kaduna state . infection with hpv is a major risk factor contributing to the development of cervical intraepithelial neoplasia and invasive cervical carcinoma . women aged 4549 years and those who had their sexual debut when aged 2023 years had the highest seroprevalence of hpv . infection with hpv varied with the women s reproductive characteristics , sexual behavior , and sociodemographic factors but did not reach statistically significant levels except for educational status , tribe , and religion . in view of the high prevalence of antibodies to hpv detected in this study , which denotes a high infection rate in the area , we recommend introduction of a subsidized hpv vaccine in our national immunization schedule in other to prevent the current scourge of cervical cancer in nigeria . two prophylactic vaccines , ie , gardasil ( merck and co , inc . , whitehouse station , nj , usa ) and cervarix ( glaxosmithkline , london , uk ) , that are safe and effective against anogenital hpv,53 are currently available for the prevention of genital hpv infection.1 in view of the results of the present study and those of another recent study showing low knowledge of hpv and its vaccines among nigerian mothers with a high awareness for cervical cancer but little knowledge of its link to hpv,12 we recommend improving knowledge at the population level by education on the mode of transmission of the virus and the risks associated with the virus as a cause of cervical cancer . in addition , cervical cancer screening programs need to be put in place in nigeria , given that cervical cancer has been shown to disproportionately affect african american women , who are nearly twice more likely than european american women to die of the disease.22,54 it is important to screen women for anti - hpv , and if positive , detect the genotypes . those found to be infected with the high - risk genotypes should have papanicolaou screening annually in order to prevent cervical cancer . the majority of the women who took part in this study could not communicate in the english language and used an interpreter , so some of the data might not accurately reflect their sociodemographic , reproductive characteristics , or sexual behavior . in addition , some were not willing to disclose all information concerning their reproductive characteristics and sexual behavior . also , we were not able to determine the presence of hpv dna in hpv igg - positive serum .

backgroundhuman papillomavirus ( hpv ) is the cause of 90%95% of squamous cell cancers . persistent infection with high - risk hpv can lead to development of precancerous lesions of the cervix in 5%10% of infected women , and can progress to invasive cervical cancer 1520 years later . this study was conducted to determine the seroprevalence of hpv immunoglobulin g ( igg ) antibodies among women of reproductive age attending a reproductive health clinic at ahmadu bello university teaching hospital , zaria , nigeria.methodsthe study was descriptive , cross - sectional , and experimental , combining the use of a structured questionnaire and analysis of serum samples obtained from 350 consecutive consenting women . the serum samples were analyzed for igg antibodies to hpv by enzyme - linked immunosorbent assay.resultswe found a seroprevalence of 42.9% ( 150/350 ) for igg antibodies to hpv in these women . women aged 4549 years and those who had their sexual debut aged 2023 years had the highest hpv seroprevalence , ie , 50% ( 57/114 ) and 51.1% ( 46/90 ) , respectively . presence of antibodies varied according to sociodemographic factors , but was significantly associated with educational status , tribe , and religion ( p<0.05 ) . human papillomavirus infection was not significantly associated with the reproductive characteristics and sexual behavior of the women . antibodies to hpv were detected in 50.0% ( 9/18 ) of women with a family history of cervical cancer and in 30.8% ( 4/13 ) of those with a history or signs of whim ( warts , hypogammaglobulinemia , immunodeficiency , myelokathexis ) syndrome as a genetic disorder ( p>0.05).conclusionfurther studies are needed to determine the hpv serotypes and evaluate the risk of natural development of hpv - related malignancies among women in the study area .

pubmed

today s world witnesses rapid innovations and developments in the area of health care ( 1 ) , so that medical knowledge has expanded rapidly over the past fifty years ( 2 ) and the number of scientific papers published has doubled over 10 years ( 3 ) . on the other hand , studies indicate that in the patient s beside , 5 questions per every one hospitalized patient and about 2 questions per every one outpatient are raised for the physicians ( 4 ) . knowledge transfer from research to routine clinical practice is one of the main obstacles preventing from benefiting medical sciences ( 5 ) . some have assessed that more than 40% of the patients do nt receive care in accordance with the best scientific evidences and 20% of patients receive care which is not sufficient or harmful ( 6 ) . generally there is a gap between what physicians do in their routine practice and what they must do ( 7 ) . evidence based medicine ( ebm ) is a new paradigm and philosophy which attempts to direct clinical services and care toward effective , efficient services with minimal complications and errors . in addition , ebm is influential in filling the knowledge gap in helping physicians to find the best evidence for patients care . thus , it is a systematic approach for solving clinical problems which allows integration of available best evidence with clinical experience and preferences and values of the patients ( 8) . this new paradigm may confirm or reject previous acceptable practice methods or may suggest new care methods which are more effective and efficient and less vulnerable ( 9 ) . the main challenge today is transfer and translation of knowledge which help physicians to make the best clinical decisions based on the best evidence in the minimum time ( 10 ) . using the best scientific evidence , sources can be distributed equitably and demands for ineffective treatment would be removed and effective services would be assured ( 11 ) . regarding the facts mentioned it seems that common sources are not effective enough for obtaining information on the current method . as it was noted , evidence based medicine is integration of the best available evidence with clinical experiences and patients preferences , and thus it is a solution for problem of lack of update information for physicians ( 12 ) . despite of these benefits , currently there is several factors affect the use of evidence - based medicine and evidence - based policy ( 13 , 14 ) . regarding the role and importance of paradigm of evidence - based practice and its remarkable impact on the effectiveness and efficiency of clinical services and healthcare , development of an integrated system seems necessary in order to manage dispersed data and ensure using evidences in clinical decision making . hence , in this paper a model is presented in order to produce and implement evidence in the form of a system . in order to achieve higher goals such as promotion of clinical effectiveness , patient safety , effectiveness and improvement of health outcomes , national evidence - based health care system insures the best evidence is generated , stored , used , shared and finally monitored and evaluated in order to provide effective and safe services in a systematic process and according to needs of different stakeholders . national health care system is one of the main subsystems of the health system which is responsible for providing health service and health care . in fact , subsystem of health service delivery is regarded as the most objective and most tangible function of the health system and it reflects impacts of other health system functions . the ultimate goal of health care system in every country is maintaining and improving community health . achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . on the other hand , among these functions , stewardship is considered as the central function of the health system and the trusted and reliance point of health systems in achievement of their goal . this function means determining and enhancing excusive rules and providing directions and strategies for all actors of the system and accepting accountability at highest level which ministry of health and medical education on behalf of the government is responsible for this task . stewardship status and social accountability of the governments necessitates that national health care system to provide the best quality health care and service for the people . clinical effectiveness. in order to achieve clinical effectiveness , the best practice should be realized in the country . the requirement for the best practice is insuring reference to the best local evidence in different parts of the health care . the system which provides reference to the best local evidence and guarantees evidence based decision making ( ebdm ) in practice is called evidence based health care system ( ebhcs ) . in other words , a system which is able to provide local knowledge required by evidence based clinical decision making ( ebcdm ) and guarantee and institutionalize its application among care providers , national health care system is one of the main subsystems of the health system which is responsible for providing health service and health care . in fact , subsystem of health service delivery is regarded as the most objective and most tangible function of the health system and it reflects impacts of other health system functions . the ultimate goal of health care system in every country is maintaining and improving community health . achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . on the other hand , among these functions , stewardship is considered as the central function of the health system and the trusted and reliance point of health systems in achievement of their goal . this function means determining and enhancing excusive rules and providing directions and strategies for all actors of the system and accepting accountability at highest level which ministry of health and medical education on behalf of the government is responsible for this task . stewardship status and social accountability of the governments necessitates that national health care system to provide the best quality health care and service for the people . clinical effectiveness. in order to achieve clinical effectiveness , the best practice should be realized in the country . the requirement for the best practice is insuring reference to the best local evidence in different parts of the health care . the system which provides reference to the best local evidence and guarantees evidence based decision making ( ebdm ) in practice is called evidence based health care system ( ebhcs ) . in other words , a system which is able to provide local knowledge required by evidence based clinical decision making ( ebcdm ) and guarantee and institutionalize its application among care providers , this paper is a study of comparative type which has been written in three stages : investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard in terms of service delivery and medical educationinvestigation and recommendation of strategies and methods which make model implementation more feasible in the country . investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard in terms of service delivery and medical education investigation and recommendation of strategies and methods which make model implementation more feasible in the country . in order to investigate status of evidence based practice system , following related literature review , some developed countries including england , usa , canada , australia , and scotland were selected which were leading countries in this regard . using search engines of google scholar , pubmed and implementation science , key terms of evidence based medicine , evidence based practice and evidence based national system were searched accompanied by the name of the countries . used sources included studies related to introduction and analysis of data bases and system associated to responsible agencies in respective countries . evidence based practice were studied in the respective countries in terms of dimensions of organization and stewardship , knowledge products , distribution and support of evidence application and the way of performance monitoring . results of these studies are given in table 2 . in order to investigate and analyze the status in the country , 12 of the scholars , instructors and experts related to the subject were selected using purposeful sampling and focus group discussion was used in order to analyze and classify . strengths and weak points and barriers to evidence based practice in the same areas ( organization and stewardship , used knowledge products , distribution and support of evidence application , and performance monitoring ) as well as knowledge of the physicians on the evidence based practice and motives of the physicians for change in practice system were evaluated and analyzed . finally , focus group discussion was used in order to investigate strategies which are applicable and feasible in iran , and strategies were studied in terms of the following aspects : needed structureprocessesknowledge products and outputsmotivationneeded support regulationperformance monitoring knowledge products and outputs needed support regulation performance monitoring delphi technique was used so as to finalizing the country s status and results of the focus group discussion were sent to 46 experts and it was finalized following twice modification . evidence based health care system in england , usa , canada , australia , new zealand , and scotland were studied in terms of the organization and stewardship , knowledge products , distribution and support of evidence application and performance monitoring . according to studies , insuring an evidence based practice culture in the country requires a system called national evidence - based health care system which consists of three subsystems as follows : national system of clinical knowledge management , national evidence - based practice systemintegrated national network of clinical effectiveness . national system of clinical knowledge management , national evidence - based practice system integrated national network of clinical effectiveness . national clinical knowledge management system ( nckms ) includes activities , actions , processes and structures which promote , facilitate and guarantee clinical knowledge management in the country ( fig . 1 ) . this part of the national evidence based health care system attempts to provide the best evidence for evidence based decision making through analysis , synthesis and localization of the fragmented raw data , if necessary . when subject of insuring clinical knowledge management through frameworks of the national system of clinical knowledge management is raised , it means insuring monitoring and providing possibility for regular control of the new data which are published in scattered manner . the outcome of processes and actions carried out in the national system of the clinical knowledge management regarding dominant social , economic , cultural circumstances and values of the country , leads to products which are called knowledge products . these products which are considered as the main constituents of the system in units of the clinical knowledge management , are produced based on two approaches : reactive and proactive . reactive approach is the approach in which reaction is given regarding current problems and difficulties in the health system of the country and by developing and distributing appropriate knowledge products it is attempted to solve the problems . proactive or prospective approach is the approach in which clinical knowledge management is done in parallel to knowledge push in the world . in other words , monitoring of the new information is done with a proactive approach regarding national priorities in different clinical levels in order to match knowledge resulting from this information with national needs of the country . clinical knowledge management units , multi - disciplinary teams of clinical knowledge management , and clinical knowledge managers are considered as the most important pillars of the national system of clinical knowledge management . in addition , this system provides structure , frameworks , incentives and requirements so as to achieve its goals so that production of knowledge products needed in the country are facilitated , promoted and insured . knowledge products produced in this system include pay knowledge , health technology reporting , clinical guidelines , and systematic reviews . in case knowledge products possess considerable value added , they are considered in the national committee on clinical policy making and are imparted as a clinical policy. in this case , its observation would be mandatory for all provides on public sector and non - governmental sector and licensing system at individual level and validating system at organizational level support such policies . national evidence based practice system ( nebps ) includes actions , activities , processes and structures which promote , facilitate and guarantee usage of clinical policies or knowledge products produced at national level ( table 3 ) . as it can be seen from table 3 , national evidence based practice system is composed of various components . in this system , evidence based provides in evidence based health centers that their structure , processes and infrastructures facilitate , promote and guarantee evidence based decision making , are seeking for establishment of such a system that institutionalize delivery of the most effective services to patients through production and application the local knowledge products . evidence based care providers make reference to knowledge products produced by national system of clinical knowledge management and local knowledge products produced by them in order to make decision . providers in this system have a reactive approach to knowledge production since no clinical decision - making body would be able to acquire all the needed knowledge and predict all clinical questions needing answer . thus , both evidence based care providers and clinical knowledge managers produce evidence , but there is essential difference in features and characteristics of the process of evidence production by these two groups ( table 4 ) . evidence demonstrated that less than 20% of the clinical actions and decisions of these providers are based on the best acceptable evidence ; while evidence based providers have the necessary skill for achieving lifelong learning and thus , they are constantly trying to enhance their personal practice and increase effectiveness and safety of their interventions . hence , evidence based care providers are divided into three levels based on their performance in production and application of the knowledge products , that is , care providers of level one , two and three . in this system , in addition to classification of care providers , service provider centers are also classified based on their evidence based units performance . this system is part of national integrated clinical effectiveness network ( nicen ) which facilitates evidence based practice in the country . in this system , there such components as electronic evidence based performance portfolio , certificate and characteristics of the evidence based centers and departments , educational service on principles of evidence based decision making and evidence based practice . national evidence based practice system ( nebps ) includes actions , activities , processes and structures which promote , facilitate and guarantee usage of clinical policies or knowledge products produced at national level ( table 3 ) . as it can be seen from table 3 , national evidence based practice system is composed of various components . in this system , evidence based provides in evidence based health centers that their structure , processes and infrastructures facilitate , promote and guarantee evidence based decision making , are seeking for establishment of such a system that institutionalize delivery of the most effective services to patients through production and application the local knowledge products . evidence based care providers make reference to knowledge products produced by national system of clinical knowledge management and local knowledge products produced by them in order to make decision . providers in this system have a reactive approach to knowledge production since no clinical decision - making body would be able to acquire all the needed knowledge and predict all clinical questions needing answer . thus , both evidence based care providers and clinical knowledge managers produce evidence , but there is essential difference in features and characteristics of the process of evidence production by these two groups ( table 4 ) . evidence demonstrated that less than 20% of the clinical actions and decisions of these providers are based on the best acceptable evidence ; while evidence based providers have the necessary skill for achieving lifelong learning and thus , they are constantly trying to enhance their personal practice and increase effectiveness and safety of their interventions . traditional care providers have the least self - learning and therefore effort . hence , evidence based care providers are divided into three levels based on their performance in production and application of the knowledge products , that is , care providers of level one , two and three . in this system , in addition to classification of care providers , service provider centers are also classified based on their evidence based units performance . this system is part of national integrated clinical effectiveness network ( nicen ) which facilitates evidence based practice in the country . in this system , there such components as electronic evidence based performance portfolio , certificate and characteristics of the evidence based centers and departments , educational service on principles of evidence based decision making and evidence based practice . preserving and promoting service quality and health care is one of the major tasks of the governments . trustee s role and social responsibility of the ministry of health and medical education as a custodian of the country s health system bounds it to provide infrastructures , processes and frameworks so that national service delivery system offer services and health care with the highest quality(20 ) . clinical effectiveness is one of quality aspects , requirement of which is access to the best evidence . thus evidence base health care system ensures that the best evidence are produced , stored , disseminated , used and shared for providing effective and safe services through a systematic process according to needs of different stakeholders and finally it is monitored and evaluated . it can not be achieved without active participation of ministry of health and medical education and other involved organizations in health policy making . investigation of consideration process and emphasis on the health quality and care in uk and usa indicates that without imposing macro interventions at national level , quality improvement and especially clinical effectiveness promotion as the core of quality would not be possible(20 ) . for example , report by institute of medicine ( iom ) in usa in 2001 mentioned several basic interventions for quality promotion all of which suggest involvement of health system stewardship in this process and following cases can be mentioned : prohibition of approval of drugs with name , packaging , color and appearance similar to it , change in payment system , search management and access to the best evidence , changes in the educational system , mandatory use of computerized physician order entry ( cpoe)- and use of reminders(21 ) . lack of legal requirement to rely on the best available evidence , insufficient skills in the use of evidence in daily practice , evidence - based decision - making , inability to produce documentary evidence , lack of financial and non - financial incentives to produce evidence according to local conditions and similar experiences in countries like australia(22 ) in the nhmrc program(22 ) , uk in nice program(23 ) , scotland in sign program(24 ) , usa in ahrq program(25 ) , canada in cma program(26 ) and new zealand in nzgg program(27 ) demonstrate that only presence of national plan and macro interventions can guarantee evidence production and application . implementation of the evidence based practice requires a system and this system needs a set of micro and macro strategies enabling facilitations , promotion and guaranteeing production and application of the clinical knowledge in the country . thus , the government should adopt policies and interventions for changing behavior in three levels of promotional , facilitation and guarantying . policies and interventions should influence on both at individual and organization level and certainly implementation of such interventions needs external and internal cooperation of the various stakeholders in the country . ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc ) have been completely observed by the authors .

background : regarding the role and importance of paradigm of evidence based practice and its remarkable impact on the effectiveness and efficiency of clinical services and healthcare , development of an integrated system seems necessary in order to manage dispersed data and ensure using evidence in clinical decision making , thus the aim of this study was designing a model for implementing national system of evidence based health care in iran.methodos:this paper is a study of comparative type which has been written in three stages : investigation of structure and process of evidence based practice in selected countries , investigation and analysis of current status in iran in this regard and recommendation of strategies which make model implementation feasible in the country . such methods as review of literature , focus group discussion and delphi technique were used for investigation.results:according to studies , insuring an evidence based practice culture in the country requires a system called national evidence based health care system which consists of three subsystems including national system of clinical knowledge management , national evidence - based practice system and integrated national network of clinical effectiveness.conclusion:the ultimate goal of health care system in every country is maintaining and improving community health . achievement of this goal depends on effectiveness of delivered services and consistency of the services with national and local priorities . in order to achieve clinical effectiveness , the best practice should be realized in the country , implementation of which requires a set of macro and micro strategies enabling facilitation , promotion or guaranteeing clinical knowledge application in the country .

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human metapneumovirus ( hmpv ) is a negative single - stranded rna virus of the family paramyxoviridae and closely related to the avian metapneumovirus ( ampv ) subgroup c [ 1 , 2 ] . hmpv is an important aetiological agent of respiratory tract infection ( rti ) in infants , or senior and immunocompromised individuals . this infection caused different symptoms ranging from influenza like syndromes ( i.e. , fever , cough , and rhinorrhea ) to severe lower respiratory tract infection . previous studies have shown that many children exposed to this virus and also easily to be reinfected as common [ 35 ] . therefore , hmpv is becoming as a major concern in child respiratory tract viral infection . genome sequencing and comparative analysis provides us a useful approach to analyze the pathogenicity of organisms . moreover , this analysis might also provide us an approach to understand its evolution history and cell - host interaction . as previously reported hmpv genome is approximately 13 kb in length , and the gene composition from 3 terminal to 5 terminal is n - p - m - f - m2 - 1/m2 - 2-sh - g - l [ 6 , 7 ] . comparative analysis suggests that its genomic organization is similar to human respiratory syncytial virus ( hrsv ) , which just lacks 2 nonstructural genes , ns1 and ns2 . moreover , hmpv has been demonstrated the existence with two main genetic lineages termed as subtype a and b , which also containing within them the subgroups a1/a2 and b1/b2 , respectively . the genetic diversity analysis shows the a2 sublineage exhibits the greatest diversity among all the sublineages of hmpv . as we all know , there are differences in the frequency of occurrence on synonymous codons in coding dna , which termed as synonymous codon usage bias . briefly , there are 64 different codons ( 61 codons encoding for amino acids plus 3 stop codons ) in each organism , but only 20 different translated amino acids . these alternative codons for the same amino acids are termed as synonymous codons . in general , codon usage variation may be the product of natural selection and/or mutation pressure for accurate and efficient translation in various organisms [ 810 ] . synonymous codon usage bias on virus can provide us with a better understanding on the evolution profile , gene expression , and virus - host interaction [ 1114 ] . however , there is still lacking about codon usage pattern of hmpv genome and its major influence factors . herein , we firstly performed the comparative analysis of synonymous codon usage in hmpv genomes and analyzed their influencing factors . this study will provide a new insight to understand the pathogenicity and its evolution history of hmpv . in this study , a total 17 complete hmpv genomes which representing two genotypes were retrieved from ncbi ( http://www.ncbi.nlm.nih.gov/ ) until december , 2011 . the serial number ( sn ) , genbank number , genotype , and other information moreover , 10 ampv genomes sequences were retrieved from ncbi database as reference frame ( in supplemental table 1 supplementary material available online at doi:10.1155/2012/460837 ) . to investigate the characteristics of synonymous codon usage , relative synonymous codon usage ( rscu ) values of each complete coding region in 17 hmpv genomes and the rscu value of each codon for their amino acid was calculated as previously described . a codon with an rscu value of more than 1.0 has a positive codon usage bias , while a value of less than 1.0 has a negative codon usage bias . when the codon with rscu values close to 1.0 , it means that this codon is chosen equally and randomly . the codon usage data of human cell and bird cell were obtained from the codon usage database online ( http://www.kazusa.or.jp/codon/ ) . the effective number of codons ( enc ) is used to measure deviation from expected random codon usage of hmpv and is independent of hypotheses involving natural selection . if only one codon is used for each amino acid , this value would be 20 , while all of codons are used equally , it will be 61 . moreover , the index of gc3s was used to calculate the fraction of the nucleotides g + c content at the synonymous third codon position ( excluding aug [ met ] , ugg [ trp ] , and the termination codons ) . multivariate statistical analysis can be used to explore the relationships between variables and samples . in this study , correspondence analysis was used to investigate the major trend in codon usage variation among genomes . in this study , the complete coding region of all 17 hmpv genomes was represented as a 59 dimensional vector , and each dimension corresponds to the rscu value of one sense codon ( excluding met , trp , and the termination codons ) . correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . in order to investigate the synonymous codon usage of hmpv , we calculated various rscu values of various codons from 17 different strains , including different genotypes . as shown in table 3 , the preferred codons in hmpv are gca , aga , aau , gau , ugu , caa , gaa , gga , cau , cau , auu , uua , aaa , uuu , cca , uca , agu , aca , uau , guu . interestingly , all preferred codons in hmpv genomes are ended with a / u , while none of them is ended with g / c . this result suggests that hmpv genome has a great synonymous codon usage bias , and this phenomenon might highly associate with the nucleotide composition in its genomes . as shown in table 2 , the g + c content of hmpv genome is 36.91% , which shares similar extent with another rna virus . there are over 68.57% codons are ended with a / u among hmpv genomes , and 40.87% codons are a3 end , and 27.7% codons are u3 end . this high abundance of a / u nucleotides is consistent with all preferred codons , which are ended with a / u . this phenomenon reflects that nucleotide composition is the main force to affect the codon usage bias in hmpv genome . moreover , as shown in table 2 , the enc values among hmpv genomes show a range from 45.127 to 48.28 , and its average value of 45.785 and sd value of 0.8458 . the stable enc values suggest that their genomic compositions are much conserved among hmpv genomes . natural selection and mutation pressure have been considered to be two key factors which have effect on codon usage patterns of organisms . in order to investigate whether mutation pressure or natural selection as a determinative factor for codon usage mutation in hmpv , we calculated correlation relation between a% , u% , g% , c% , gc% and a3% , u3% , g3% , c3% , gc3% . as shown in table 4 , there exhibits a very complex correlation map observed in nucleotide compositions . in detail , u3% has a significant positive correlation with u% ( r = 0.7821 , p < 0.01 ) , while shared negative correlation with c% ( r = 0.7153 , p < 0.01 ) and gc% ( r = 0.7474 , p < 0.01 ) . c3% has positive correlation with c% ( r = 0.8331 , p < 0.01 ) and gc% ( r = 0.7880 , p < 0.01 ) , and negative correlation with a% ( r = 0.6199 , p < 0.01 ) and g% ( r = 0.5846 , p < 0.05 ) . gc3% has significant positive correlation with c% ( r = 0.7178 , p < 0.01 ) and gc% ( r = 0.8052 , p < 0.01 ) , but has negative correlation with u% ( r = 0.7342 , p < 0.01 ) . interestingly , the gc3% shows positive correlation with c% , while shows negative correlation with g% . we calculated the gc3 skew by using formula as cg3 skew = ( c3 g3)/(c3 + g3 ) . the gc3 skew of hmpv range s from 0.371 to 0.592 , which reveals that gc composition involved in the codon usage bias . these data suggest that the nucleotide constraint might play an important role in influencing synonymous codon usage bias . furthermore , the correlation analysis of first two principle axes ( f1 and f2 ) in hmpv and its nucleotide contents were performed ( table 5 ) . apparently , the first principle axis ( f1 ) has a significantly negative correlation with u3% , and negative correlation with gc3% . this result suggests that nucleotide u3% and gc3% are the major factor influencing the synonymous codon usage pattern in hmpv genome . moreover , we observed the second principle axis ( f2 ) shared a significant positive correlation with g3% , and negative correlation with c3% and gc3% . therefore , compositional constraint is a major factor which is involved in shaping the pattern of synonymous codon usage bias in hmpv genome . to investigate the effect of different hpmv genotypes on synonymous codon usage , we analyzed the codon usage bias of different genotypes with correspondence analysis . from the correspondence analysis , the first dimension variable f1 and the second dimension variable f2 can reflect 43.27% and 33.38% of total mutation , respectively . as the plot of correspondence analysis shown ( figure 1 ) , each genotype is mainly separated and clustered into two clades . however , the sublineage of each genotype did not exhibit any significant difference among them . but this might be due to the limited number of hpmv genomes available in current study . therefore , the phylogenetic distant might effect on the variation of synonymous codon usage in hmpv , and this difference might reflect on their biological effect , such as viral replication , virulence , and so forth . from the enc - gc3% plot analysis ( figure 2 ) , the plots of each hmpv genomes are all under the expected curve , none of them shows above the curve . moreover , there are still some other factors that can effect on the codon usage bias of hmpv . in the current study as shown in table 3 , the pattern of synonymous codon usage in hmpv shows a complementary profile , which shows in human cell . in detail , hmpv and human host cell shared only 1 preferred codon ( aga ) , which encoded for arginine , while there are 17 different preferred codons between them . as a reference frame , ampvs were enrolled in this study , and it also shows a complementary pattern with its host , bird cell . the comparative analysis among hmpv and human host cell , hmpv and ampv host bird cell , and hmpv and ampv were analyzed . to compare the complementary ability of hmpv with bird cell , there are more overlays ( 6 preferred codons overlay ) than hmpv with its human host cell ( only 1 preferred codons overlay ) . this result shows human cell has much more complementary pattern with hmpv than bird cells . this might be more suitable for hmpv survive and persist infect in human host environment . this result also suggests that host factor plays an important role in codon usage bias in hmpv . this complementary trend will benefit for virus replication instead of competitive with its host and it might help us to understand the mechanism of hmpv persistent infection . interestingly , hmpvs are shares with more than 15 preferred codons to ampv , which might be due to a close phylogenetic distance . synonymous codon usage analysis can reveal much about virus genome . to understand the extent and causes of codon usage bias is essential for studying the viral evolution , particularly the interaction between viruses and host immune response . in this study as we know , the variation and evolution of virus generally happened in the changes of nucleotide composition . therefore , the nucleotide composition bias is the main force to influence the synonymous codon usage patterns . in this study first of all , in hmpv , all preferred codons are ended in a / u nucleotide , which occupied the majority of nucleotide composition in hmpv genome . this phenomenon confirmed that nucleotide composition was the main force in shaping the pattern of codon usage . secondly , enc was used to quantify the codon usage bias , which is one of the best overall estimators of absolute synonymous codon usage bias . in this study , we observed enc of these genomes fluctuated from 45.13 to 48.28 with a mean 45.78 0.85 . this enc value of hmpv is consistent with other previously reported rna virus in the same family paramyxoviridae , ranging from 43.8 to 55.1 , that is , measles virus 55.1 , mumps virus 54.3 , parainfluenza-3 virus 43.8 , and respiratory syncytial virus 44.3 [ 8 , 24 , 25 ] . moreover , the enc of hmpv is more close to respiratory infection agents , rsv and parainfluenza-3 , which reveals that the similar extent of codon usage bias among viruses might have similar infection syndrome . this observation helps us to address an interesting assumption that synonymous codon usage bias of virus might associate with its pathogenicity . mutation pressure and natural selection are generally treated as the main factors that account for codon usage bias in different organisms . enc - gc plot was considered as a part of the general strategy to investigate patterns of synonymous codon usage [ 9 , 18 , 26 ] . herein , all the plots are laid below the expected curve , suggesting that codon usage bias in all these 17 hmpv genomes was principally influenced by mutation bias , which consistent with that mutation pressure rather than natural selection is the most important determinant of the codon usage in human rna virus [ 8 , 2731 ] . this observation can be explained as the mutation rates in rna viruses much higher than those in dna viruses . mutation pressure is the main force in shaping synonymous codon usage bias of rna virus . however , based on correspondence analysis , we observed an interesting phenomenon that codon usage bias in hmpv showed distinct differences among different phylogenetic types . it might suggest that codon usage bias plays an important role in hmpv evolution history . this similar phenomenon also observed in several other viruses , this might reflect that phylogenetic difference is a common influencing factor in shaping codon usage bias [ 25 , 27 , 3034 ] . therefore , we hypothesize that the difference of codon usage bias might influence its virulence in different genotypes . in this study , we also observed that hmpv showed a complementary trend with human cells by comparing the codon usage . this complementary will be benefit for the survive of virus , which can keep replication by using the nonpreferred codons in the host cell without competition , and this could be one of the mechanisms of virus persistent infection in the human environment . moreover , this pattern might also be caused by the longitude selection and evolution between human hosts with virus . therefore , this characteristic is important for hmpv keeping balanced with their host on the codon usage side , and also for understanding the cell - host interaction and viral evolution . in summary , we firstly reported the synonymous codon usage pattern in hmpv genomes and revealed that mutation pressure is the main force in shaping its codon usage bias . phylogenetic difference and host factors are also discussed , and this information can provide better understanding on the molecular evolution and its pathogenicity of hmpv .

human metapneumovirus ( hmpv ) is an important agent of acute respiratory tract infection in children , while its pathogenicity and molecular evolution are lacking . herein , we firstly report the synonymous codon usage patterns of hmpv genome . the relative synonymous codon usage ( rscu ) values , effective number of codon ( enc ) values , nucleotide contents , and correlation analysis were performed among 17 available whole genome of hmpv , including different genotypes . all preferred codons in hmpv are ended with a / u nucleotide and exhibited a great association with its high proportion of these two nucleotides in their genomes . mutation pressure rather than natural selection is the main influence factor that determines the bias of synonymous codon usage in hmpv . the complementary pattern of codon usage bias between hmpv and human cell was observed , and this phenomenon suggests that host cells might be also act as an important factor to affect the codon usage bias . moreover , the codon usage biases in each hmpv genotypes are separated into different clades , which suggest that phylogenetic distance might involve in codon usage bias formation as well . these analyses of synonymous codon usage bias in hmpv provide more information for better understanding its evolution and pathogenicity .

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as more and more genomes are automatically sequenced , comprehensive protein annotation is a needed step after gene identification . even in good annotated genomes ( human , mouse ) about 30% of all proteins are not functionally identified ( 13 ) , and thus often a similarity search will not be sufficient . here , we present a suite of protein tasks , protsweep , domainsweep and 2dsweep , which perform analysis from sequence similarity to small domains and structural elements . this includes similarity searches against protein sequence databases and specialized motif collections , prediction of secondary structural elements , attributing each sequence to known super - families , protein localization prediction , physicochemical protein characteristics and domain functional assignation . our strategy for assigning relevant functional roles is based on the joint use of both global ( homology similarity ) and local ( domain and motif ) sequence similarities ( 4 ) . several query sequences can be uploaded by the usual copy & paste procedure into the input box using fasta format . if more than one sequence is to be queried , a multiple fasta file can be used . then the user will be redirected to an application page , and the runbutton can start the task . additionally , there is a link to an online help , indicated with a ? , with the following topics : short description , programs employed , algorithm , output , additional options and acknowledgments . results can be received by selecting the tab go to results page. the results are provided as html for visual inspection or can be downloaded as xml for storage in private databases . in case of error when clicking on the application name in the results manager page , a log - file is displayed where more human readable error messages can be found . standard protein databases used by the tasks like uniprot / swissprot , uniprot / trembl and refseq are automatically updated whenever new versions become available . concerning ensembl , the situation is more complex . due to possible inconsistencies between the different ensembl api versions , which are used in the tasks , it is not possible to automatically update the ensembl data and this needs to be done by hand . the different databases and the way they were used in these pipelines are described in table 1 . table 1databases used in the different pipelines , and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep ( non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage = 80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines , and the programs and parameters used to search them standard protein databases used by the tasks like uniprot / swissprot , uniprot / trembl and refseq are automatically updated whenever new versions become available . concerning ensembl , the situation is more complex . due to possible inconsistencies between the different ensembl api versions , which are used in the tasks , it is not possible to automatically update the ensembl data and this needs to be done by hand . the different databases and the way they were used in these pipelines are described in table 1 . table 1databases used in the different pipelines , and the programs and parameters used to search themserverdatabaseslinksprogramparametersprotsweepuniprot / swissprothttp://www.ebi.ac.uk / swissprotblastp - nofilter -exp = 10.0uniprot / tremblhttp://www.ncbi.nlm.nih.gov / refseq / blastp - nofilter -exp = 10.0refseqftp://ftp.ebi.ac.uk / pub / databases / trembl / sptrembl / blastp - nofilter -exp = 10.0ensemblhttp://www.ensembl.org / blastp - nofilter -exp = 10.02dsweepdssphttp://www.sander.ebi.ac.uk / dssp / blastp - exp = 0.001 -extension = 10 - nogappedalignnrpep ( non - redundant ncbi protein database)ftp://ftp.ncbi.nih.gov / blast / db / fastapsiblast - b20000 -a5 -j2 -e0.001uniprot / swissprot + uniprot / trembl + updateshttp://www.expasy.ch / sprot / sprot - top.html http://www.ebi.ac.uk/swissprotmsfgenerator-exp = 0.001 -overlap = 75 -customrange = 5 custompercentage = 80,60,50,45,40,35domainsweepprositeftp://ftp.expasy.ch / databases / prositemotifs & pfscandefaultpfamhmmhttp://www.sanger.ac.uk / software / pfam / hmmscan - lib = pfam.hmm -dprintsftp.bioinf.man.ac.ukhmmscan - lib = prints.hmm -dsmarthttp://smart.embl - heidelberg.de / hmmscan - lib = smart.hmm -dtigrfamshttp://www.tigr.org / tigrfams / index.shtml / hmmscan - lib = tigrfams.hmm -dscophttp://scop.mrc - lmb.cam.ac.uk / scopscopscandefaultblocksftp://ftp.ncbi.nih.gov / repository / blocks / unixblockssearcher - cutoff = 0.01 dinterprohttp://www.ebi.ac.uk / interpro / srs queriesprodomhttp://www.toulouse.inra.fr / prodom.htmlprodomblastdefault databases used in the different pipelines , and the programs and parameters used to search them protsweep is an approach to the functional characterization of unknown proteins based on a cascade of similarity searches . it is well known that protein databases do not completely overlap and differ in their annotation quality ( 5 ) . this task takes into account the significant differences among databases ( supplementary table 1 ) to improve the quality of the protein characterization . it selects the order in which the databases have to be searched and combines the annotation found depending on the results . protsweep classifies proteins into the following categories : identical , homolog , similar , weakly similar and putative proteins . the query protein starts the blast ( 6 ) cascade against swissprot ( 7 ) first ( figure 2 ) . we do take into account three parameters to classify the blast hits : ( i ) percentage of identity , ( ii ) spercent. the two last parameters are related to the length of the total alignment , being qpercent the percentage of the query sequence length covered in the alignment with the database hit and spercent the percentage of the hit ( subject ) sequence length covered by the alignment ( figure 2 ) . depending on the classification of the blast hits according to these parameters and the hit protein annotation if the hit has 100% qpercent and spercent and more than 98% identity , it is considered an identical protein and the swissprot i d will be searched in ensembl ( 8) . if it is successful , all information from both databases will be combined ( supplementary table 2 ) and stored in the xml output . if the i d can not be found in ensembl then a blast search is performed with the query protein against ensembl . the best ensembl hit is selected and compared against the swissprot hit using the smith if the identity between sequences is greater than 98% , then the information from both sources and the blast alignment will be added to the final output , if the identity is less , only swissprot annotation and the alignments will be added to the xml . if the qpercent and spercent is between 80% and 98% and the identity is between 85% and 98% , the hit is classified as homologous and follows the same strategy with ensembl as already described ( figure 2 ) . in case , spercent are greater than 85% , then the blast cascade continues with sptrembl and refseqprot . in the case that no identical or homologous hits can be found in any of the databases , the best similar hit among the three databases is selected and classified as similar , weakly similar or putative ( figure 2 ) . if the protein is characterized , information concerning the coding gene , about the splicing variants and orthologous genes is also provided . depending on the degree of homology , protein function , transcript of origin , genomic localization , and go annotation or partial similarities will also be shown . hypothetical proteins will only be presented in the result when no other information about identical or homologous proteins can be found in any of the databases ( supplementary figure 1 ) . the web output of protsweep ( supplementary figure 1 ) is divided in five sections : ( i ) general information , ( ii ) identified protein and transcripts , ( iii ) features and functions , ( iv ) genomic localisation and ( v ) homology to other organisms / genes . the information provided in each of these sections is provided in figure 2 and supplementary table 2 . the user has immediate access to all complete application outputs and database entries via hyperlinks . at the bottom of the html output there is a link to the explanatory legend as well as to the xml output containing all the generated information . domainsweep identifies the domain architecture within a protein sequence and therefore aids in finding correct functional assignments for uncharacterized protein sequences ( figure 3 ) . it employs different database search methods to scan a number of protein / domain family databases . among these models , in increasing complexity , are : prodom ( 10 ) , automatically generated protein family consensus sequences , prosite ( 11 ) regular - expression patterns , blocks ( 12 ) , ungapped position - specific scoring matrices of sequence segments , prints ( 13 ) sequence motifs , prosite profiles ( 7 ) , gapped position - specific scoring matrices and hidden markov models like pfam ( 14 ) , smart ( 15 ) , tigrfams ( 16 ) and scop ( 17 ) . each database covers a slightly different , but overlapping set of protein families / domains . each model has its own diagnostic strengths and weaknesses and for each of these protein / domain family databases used we have established different thresholds . for example , in the case of the database pfam - a , we compare the input sequence against the hidden markov model profile of each pfam protein family . in principle , it is possible to decide the significance of a match upon its e - value . however , there are a few complications such as that there is no analytical results available for accurately determining e - values for gapped alignments , especially profile hmm alignments . we use as threshold the trusted cut - off value ( tc ) existing for each pfam family . this value is the lowest score for sequences included in the family ( e.g. in the full alignment ) . therefore , we consider a hit very significant if scores better than the trusted cut - off and at the same time has a significant e - value . in the case of scop , we use the scop filtering mechanism to look for consistency in the hmmscan output , and filtering out inconsistent hits . in the case of smart we use only the e - value . for each of the protein / domain databases used afterwards domainsweep takes all true positive hits of all individual database searches for further data interpretation . domain hits are listed as significant : if two or more hits belong to the same interpro family . the task compares all true positive hits of the different protein family databases grouping together those hits , which are members of the same interpro family / domain.if the motif shows the same order as described in prints or blocks . both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order . the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry . only hits in correct order the task compares all true positive hits of the different protein family databases grouping together those hits , which are members of the same interpro family / domain . if the motif shows the same order as described in prints or blocks . both databases characterize a protein family with a group of highly conserved motifs / segments in a well - defined order . the task compares the order of the identified true positive hits with the order described in the corresponding prints or blocks entry it is clear that any automatically produced sequence analysis implies a reasonable compromise between sensitivity and selectivity , and that no ideal recognition threshold exists that would allow for perfect separation of true and false similarities . our thresholds tend to be rather conservative and stringent and thus the possibility of extending false positives is very limited . the output in the web consists of two groups of graphs , those corresponding to the significant and putative hits , and one table output containing all reported protein domains ( supplementary figure 2 ) . putative hit a cartoon of the sequence with the domain corresponding to the match , the hit i d , description , begin , end and gene ontology ( go ) annotation . the user has immediate access to all complete application outputs and database entries ( via hyperlinks ) by clicking on the corresponding part of the picture . at the bottom of each graph the table output contains all hits , ids , descriptions and links to the original output . the xml output containing all the generated information is available via hyperlink at the bottom of the task output . 2dsweep identifies the structural domains in the protein and therefore aids in finding structural elements . it reports on predictions for alpha - helix , beta - strand , coiled - coil and helix - turn - helix motifs , transmembrane regions , signal sequences , hydrophobicity , antigenicity , protease cleavage sites and more . when predicting the secondary structure of a protein , it is useful to exploit the features of several available prediction algorithms rather than to rely on a single program . unfortunately , combining prediction methods on a large scale is complicated by the fact that prediction programs have very different input requirements and output formats . some of them perform much better when they have a multiple sequence alignment covering different degrees of similarity as input instead of a single sequence . we have developed msfgenerator , a program , which creates a multiple sequence alignment for a single protein sequence according to user , defined rules ( supplementary data msf ) . it performs a blast search against a non - redundant protein database following different strategies that will generate different kind of alignments ( supplementary data msf , figure 4 ) . the output of msfgenerator is an alignment in msf format ( multiple sequence file ) . the generated msf will be used as input for four different structure prediction programs : psipred ( 18 ) , jnet ( 19 ) , prof ( 20 ) , and dsc ( 21 ) . psipred is a two - stage neural network that bases its prediction on position specific scoring matrices , jnet is a neural network method that works by utilizing an alignment as input , alongside psiblast ( 22 ) and hmm profiles . dsc is based on decomposing secondary structure prediction into basic concepts and then uses simple and linear statistical methods to combine them . since dsc is known to perform worse than the other prediction methods employed in 2dsweep , the usage of dsc is optional . figure 4.2dsweep flowchart . as a second concept , 2dsweep searches for dssp ( definition of secondary structure of the protein , ( 23 ) annotation for the input protein . found it extracts secondary structure elements ( if any ) from the structure definition of the dssp database . if there is more than one element covering the same sequence region , 2dsweep uses a simple majority vote to determine the structure at each position . the result of this procedure is shown together with the prediction of the different secondary structure prediction tools . first , the distribution of small , charged and hydrophilic amino acids are shown and probable antigenic regions are indicated . furthermore , the task searches for transmembrane helices and intervening loop regions using four different methods : tmhmm ( 24 ) , das ( 25 ) , tmap ( 26 ) and tmpred . in eukaryotic protein sequences finally , information is given about molecular weight , isoelectric point , the distribution of protease cleavage - sites , and the possible sub - cellular localization of the protein . the web output of 2dsweep ( supplementary figure 3 ) is divided in five sections : ( i ) general information , ( ii ) secondary structure , ( iii ) features and ( iv ) cleavage sites . the information provided in each of these sections is shown in figure 4 and supplementary table 3 . the complete results can be viewed by clicking on the corresponding part of the picture . at the bottom of each graph there is a link to the corresponding explanatory legend . as in the other tasks the xml output containing all the generated information is available via a hyperlink at the bottom of the task output these servers have been implemented using the w3h task framework ( 27 ) , which allows the execution of compound jobs using work and data flow descriptions in a heterogeneous bioinformatics environment using meta - data information . the system regulates the dataflow by specifying dependency rules between the used applications in the meta - data , which allows the design of high complexity bioinformatics tasks , and stores the results of the different applications together with the new results computed during the process . the final output of the task is an xml file which contains all relevant information generated . the xml information is transformed by means of w2h 's ( 28 ) post - processing mechanism into an html page for the task report using the extensible style - sheet language transformations ( xslt ; http://www.w3.org/tr/xslt for facilitating a final visual inspection of the results . furthermore , the xml output can be also required and used for further analysis ( i.e. direct integration in user 's databases , additional pipeline analysis ) . all public databases used by these servers are installed under the sequence retrieval system ( srs ) at the dkfz ( 29 ) . the dkfz srs server contains more than 500 databases that are automatically updated whenever new releases become available ; this means that the webservers will be using the very last version of each database . therefore , as new and improved algorithms and methodologies are developed , they are incorporated into the protein analysis process without having to redesign the entire task . it is also possible to incorporate specific sets of databases as they become available , and to implement arbitrary configuration parameters . the development of the three pipelines presented here , has been user - driven from the beginning . their functionalities are continually being updated and extended in response to requests and suggestions emerging from our core users like lifedb ( 30,31 ) , where these servers are actively used in their protein analysis and annotation . we are currently developing checks especially through the application of filtering strategies and algorithms that will take into account the relationships between domain structure and homology searches . at the moment we are starting to develop a filtering system for the homology searches results taking into account the different quality of annotation in different protein databases with the idea to assign confidence levels and cross - checking results between tasks . we are additionally working on the implementation of directed text mining using the keywords of the proteins description .

the wealth of transcript information that has been made publicly available in recent years has led to large pools of individual web sites offering access to bioinformatics software . however , finding out which services exist , what they can or can not do , how to use them and how to feed results from one service to the next one in the right format can be very time and resource consuming , especially for non-experts.automating this task , we present a suite of protein annotation pipelines ( tasks ) developed at the german cancer research centre ( dkfz ) oriented to protein annotation by homology ( protsweep ) , by domain analysis ( domainsweep ) , and by secondary structure elements ( 2dsweep ) . the aim of these tasks is to perform an exhaustive structural and functional analysis employing a wide variety of methods in combination with the most updated public databases . the three servers are available for academic users at the husar open server http://genius.embnet.dkfz-heidelberg.de/menu/biounit/open-husar/

pubmed

delirium is a common and severe complication among elderly patients and is associated with increased morbidity and mortality , prolonged hospital stay , increased risk of post - discharge institutionalization and dementia . identifying accurate biomarkers for delirium may shed light on the pathophysiology and may help to improve delirium recognition and care . oxidative stress and disturbances in serotonergic and dopaminergic neurotransmission might all be involved in the pathophysiology of delirium and probably act together . within the central nervous system , tetrahydrobiopterin ( bh4 ) functions as an essential cofactor in enzymatic reactions responsible for the production of serotonin and dopamine . in addition , bh4 is a cofactor for nitric oxide synthase ( nos ) that catalyzes the production of nitric oxide ( no ) and citrulline from arginine . , when bh4 is partially deficient , some cellular sources of nos may generate superoxide ( o2 ) instead of no and citrulline . in patients with delirium , bh4 status has only been investigated after elective cardiac surgery . in order to assess bh4 status , we measured amino acid levels and subsequently calculated the phenylalanine / tyrosine ( phe / tyr ) ratio . this ratio is an indicator for the bh4 status as it reflects the activity of the enzyme phe hydroxylase , an enzyme that uses bh4 as a cofactor . furthermore , we determined the ratios of tryptophan ( trp ) , phe and tyr to the other large neutral amino acids ( lnaas ) . trp is the precursor of serotonin , while phe and tyr are the precursors of dopamine . the lnaas ( trp , phe , tyr , valine , isoleucine and leucine ) compete with each other for transport across the blood - brain barrier . therefore , a decreased trp / lnaas ratio is suggestive for a decline in the amount of trp that enters the brain and consequently for reduced synthesis of serotonin . moreover , we measured plasma levels of the dopamine metabolite homovanillic acid ( hva ) , approximately 30% of which is estimated to originate from dopamine neurons in the central nervous system and which is therefore thought to be a reliable indicator for central dopamine activity . finally , we measured plasma levels of arginine and citrulline to investigate the production of no by nos . the aim of the study was to investigate bh4 status , potential disturbances in serotonergic and dopaminergic neurotransmission and the production of no in patients with and without delirium . the present study was performed within the delirium in the old ( dito ) study in which mean levels of neopterin , interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium . in the dito study , a cross - sectional study , we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . exclusion criteria were a diagnosis of lewy body dementia , parkinson 's disease , neuroleptic malignant syndrome , tardive dyskinesia , ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines , aphasia , insufficient understanding of the dutch language and a mini - mental state examination ( mmse ) score < 10 points out of 30 . we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group . written informed consent was obtained from all participants . in case of delirium or cognitive impairment at the time of admission , informed consent was obtained from a representative of the patient . all participants were observed daily by the nursing and medical staff and by members of the research team until discharge . to screen for a change in behavior , the 13-item delirium observation screening scale was used during the first 5 days of admission . the diagnosis of delirium was made by a geriatrician , according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) , and was based on the psychiatric examination of the patient , the medical and nursing records , including the delirium observation screening scale scores , and information given by the patient 's closest relative . when the diagnosis of delirium was doubtful , the case was discussed with the geriatric consultation team to gain consensus . cognitive functioning was assessed in absence of delirium using the mmse . when it was impossible to score the mmse during admission because the patient was too ill , cognitive function was discussed with a clinician or assessed with information from the available medical records . when the clinical opinion was that the patient would have a mmse score 10 , the patient was not excluded from the study . this index encompasses 19 medical conditions , including dementia , and each condition is weighted with a score of 1 - 6 by severity . physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index . instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living . blood samples of all patients were collected within 48 h after admission . when a patient developed delirium during the hospital stay , new blood samples were collected within 24 h after the onset of the delirium and non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid . after blood sampling , the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells . within 3 h , plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described . plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection , as previously described for the determination of serotonin . medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics . biochemical parameters with a skewed distribution were logarithmically transformed ( all amino acids , amino acid ratios and hva ) . univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids , amino acid ratios and hva ( dependent variables ) and the presence of delirium additional analyses were performed for all amino acids , amino acid ratios and hva after also adding mmse score to the models . a two - tailed p < 0.05 was defined as statistically significant . statistical package for the social sciences , version 21.0 ( spss inc . , the present study was performed within the delirium in the old ( dito ) study in which mean levels of neopterin , interleukin-6 and insulin - like growth factor-1 were compared between patients with and without delirium . in the dito study , a cross - sectional study , we included patients who were admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . exclusion criteria were a diagnosis of lewy body dementia , parkinson 's disease , neuroleptic malignant syndrome , tardive dyskinesia , ongoing treatment with antipsychotics or other psychiatric medications except haloperidol and benzodiazepines , aphasia , insufficient understanding of the dutch language and a mini - mental state examination ( mmse ) score < 10 points out of 30 . we excluded patients with a mmse < 10 because it can be quite difficult to distinguish between features of severe dementia and delirium at admission as well as to measure improvement of cognitive function in this group . written informed consent was obtained from all participants . in case of delirium or cognitive impairment at the time of admission , informed consent was obtained from a representative of the patient . all participants were observed daily by the nursing and medical staff and by members of the research team until discharge . to screen for a change in behavior , the 13-item delirium observation screening scale was used during the first 5 days of admission . the diagnosis of delirium was made by a geriatrician , according to the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) , and was based on the psychiatric examination of the patient , the medical and nursing records , including the delirium observation screening scale scores , and information given by the patient 's closest relative . when the diagnosis of delirium was doubtful , the case was discussed with the geriatric consultation team to gain consensus . when it was impossible to score the mmse during admission because the patient was too ill , cognitive function was discussed with a clinician or assessed with information from the available medical records . when the clinical opinion was that the patient would have a mmse score 10 , the patient was not excluded from the study . this index encompasses 19 medical conditions , including dementia , and each condition is weighted with a score of 1 - 6 by severity . physical functionality was assessed using the 6-item katz activities of daily living scale and the barthel index . instrumental functionality was assessed using the 7-item older americans resource scale for instrumental activities of daily living . blood samples of all patients were collected within 48 h after admission . when a patient developed delirium during the hospital stay , new blood samples were collected within 24 h after the onset of the delirium and non - fasting blood was collected preferably between 8 and 10 a.m. in an 8-ml tube containing ethylene diamine tetraacetic acid . after blood sampling , the tubes were stored at room temperature to prevent changes in the transfer of amino acids between plasma and blood cells . within 3 h , plasma amino acid levels were determined by high - performance liquid chromatography with automated pre - column derivatization with ortho - phthalaldehyde as previously described . plasma hva levels were determined by reversed - phase high - performance liquid chromatography and electrochemical detection , as previously described for the determination of serotonin . medians and interquartile ranges were determined for continuous participant characteristics and proportions for categorical characteristics . biochemical parameters with a skewed distribution were logarithmically transformed ( all amino acids , amino acid ratios and hva ) . univariate one - way analysis of variance was used to investigate the association between mean levels of amino acids , amino acid ratios and hva ( dependent variables ) and the presence of delirium additional analyses were performed for all amino acids , amino acid ratios and hva after also adding mmse score to the models . table 1 presents the baseline characteristics of the 86 participants who were included in the study . of the 23 patients diagnosed with delirium , 21 the mean levels and corresponding 95% confidence intervals ( cis ) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3 . in adjusted models , mean levels of arginine were significantly lower in patients with delirium ( 34.8 mol / l , 95% ci 28.8 - 42.0 ) than in those without ( 45.2 mol / l , 95% ci 40.6 - 50.5 ) ( p = 0.022 ) ( fig . mean phe / tyr ratios were significantly higher in patients with delirium ( 1.34 , 95% ci 1.19 - 1.51 ) than in patients without delirium ( 1.14 , 95% ci 1.06 - 1.22 ) ( p = 0.028 ) ( fig . 1 ) . in addition , mean trp / lnaas ratios were significantly lower in patients with delirium ( 4.90 , 95% ci 4.19 - 5.74 ) than in those without ( 6.12 , 95% ci 5.58 - 6.71 ) ( p = 0.021 ) ( fig . no associations between the other amino acids and ratios and delirium were found , although citrulline ( fig . 1 ) and trp levels were at the border of significance lower in patients with delirium than in those without ( p = 0.052 and p = 0.067 , respectively ) . n = 1 [ not enough plasma ] ; no delirium , n = 1 [ measurement failed ] ) . mean hva levels were not statistically significantly different between patients with delirium ( 123.0 nmol / l , 95% ci 93.3 - 162.6 ) and patients without delirium ( 93.3 nmol / l , 95% ci 79.4 - 109.4 ) ( p = 0.098 ) . in the models additionally adjusted for mmse score , the association between arginine and delirium did not reach statistical significance ( delirium : mean 36.0 mol / l , 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l , 95% ci 39.8 - 50.5 , p = 0.107 ) . mean phe / tyr ratios remained borderline significantly higher in patients with delirium ( 1.34 , 95% ci 1.16 - 1.55 ) than in those without ( 1.15 , 95% ci 1.07 - 1.24 ) ( p = 0.089 ) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium ( 5.00 , 95% ci 4.15 - 6.01 ) compared to those without ( 6.15 , 95% ci 5.58 - 6.78 ) ( p = 0.062 ) . table 1 presents the baseline characteristics of the 86 participants who were included in the study . of the 23 patients diagnosed with delirium , 21 the mean levels and corresponding 95% confidence intervals ( cis ) of the investigated biochemical parameters in patients with and without delirium are presented in tables 2 and 3 . in adjusted models , mean levels of arginine were significantly lower in patients with delirium ( 34.8 mol / l , 95% ci 28.8 - 42.0 ) than in those without ( 45.2 mol / l , 95% ci 40.6 - 50.5 ) ( p = 0.022 ) ( fig . mean phe / tyr ratios were significantly higher in patients with delirium ( 1.34 , 95% ci 1.19 - 1.51 ) than in patients without delirium ( 1.14 , 95% ci 1.06 - 1.22 ) ( p = 0.028 ) ( fig . 1 ) . in addition , mean trp / lnaas ratios were significantly lower in patients with delirium ( 4.90 , 95% ci 4.19 - 5.74 ) than in those without ( 6.12 , 95% ci 5.58 - 6.71 ) ( p = 0.021 ) ( fig . no associations between the other amino acids and ratios and delirium were found , although citrulline ( fig . 1 ) and trp levels were at the border of significance lower in patients with delirium than in those without ( p = 0.052 and p = 0.067 , respectively ) . hva data were missing for 2 patients ( delirium , n = 1 [ not enough plasma ] ; no delirium , mean hva levels were not statistically significantly different between patients with delirium ( 123.0 nmol / l , 95% ci 93.3 - 162.6 ) and patients without delirium ( 93.3 nmol / l , 95% ci 79.4 - 109.4 ) ( p = 0.098 ) . in the models additionally adjusted for mmse score , the association between arginine and delirium did not reach statistical significance ( delirium : mean 36.0 mol / l , 95% ci 28.7 - 45.1 vs. no delirium : mean 44.8 mol / l , 95% ci 39.8 - 50.5 , p = 0.107 ) . mean phe / tyr ratios remained borderline significantly higher in patients with delirium ( 1.34 , 95% ci 1.16 - 1.55 ) than in those without ( 1.15 , 95% ci 1.07 - 1.24 ) ( p = 0.089 ) and mean trp / lnaas ratios remained borderline significantly lower in patients with delirium ( 5.00 , 95% ci 4.15 - 6.01 ) compared to those without ( 6.15 , 95% ci 5.58 - 6.78 ) ( p = 0.062 ) . in the present study we found disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium when compared to patients without delirium . as far as we are aware , this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients . in order to assess the bh4 status , we measured the phe / tyr ratio . in patients with delirium we found an increased ratio , suggesting a deficiency in the essential cofactor bh4 for the production of serotonin , dopamine and no . decreased bh4 availability has already been found in other neuropsychiatric disorders such as alzheimer 's disease , parkinson 's disease and schizophrenia . our finding is not in agreement with the results of a previous delirium study which showed that levels of bh4 and phe / tyr ratios did not differ between patients with and without delirium . however , that study included a relatively younger group of patients undergoing elective cardiac surgery . in the present study , serotonergic neurotransmission was investigated with the trp / lnaas ratio and the phe / tyr ratio . we found that patients with delirium had a decreased trp / lnaas ratio , which might suggest reduced serotonin production in the central nervous system . this hypothesis is strengthened by the finding that patients with delirium had an elevated phe / tyr ratio , which might suggest deficiency in the essential cofactor bh4 in the production of serotonin . in previous studies , controversial results have been reported . several studies found a reduced trp / lnaas ratio during delirium , whereas two studies reported no difference in this ratio between patients with and without delirium . the study performed by flacker and lipsitz included only patients with mild illnesses not requiring hospitalization . therefore , the findings may not be generalizable to acutely ill patients who needed medical care in hospital . the study performed by van der cammen et al . included delirium patients with alzheimer 's disease . it might be possible that in those patients a disturbance in cholinergic neurotransmission played a more important role in the development of delirium than disturbances in other pathophysiological pathways . furthermore , we found no differences in phe / lnaas ratios , tyr / lnaas ratios and hva levels between patients with and without delirium , suggesting that dopaminergic neurotransmission is not impaired during delirium . the finding that plasma hva levels are not significantly increased in patients with delirium compared to patients without delirium is not in agreement with earlier results . however , those studies were performed in patients with alzheimer 's disease and patients undergoing cardiac surgery , and therefore the results may not be generalizable . ramirez - bermudez et al . found that cerebrospinal fluid hva levels correlated with psychotic symptoms of delirium ( hallucinations and delusions ) in neurological patients . it might also be possible that we did not find an association between the dopaminergic markers and the presence of delirium because we included patients both with and without psychotic features . in our study , we also found reduced plasma arginine levels and borderline statistically significantly reduced citrulline levels in patients with delirium . considering the cross - sectional study design first , it is possible that patients with delirium had a pre - existing arginine deficiency which might have resulted in a reduced production of citrulline by nos ( fig . second , when bh4 is partially deficient , as our results do suggest , some cellular sources of nos may generate o2 instead of citrulline and no from arginine ( fig . if this latter scenario is true for delirium , this would also suggest an increased status of oxidative stress , since it favors peroxynitrite formation ( fig . if peroxynitrite is not scavenged by antioxidants , it may cause oxidative damage to cellular macromolecules , which has already been hypothesized to occur in alzheimer 's disease . however , both amino acids have been investigated previously by osse et al . in patients with delirium after cardiac surgery , but they found no differences in arginine and citrulline levels between patients with and without delirium . since they also reported no difference in bh4 status between patients with and without delirium , first , our findings were obtained in a relatively small group of patients ; therefore , confirmation in a larger population is recommended . second , it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters . in this study , we adjusted for the charlson comorbidity index , which includes dementia , and our estimates remained statistically significant . however , for our additional analysis mmse scores were not available for all patients ; therefore , we can neither confirm nor deny that the presence of a comorbid cognitive disturbance , not diagnosed as dementia ( yet ) , was a confounding factor . it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium , just like delirium symptoms . in the present study , blood sampling and delirium occurred on the same day , but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling . finally , some potential participants were not included in the study and this may have resulted in some selection bias ; however , since this was random and occurred in both patients with and without delirium , we think that our results are only minimally influenced by this . first , the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms . second , we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial . first , our findings were obtained in a relatively small group of patients ; therefore , confirmation in a larger population is recommended . second , it might be speculated that the degree of the patients ' cognitive functioning influenced the mean levels of the biochemical parameters . in this study , we adjusted for the charlson comorbidity index , which includes dementia , and our estimates remained statistically significant . however , for our additional analysis mmse scores were not available for all patients ; therefore , we can neither confirm nor deny that the presence of a comorbid cognitive disturbance , not diagnosed as dementia ( yet ) , was a confounding factor . it is possible that the levels of biochemical markers are dependent on delirium duration and severity or even fluctuate during the day in patients with delirium , just like delirium symptoms . in the present study , blood sampling and delirium occurred on the same day , but there is a possibility that the patients had no delirium symptoms at the moment of blood sampling . finally , some potential participants were not included in the study and this may have resulted in some selection bias ; however , since this was random and occurred in both patients with and without delirium , we think that our results are only minimally influenced by this . first , the intensive monitoring of clinical symptoms of patients with delirium until discharge and the dsm - iv diagnosis by a geriatrician makes it less likely that we missed delirium or misdiagnosed symptoms . second , we did not focus on one but on several possible pathways that might lead to delirium as it has been suggested that the pathophysiology is multifactorial . in this study in older , acutely ill hospitalized patients , we found that patients with delirium had higher phe / tyr ratios , lower trp / lnaas ratios and lower levels of arginine and citrulline than patients without delirium . these findings might suggest that decreased bh4 availability , disturbed serotonergic neurotransmission and an increased status of oxidative stress may have played a role in the pathogenesis of delirium in our patient group . since as far as we know this is the first delirium study investigating bh4 status and levels of arginine and citrulline in acutely ill elderly hospitalized patients , confirmation of our results in a larger , comparable population is recommended . moreover , more research is needed to explore the potential differences in the pathophysiology of delirium in patients with and without cognitive disorders .

backgroundoxidative stress and disturbances in serotonergic and dopaminergic neurotransmission may play a role in the pathophysiology of delirium.aimsin this study , we investigated levels of amino acids , amino acid ratios and levels of homovanillic acid ( hva ) as indicators for oxidative stress and disturbances in neurotransmission.methodsplasma levels of amino acids , amino acid ratios and hva were determined in acutely ill patients aged 65 years admitted to the wards of internal medicine and geriatrics of the erasmus university medical center and the ward of geriatrics of the havenziekenhuis , rotterdam , the netherlands . differences in the biochemical parameters between patients with and without delirium were investigated by analysis of variance in models adjusted for age , gender and comorbidities.resultsof the 86 patients included , 23 had delirium . in adjusted models , higher mean phenylalanine / tyrosine ratios ( 1.34 vs. 1.14 , p = 0.028 ) , lower mean tryptophan / large neutral amino acids ratios ( 4.90 vs. 6.12 , p = 0.021 ) and lower mean arginine levels ( 34.8 vs. 45.2 mol / l , p = 0.022 ) were found in patients with delirium when compared to those without . no differences were found in hva levels between patients with and without delirium.conclusionthe findings of this study suggest disturbed serotonergic neurotransmission and an increased status of oxidative stress in patients with delirium .

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overweight and obesity prevalence rates are increasing worldwide . about 1.4 billon adults were overweight in 2008 , of whom 200 million men and 300 million women were considered to be obese . in the netherlands , prevalence rates of overweight ( and obesity ) are 60% ( 14% ) in men and 44% ( 13% ) in women . high prevalence rates have been found specifically among ethnic minorities and low socioeconomic status ( ses ) groups [ 1 , 3 ] . these health risks include cancer , cardiovascular diseases like hypertension and diabetes , and problems with mobility [ 57 ] . the who recommends adults aged 1864 to engage in moderate - intensity aerobic physical activities such as walking , cycling , or doing household chores for at least 150 minutes a week . active transport ( i.e. , walking or cycling from a to b ) can thus help achieve sufficient levels of physical activity [ 9 , 10 ] . this review of reviews showed that age and overweight were inversely correlated with physical activity , whereas the male gender and ethnic origin ( white ) were positively correlated with being active . generally , these correlates were also found for active transport among low - income populations . in the past decade particularly , studies from the usa and australia have found positive relations between physical activity and neighbourhood walkability [ 13 , 14 ] . most of these studies , however , have been performed among high - ses populations who tend to live in neighbourhoods with relatively high walkability . moreover , high - ses populations have shown higher levels of physical activity and lower prevalence rates of overweight compared to low - ses populations . it is not yet clear whether the findings from these studies are also valid for low - ses populations living in deprived neighbourhoods . detailed information on the relation between neighbourhood characteristics and physical activity among subgroups within this population is currently lacking . it is hypothesized that more favourable neighbourhood characteristics are associated with higher levels of activity . past studies that focused on the effect of neighbourhood walkability on active transport have shown that associations are reduced after controlling individual demographic characteristics . however , in addition to the confounding role of demographic characteristics , knowledge about interactions between demographic variables and neighbourhood characteristics is required in order to distinguish subgroups that are more vulnerable or responsive to environmental features than others . inhabitants of dutch deprived neighbourhoods vary in ethnic background and are often characterized by a low socioeconomic status . as a result , the prevalence rate of overweight is higher , and level of physical activity is lower compared to those of the average dutch population [ 17 , 18 ] . this makes inhabitants of dutch deprived districts an important target group for physical activity interventions . furthermore , previous research has shown that it is difficult to address ethnic minorities , and guidelines for this specific group are lacking . more insight into the personal and neighbourhood factors that are associated with active transport could contribute to the development of effective interventions targeting ethnic minorities . therefore , this study investigates which personal and neighbourhood environmental characteristics are associated with active transport among inhabitants of dutch deprived neighbourhoods . in addition , interactions between personal and neighbourhood environmental characteristics will be tested to explore differential associations of neighbourhood walkability with active transport within subgroups . briefly , urban40 is a longitudinal study that evaluates the health impact of area - based interventions to improve housing , employment , education , social integration , and safety in the 40 most severely deprived neighbourhoods in the netherlands , also known as the dutch district approach . in 2007 , the dutch government made a list of the 140 most deprived neighbourhoods in the netherlands , based on scores on eighteen different registry - based indicators of physical and socioeconomic deprivation as well as physical and social problems reported by residents . from this list , twenty neighbourhoods were selected that belonged to the 140 most deprived dutch neighbourhoods . in each district , 250 randomly selected adults aged 18 and older received a letter in which they were invited to participate , including a reply card that they could return free of charge . trained interviewers contacted the residents who sent a reply card and arranged home visits for the interview . up to two reminder letters were sent when people did not respond via the reply card . in addition , trained interviewers from an interview agency specialized in research among multicultural populations recruited participants by unplanned home visits ( ringing doorbell at the addresses on the original list ) . the trained interviewers were tailored to gender and ethnicity ( spoken language , e.g. , dutch , english , turkish , classical arabic , or arab - berber ) of the residents . financial constraints limited recruitment of respondents by unplanned home visits in all districts , favouring larger districts . respondents filled out the questionnaire by themselves , but they could also ask the assistant that brought the questionnaire to help them , if needed in their own language ( dutch , english , turkish , classical arabic , or arab - berber ) . in particular the participants that were of non - dutch origin made use of this option ; they were orally interviewed and the assistant filled in the answers . a total of 5000 respondents were invited to participate . of the 374 participants that returned the reply card , 299 filled out the questionnaire . this resulted in a total study population of 740 adults that were recruited between may 2010 and november 2011 . after deletion of cases without data on key variables ( n = 118 ) , a final sample of 622 participants remained . active transport was defined as the minutes per week spent on walking or cycling from a to b. the validated squash questionnaire for physical activity measured active transport by asking respondents to think about a regular week in the past months . respondents then indicated how many days per week they engaged in several forms of physical activity and how many minutes they engaged in them . subsequently , they could fill in the days , hours , and minutes spent on active transport . total minutes of walking for active transport per day were calculated by multiplying the number of hours reported with sixty and adding them to the minutes reported . subsequently , minutes of active walking per week were calculated by multiplying the minutes per day with the reported number of days per week . this number of minutes of walking for active transport per week was used in the analyses . minutes of cycling for active transport per week were calculated according to the same steps . neighbourhood environmental characteristics were assessed by use of the neighbourhood walkability scale ( news ) . three scales with twelve questions in total investigated the access to services ( three items ) ( = 0.71 ) ( e.g. , shops are within easy walking distance from my home ) , neighbourhood surroundings ( four items ) ( = 0.66 ) ( e.g. , the natural surroundings in my neighbourhood are beautiful ) , and safety from crime ( five items ) ( = 0.66 ) ( e.g. , because of criminality it is unsafe to walk in my neighbourhood during the day ) . three items of the safety from traffic scale were included separately in the analyses because of the low reliability of the scale ( < 0.6 ) . the items were there is much traffic on nearby streets which make walking in the neighbourhood difficult or unpleasant ; the speed of traffic in nearby streets is normally low ( 30 km / h or less ) ; and most car drivers exceed maximum speed when driving through the neighbourhood . the answers were given on a four - point likert scale ( completely disagree a higher score corresponded with better access to services , better neighbourhood surroundings , and more safety from crime and traffic . personal characteristics that were taken into account were age , sex , ethnicity , and body mass index ( bmi ) . ethnicity of the respondent was operationalized by assessing the country of birth of both parents . if a respondent had at least one parent who was born outside the netherlands , he or she was considered to be a migrant . neighbourhood status was included as a confounder , to correct variations in degree of deprivation among neighbourhoods . neighbourhood status was determined using the neighbourhood status score ( nss ) of the dutch social cultural planning office ( scp ) . this score is based on the income , education level , and employment status of the inhabitants and indicates the degree of social deprivation per neighbourhood . lower scores correspond with a larger degree of social deprivation , whereas high scores indicate a more favourable socioeconomic status . characteristics of the target population were investigated by means of descriptive analyses . independent samples t - tests were performed to investigate the difference in scores on the news and in minutes of active transport per week between the following subgroups : respondents equal to or younger than the median age of 43 years and respondents older than 43 years : men and women ; native dutch respondents and migrants ; and respondents with a bmi lower than 25 and respondents with a bmi higher than 25 . bivariate correlations were calculated to explore the associations between personal characteristics , and minutes per week spent in active transport and the associations between news scales and minutes spent engaging in active transport . in order to gain more insight into these correlations and to adjust for the difference in effect between baseline characteristics multivariate linear regression analyses were performed . the full main effects model consisted of nss , the personal characteristics ( age , sex , ethnicity , and bmi ) , and the neighbourhood characteristics . interactions between personal characteristics and neighbourhood variables were investigated by adding each interaction term separately to the full main effects model . most of the participants were dutch , the migrants were mainly non - western ( 87.45% ; not tabulated ) . the nss of the twenty participating districts varied between 3.64 ( highly deprived ) and 0.15 ( moderately deprived ) with a mean of 1.82 and a sd of 1.03 . table 2 shows the scores for the neighbourhood characteristics and the minutes of active transport per week for subgroups of the population . on average , residents of deprived neighbourhoods walked 33 minutes and cycled for 32 minutes per week for active transport . the younger participants were found to be more active in terms of walking as well as cycling than the older participants . migrants were more active in terms of walking , whereas the dutch biked more often . participants with a bmi below 25 were more active than those overweight , with a significant difference in activity levels for cycling . respondents older than 43 years perceived the neighbourhood surroundings as more positive than the younger respondents . women experienced better access to services than men . native dutch participants perceived better access to services , better neighbourhood surroundings , and felt safer from crime than migrants . furthermore , they experienced less traffic on nearby streets that made walking difficult than migrants . participants who lived in a neighbourhood with a relatively higher status within this population perceived all neighbourhood characteristics as more favourable than the people from districts with a lower nss . multivariate regression analyses showed that minutes of walking decreased significantly by one minute for each year increase in age . nss was also found to be a significant associate of walking ; respondents from higher status neighbourhoods walked less than respondents from lower status neighbourhoods . perceived speed of traffic was the only neighbourhood variable found to be a statistically significant associate of walking . interactions between ethnicity and the perception of traffic exceeding maximum speed ( : 0.40 ; p < 0.001 ) ; sex and the perception of traffic exceeding maximum speed ( : 0.26 ; p < 0.05 ) ; and between age and speed of traffic ( : 0.53 ; p < 0.01 ) were statistically significant . subgroup analyses indicated that migrants and women reported more walking for transport when cars were perceived to not exceed maximum speed ( : 0.18 ; p < 0.01 and : 0.13 ; p < 0.05 , resp . ) , whereas this perceived environmental factor was unrelated to walking of native dutch and male respondents ( : 0.06 ; ns and : 0.04 ; ns , resp . ) . stratification for age showed that low perceived speed of traffic was significantly associated with more walking in respondents of 43 years or younger ( : 0.18 ; p < 0.01 ) but not in respondents older than 43 ( : 0.06 ; ns ) . every year of increase in age was associated with a decrease of one minute of cycling per week ( table 3 ) . the native dutch cycled more compared with the migrants ( : 0.11 ; p 0.01 ) . the interaction between ethnicity and neighbourhood surroundings was found to be significant ( : 0.50 ; p 0.01 ) . more attractive neighbourhood surroundings were significantly associated with more cycling per week among the migrants ( : 0.23 ; p 0.001 ) , but not in the dutch respondents ( : 0.02 ; ns ) . this study investigated which personal and neighbourhood environmental characteristics were associated with active transport among inhabitants of dutch deprived neighbourhoods . this finding is supported by a study of ogilvie et al . and could be a result of differences in fitness between the younger and the older respondents . people tend to perceive more barriers for physical activity as they age , such as physical disabilities and poorer perceived health . migrants walked more minutes per week compared with the dutch , probably because walking is considered to be an easy and cheap mode of transportation , which is less sensitive to cultural habits compared to cycling . the dutch cycled more than the migrants , probably because they are more used to cycling and have more access to this mode of transportation . higher scores on bmi correlated with lower levels of active transport , which confirms findings of pucher et al . . perceived speed of traffic was the only neighbourhood environmental characteristic that was associated with walking for transport . when the average speed of traffic was perceived as low , people reported more walking for active transport . a review of previous studies reported mixed evidence with regard to safety and active transport , which illustrates a need for clear definitions of safety in this field of research . our measurements confirm this need for proper measurements of safety from traffic , since we were forced to include the relevant questions separately in the analyses as a result of low reliability of the scale . interactions showed that with regard to ethnicity , migrants appeared to be more responsive to neighbourhood walkability than the dutch respondents . speed of traffic ( walking ) and aesthetics ( cycling ) was found to be more strongly associated with active transport among migrants than among native dutch . this could be a result of cultural differences ; migrants seem to be more in need of a safe , attractive , and stimulating environment to induce active transport . an investigation of transportation among dutch migrants showed that migrants cycled more when high quality cycle infrastructure was available and if a strong cycle culture was present in the neighbourhood . in addition , female and younger respondents were subgroups that appeared to be somewhat more responsive to traffic safety than male and older respondents . a dutch study on traffic safety found that higher levels of neighbourhood traffic safety correlated with increased odds of being active , especially in women and people aged 35 to 59 , which partly confirms our findings . personal characteristics have been shown to be stronger associates of active transport than neighbourhood characteristics , a finding which is supported by multiple other studies [ 33 , 34 ] . nevertheless , there were also studies that did find associations between neighbourhood walkability and active transport [ 13 , 14 ] . this could be explained by differences in levels of transportation facilities in the countries under study . most of these studies were conducted in the us or australia , countries that sometimes lack proper walking or cycling trails . however , the netherlands especially is already well - equipped for facilitation of walking and cycling , so relative differences in neighbourhood characteristics may only have a minimal impact on active transport . this implies that results of these studies are likely to be only valid for the population and country in which it was conducted , as was suggested in previous studies [ 12 , 34 ] . the low variability of neighbourhood characteristics within the deprived districts in our study may have decreased the possibility to find strong environmental relationships even more . it is also possible that the characteristics investigated in this study did not cover all environmental aspects that might be relevant in active transport . nevertheless , interaction effects indicated that within some groups of the population under study , neighbourhood characteristics are important and need to be considered in efforts to promote physical activity . since the data collected for this study was cross - sectional , statements about causality can not be made . second , the overall response rate was low , although it was equivalent to those of comparable studies of the past years [ 10 , 12 , 35 ] . third , self - reported measures of active transport were used , which could have been prone to recall bias and socially desirable answers . fourth , an abbreviated version of the news questionnaire was used , which may be the reason for the one scale on safety from traffic being considered unreliable . despite some limitations , this study adds valuable information to current evidence in particular because of the focus on a low - ses population , on the understudied cycling behaviour , and the ethnic composition of the study population . participants were hard to reach and are therefore often neglected in research , among others because of possible cultural and language differences between the interviewer and respondents . intensive recruitment strategies such as matching of interviewer and participant and executing the study in the respondents ' mother tongue were used to overcome this difficulty . the setting of deprived neighbourhoods was also a valuable characteristic , since they are the focus of governmental policies and interventions . insight into the associations between neighbourhood characteristics and patterns of physical activity and their determinants in these districts could provide more information about the contents and implementation of such policies and interventions . results of the current study may be used to design experimental research in order to test causality of the findings . eventually , this may lead to evidence for effective intervention development for the subgroups most in need of interventions to increase active transport and subsequently levels of physical activity .

background . active modes of transport like walking and cycling have been shown to be valuable contributions to daily physical activity . the current study investigates associations between personal and neighbourhood environmental characteristics and active transport among inhabitants of dutch deprived districts . method . questionnaires about health , neighbourhoods , and physical activity behaviour were completed by 742 adults . data was analysed by means of multivariate linear regression analyses . results . being younger , female , and migrant and having a normal weight were associated with more walking for active transport . being younger , male , and native dutch and having a normal weight were associated with more cycling for active transport . neighbourhood characteristics were generally not correlated with active transport . stratified analyses , based on significant person - environment interactions , showed that migrants and women walked more when cars did not exceed maximum speed in nearby streets and that younger people walked more when speed of traffic in nearby streets was perceived as low . among migrants , more cycling was associated with the perceived attractiveness of the neighbourhood surroundings . discussion and conclusion . results indicated that among inhabitants of dutch deprived districts , personal characteristics were associated with active transport , whereas neighbourhood environmental characteristics were generally not associated with active transport . nevertheless , interaction effects showed differences among subgroups that should be considered in intervention development .

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optic neuritis related to ms is an acute optic neuropathy occurring in young people , especially in women . the clinical picture shows a decline in visual acuity , a pain which increases with movements of the eye , a decline in contrast sensitivity , dyschromatopsia and changes in the visual field ( 1 ) . it occurs more often unilaterally in young people , predominantly in women with an incidence of 1 - 5 per 100,000 annually ( 2 ) . the main aim of this study was to determine the onset of optic neuritis as the first ms clinical presentation . we also wanted to analyze the role and significance of morphometric changes in the optic nerve and visual field by analyzing parameters of optic coherent tomography ( oct ) and computer perimetry in these patients . one of the aims was to determine the efficiency of pulse corticosteroid therapy in those patients . a clinical , retrospective study was conducted at the eye clinic and the neurological clinic , sarajevo university clinical centre ( succ ) at the time of the march 20092011 . after the first clinical signs follower is a detailed examination of patients by neurologists and ophthalmologists . here we did not analyzed cases of ms relapses . in all analyzed patients we found a unilateral optic neuritis . we approached ms verification according to paty or brakhof modern clinically defined multiple sclerosis ( cdms ) criteria ( 3 , 4 ) . first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination . of radiological tests , mri of the brain and spinal cord were analyzed. second criterion : two ms clinical episodes , one mri manifest lesion and paraclinical symptoms. laboratory - supported ms diagnosis: two episodes , one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid. one episode , two clinically separate lesions and cerebrospinal abnormalities . first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination . of radiological tests , second criterion : two ms clinical episodes , one mri manifest lesion and paraclinical symptoms . laboratory - supported ms diagnosis : two episodes , one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid . one episode , two clinically separate lesions and cerebrospinal abnormalities . of neurological parameters we analyzed clinical manifestations of motor , sensory and sensitive disturbances , relevant to the confirmation of ms diagnosis . of ophthalmological parameters , we analyzed : visual acuity ( va ) by snellen charts , visual field ( vf ) by octopus 100 , and thickness of the peripapillary rim nerve fibres by stratus zeiss optic coherent tomography ( oct ) . . age structure of ms patients with verified optic neuritis gender structure of ms patients with verified optic neuritis the age structure analysis of the sample shows that optic neuritis was most often present in the patients aged 18 to 30 years . in the entire sample , onset of optic neuritis was more frequent in the female patients ( 70% ) compared to the male patients . the first clinical manifestation of ms in our sample statistically significant tests ( x=9,7 p=0,01 ) we have confirmed that the optic neuritis is one of the first clinical manifestation of ms , compared to the other motor , sensory and sensory events in our sample . ophthalmological parameters we followed after 7 and 15 days , after 1 , 3 , 6 and 12 months . analyzing disturbances in the computerised visual field : centrocecal and paracentral scotoma and diffuse sensitivity , we find most frequent disturbances in the centrocecal region in 50% of the cases . different kinds of scotoma in visual field in ms patients with optic neuritis by analysis of oct results in part of the nerve fibre thickness of papillary and parapapillary layers ( of the neuroretinal rim ) , we had , after three months , results showing the thinning of the nerve fibres in the whole circumference , most often in the upper quadrant . retinal nerve fibre layer ( rnfl ) by optic disc visual acuity on first examination and one month after corticosteroid therapy visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction . after the administration of pulse corticosteroid therapy , all patients with optic neuritis had a significant improvement of va , and a month upon administration it varied from 0.6 to 1.0 . in our sample of 89 patients with the verified ms diagnosis , 10 patients had a clinical picture of optic neuritis , as a first clinical sing of disease , it was statistically significant ( x= 9,7 p=0,01 ) . the optic neuritis study group describes the changes of visual functions in ms patients , which usually present as a decline in vision , blurring , decline in contrast sensitivity , color vision disturbances , as well as disturbances in the visual field ( 1 - 5 ) . in their studies , allanore y. and deretzi g.confirm the genetic impact on ms development as an autoimmune disease in some families ( 6 ) . studies were performed to analyze the influence of stress , viral and bacterial infections of respiratory tract , urinary and gastrointestinal systems on the appearance and occurrence of ms exacerbations ( 7 , 8) . in our study , the disease appeared most often in the patients aged 18 - 30 years . population get results which show that the ratio of the incidence of occurrence of ms ranged from 1.9 : 1 to 3.6:1 in favor of women ( 9 ) . a large number of studies confirm the ms and on occurrence at a younger age and more often in women ( 1 , 2 , 5 , 10 ) . balashov ke . finds a higher on incidence in the spring months in the patients without verified demyelisating lesions , which has been observed in our past practice too , and which could be an aim of our future studies ( 11 ) . cigarette smoking is also a risk factor in the development of ms and on ( 12 ) . in 50% of our subjects we had centrocecal scotoma in the visual field , and sensitivity depression in 30% . disturbances in the central areas of the visual field were verified in other studies as well ( 1 , 13 ) . visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction . all patients with an on and verified ms diagnosis monitored in this study were treated with pulse corticosteroid therapy in a dosage of 1,000 mg for three days , followed by 8 days of 1mg / kg b.w . a month after pulse corticosteroid therapy , visual acuity in all patients with on ranged from 0.6 to 1.0 . corticosteroids administered intravenously by the pulse therapy scheme prevent on relapse , but they have the same effect on definite va as corticosteroids orally administered . ontt finds that the on patients treated with prednisolone orally administered in a dosage of 1mg / kg b.w . for 14 days ontt shows that intravenously administered corticosteroids inhibit ms development for a 2-year period , bur after three years that effect disappears ( 14 , 15 ) . three months after the first on presentations , the oct analysis of retinal nerve fibre thickness and neuroretinal rim confirmed the nerve fibre thinning in all subjects . ontt shows that 50% of the patients with on develop ms over a 15-year period . ontt also finds that in on patients without mri changes , ms develops in 25% of the cases , and in the cases with one or more cns lesions that is 75% . all patients had oct - verified nerve fibre atrophy after on ( 1 , 14 ) . according to other researchers , by oct analysis it can discover in vivo atrophy of nerve fibres in patients with ms as structural damages of axons of afferent fibres of the visual pathway ( 15 , 16 ) . oct rnfl thickness average analysis studies show , by morphometric analysis of the structure of nerve fibres by oct , that there is fibre atrophy in the ms patients with and without on . however , retinal nerve fibre atrophy is more significant in the patients with on ( 16 ) . in our sample with the predominantly female gender ranging from 18 to 30 years of age , on occurred as the first clinical presentation of ms . the analysis of the computerised visual field confirmed centrocecal disturbances as the most common , and optic coherent tomography registered the thinning of the nerve fibres of the neuroretinal rim in all subjects . a month after pulse corticosteroid therapy ,

aim : to analyze the clinical signs of multiple sclerosis ( ms ) and show that optic neuritis is one of the first event , which indicates the development of disease.patients and methods : the study involved 89 cases in which it confirmed ms at the time of the march 20092011 . since ophthalmological parameters were analyzed visual acuity ( va ) , visual field ( vf ) , and retinal nerve fibre layer ( rnfl ) thickness of peripapillary rim by optic coherent tomography ( oct).results : ten(10 ) patients had on as the first clinical manifestation of the disease which was statistically significant ( x2 = 9,7 p=0,01 ) compared to the manifestation of other clinical signs of disease . in vf , centrocecal scotomas were predominant in 50% of the subjects ; the rnfl thinning of the neuroretinal rim was verified in all patients , most often in the upper quadrant . a month after pulse corticosteroid therapy , visual acuity in all patients with on ranged from 0.6 to 1.0.conclusion:on is one of the first ms clinical manifestation . in vf , the most common disturbances are in the centrocecal area . the rnfl thinning was verified in all patients with oct .

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diabetes can cause acute and long - term complications , and is as well , a global risk factor for cardiovascular disease.1 lifestyle and behavior , as well as individual genetic predisposition , influence the risk of diabetes and prevalence in a community.2 public knowledge is the basis for lifestyle and behavior in a given community.3 therefore , the knowledge particular to diabetes is a key determinant of the lifestyle of a community , and can discriminate those who are at risk from those who are free of risk . diabetes is increasingly prevalent in ethnic minority groups globally.2,4,5 recently , lorga et al6 reported that 16.7% of the karen rural community had an abnormally high blood glucose level ( > 100 mg / dl ) , and that there was a considerable proportion of prediabetic persons ( 13.04% ) in a cross - sectional survey among the karen minority . the karen ethnic rural community along the thai myanmar border is a hard - to - reach population for health promotion services . karen people use their native language and most of the senior adult population in the community can not read thai script . moreover , in a district like thasongyang , the population pattern is very dynamic and unstable because of a large migration across the border.7 karen people are indigenous to , and tend to stay along the thai - myanmar border such as area around tak province . there is limited literature to guide the assessment of health literacy and knowledge of diabetes in such a community.8 the objective of the study was to assess the knowledge of diabetes of the rural karen residents , and compare the diabetes knowledge between male and female respondents . this study was approved by the ethical board of the boromrajonani college of nursing nakhon lampang , lampang , thailand , and conducted with the verbal informed consent of participants . it was conducted in thasongyang , which is a district in the far northwest of thailand , by the narrow moei river on the border of myanmar . all participants were thai citizens but most of the participants could not read thai script . none had any prior known diagnosis of diabetes . diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang . the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language , and were completed by the interviewers . fasting plasma glucose ( fpg ) measurement was preceded by overnight fasting for 8 hours before the test . body weight and height were measured by the public health officers using a standard measuring scale . body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters . continuous variables were summarized as median , maximum , and minimum values , as the distribution was not normal . the nonparametric test for trend analysis was run by nptrend in stata version 11 ( stata corp , college station , tx ) . yes , no , and not sure answers in the questionnaires were recoded as categories in order to compare by rank sum test . a correct answer was coded to be 1 , and an incorrect answer , or the answer of not sure , was coded as zero . statistical significance was defined as p value less than 0.05 , with a 95% confidence interval . diabetes knowledge was assessed by a questionnaire that was developed by consensus between native researchers and researchers from the boromrajonani college of nursing nakhon lampang . the questionnaires were delivered in the karen language by researchers speaking both thai and the karen language , and were completed by the interviewers . fasting plasma glucose ( fpg ) measurement was preceded by overnight fasting for 8 hours before the test . body weight and height were measured by the public health officers using a standard measuring scale . body mass index was calculated by the ratio of body weight in kilograms divided by the square of the height in meters . continuous variables were summarized as median , maximum , and minimum values , as the distribution was not normal . the nonparametric test for trend analysis was run by nptrend in stata version 11 ( stata corp , college station , tx ) . yes , no , and not sure answers in the questionnaires were recoded as categories in order to compare by rank sum test . a correct answer was coded to be 1 , and an incorrect answer , or the answer of not sure , was coded as zero . statistical significance was defined as p value less than 0.05 , with a 95% confidence interval . we assessed the diabetes knowledge briefly in three parts ( 1 ) general knowledge , ( 2 ) knowledge about the risk factors , and ( 3 ) knowledge about the symptoms , diagnostic signs , and complications of diabetes mellitus . one - third of the population answered correctly ( range 27.09%41.81% ) , and another one - third gave no answer to these questions ( range 33.44%39.80% ) , whereas another one third gave a wrong answer . among the karen community , 36.45% knew that old age is a risk factor for diabetes , 37.12% knew that they had diabetic relatives , 39.80% knew that obesity can lead to diabetes , 37.12% answered that exercise can prevent diabetes , 25.09% reported that pregnant women can have diabetes , and 41.81% said they should not eat excess fatty food for fear of diabetes . regarding the symptoms , diagnosis and complications of diabetes , less than 30% knew that fasting blood glucose level should be less than 100 mg% . a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire . among the respondents , 33.78% answered that diabetic patients could have thirst and polyuria , 34.78% knew that numbness of the limbs might link to diabetes , 35.12% recognized the importance of foot care in people living with diabetes , 32.78% answered that untreated diabetes could lead to blindness , and 34.78% knew that slow wound healing might be due to diabetes . overall , one - third of the sample population gave the correct answers ( range 32.78%34.78% ) while the other two - thirds were either unsure or gave wrong answers . overall , public knowledge of diabetes among karen ethnic rural community is at the lower extreme ( figure 1 ) . diabetes knowledge was compared between male and female by median sum of the knowledge scores . overall knowledge level in female participants was also lower than in male participants ( figure 2 ) . the median sum of the knowledge scores was 6 ( interquartile range : 211 ) among males , and 3 ( interquartile range : 18 ) among the females ( p for trend = 0.009 ) . there was a significant difference in level of knowledge among females and males . we assessed the diabetes knowledge briefly in three parts ( 1 ) general knowledge , ( 2 ) knowledge about the risk factors , and ( 3 ) knowledge about the symptoms , diagnostic signs , and complications of diabetes mellitus . one - third of the population answered correctly ( range 27.09%41.81% ) , and another one - third gave no answer to these questions ( range 33.44%39.80% ) , whereas another one third gave a wrong answer . among the karen community , 36.45% knew that old age is a risk factor for diabetes , 37.12% knew that they had diabetic relatives , 39.80% knew that obesity can lead to diabetes , 37.12% answered that exercise can prevent diabetes , 25.09% reported that pregnant women can have diabetes , and 41.81% said they should not eat excess fatty food for fear of diabetes . regarding the symptoms , diagnosis and complications of diabetes , less than 30% knew that fasting blood glucose level should be less than 100 mg% . a few of the rural karen ethnic residents could correctly answer questions in the third part of the questionnaire . among the respondents , 33.78% answered that diabetic patients could have thirst and polyuria , 34.78% knew that numbness of the limbs might link to diabetes , 35.12% recognized the importance of foot care in people living with diabetes , 32.78% answered that untreated diabetes could lead to blindness , and 34.78% knew that slow wound healing might be due to diabetes . overall , one - third of the sample population gave the correct answers ( range 32.78%34.78% ) while the other two - thirds were either unsure or gave wrong answers . overall , public knowledge of diabetes among karen ethnic rural community is at the lower extreme ( figure 1 ) . diabetes knowledge was compared between male and female by median sum of the knowledge scores . overall knowledge level in female participants was also lower than in male participants ( figure 2 ) . the median sum of the knowledge scores was 6 ( interquartile range : 211 ) among males , and 3 ( interquartile range : 18 ) among the females ( p for trend = 0.009 ) . there was a significant difference in level of knowledge among females and males . the number of diabetes cases is rising worldwide , and the burden of this is more severe in low- and middle - income countries.9,10 belonging to an ethnic minority has been reported as a risk factor for the development of type 2 diabetes.2 disadvantaged people in these groups are more prone to the consequences of a silent epidemic.11 it is important to assess the public knowledge of diabetes , especially in minority ethnic populations . today , rising public health education and reduced disparity between groups are compulsory for the prevention and care of diabetes in all settings.12 risk perception of a community is the basis for healthy behavioral changes within a community.13 perception of risk among people at risk can prevent the disease thus , we assessed the perception of diabetes risk factors among the karen ethnic rural community . we found only one - third of the study sample had knowledge about diabetes risk factors ( table 2 ) . most in the karen community did not know that elderly age , being overweight , and physical inactivity are risk factors for diabetes . initiation of measures to educate this minority population , including use of mass media , and printed materials in the local language , might be a useful strategy for risk communication . knowledge of diabetes symptoms is important for early diagnosis and accessing care . recognizing the symptoms of diabetes and its complications may bring the diabetic individuals to seek health care service . symptoms of diabetes complications are common presentations , even though these might be indicators of late diagnosis , or poor glycemic control . individuals lacking knowledge of these may not seek medical care in time , and consequently will have complications of diabetes.14 we assessed such knowledge in karen ethnic community ( table 2 ) . the histogram of knowledge scoring was skewed to the left and peaked at lower end of the scoring ( figure 1 ) . in contrast , the similar histogram for public knowledge of diabetes in singapore , was reported as totally skewed to the right side.15 the knowledge distribution among the karen ethnic sample was at the lower end , and pointed out a need for intervention . in fact , diabetes and its consequent cardiovascular disease risk are higher in women.7 there is also increasing concern about gestational diabetes;16 however , our observations pointed out that diabetes knowledge among female karen participants was lower than among males . this type of health inequality has also been reported in many other diseases.17 type 2 diabetes is more common in disadvantaged groups of women , such as those with least income and who are least educated.18 risk behaviors for type 2 diabetes have been highly prevalent even in the developed setting;19 moreover , poor women in the developing world tend to delay seeking care until symptoms are too severe.17 in the particular setting of this study , it is likely that women usually work in the home at housekeeping , and become housebound and sedentary . a television education program in the local language might reach these women.19 lack of diabetes knowledge can cause missed diagnosis , late diagnosis , and higher incidence of diabetes complications . creation of a peer - group gathering place for women , such as a health education club for mothers , and exercise club for housewives , may promote women s knowledge of diabetes and sharing of information . in contrast to the current study findings , in a previous study , the public knowledge among thai people was reported to be fairly high and there was no gender gap between thai men and women.20 it is obvious that diabetes health education is poor among the karen ethnic community . this might be due to a language barrier , which is a major obstacle to educating minority groups , as in other parts of asia.21 existing evidence suggests that such a situation calls for a culturally tailored and well - designed health education strategy.22 as this was a pilot study swiftly conducted to provide important information , there were weaknesses in the current study . the questionnaire used in this study was made easily comprehensible for rural karen residents , and study findings may not be generalizable to other settings . albeit , the findings are the first of their kind to our knowledge , and are meant to assist in guiding further studies . despite these limitations , this investigation contributed to our understanding of the health disparity in an ethnic minority population . it provided insights into the need for health education interventions for the prevention of diabetes in a rural ethnic community . the result of this study showed that karen ethnic men and women were less knowledgeable about all aspects of diabetes . public education about diabetes in this setting should be started urgently and carefully , to overcome language and cultural barriers and prevent diabetes epidemic among these rural people . our study finding is expected to be a step forward , in minimizing the disparity in diabetes health education .

background and purposethe public knowledge of diabetes is important for prevention of disease . this study aimed to evaluate knowledge of diabetes , risk factors , and the common warning signs of diabetes and complications among community participants in a rural karen ethnic community.methodsparticipants were asked to answer a questionnaire regarding their knowledge of diabetes . fasting blood glucose testing , blood pressure measurement , and body mass index ( bmi ) assessment were provided to the participants . the study was conducted at thasongyang district , tak province , thailand.resultsa total of 299 karen rural residents were included in the study . the median age was 45 years and median fasting blood glucose was 88 mg / dl . the response rate to the questionnaires was 91.97% . half of the participants knew diabetes is a noncommunicable disease needing lifelong treatment . overall , one - third of the community participants could correctly answer the knowledge assessment questions regarding risk factors and common features of diabetes . whereas the other two - thirds either gave a wrong answer or were not sure . female participants had poorer diabetes knowledge than the males.conclusionthe public knowledge of diabetes , as represented by this sample of the karen ethic community , is alarmingly low . there is significant gender difference in knowledge level . culturally tailored and gender - sensitive diabetes health education interventions are urgently needed in this minority ethnic community .

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it is well established that individuals with serious mental illness ( smi ) , which include diagnoses like schizophrenia and bipolar disorder , have a much higher rate of morbidity and mortality in comparison to the general population . specifically , diabetes impacts approximately 25% of individuals with smi , 2 to 3 times the rate than the general population . while this increased rate in diabetes is a multifaceted issue , factors such as lower utilization of preventive care and use of newer antipsychotic medications are likely to be associated with higher prevalence of diabetes in this population . with a growing emphasis on preventive and population - based care set forth by the affordable care act ( aca ) , new avenues of health care delivery are being promoted . through the aca , under section 2703 , these health home programs were designed to provide services to reduce barriers to care for those with chronic conditions . given the chronic impairments and difficulties faced by those with smi , it has been hypothesized that the use of the health home will improve early recognition of developing problems and provide access to much needed preventative somatic care . furthermore , given the psychiatric nature of the impairments faced by those with smi , it was hypothesized that placing the health home within the mental health clinic these individuals receive the majority of their care would be the ideal setting to maximize exposure to health home services . this case study presents some preliminary results on a new behavioral health home ( bhh ) , which focused on the use of population - based care to identify abnormal hemoglobin a1c ( hba1c ) levels and clarify the severity of such findings thus permitting enhanced , individualized interventions for individuals with smi . it was hypothesized that through the use of bhh services , patients would receive better quality of somatic care and that gaps in care would be readily identified . this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine . the division consists of 2 large outpatient mental health clinics , adult and child assertive community treatment ( act ) team , and an adult psychiatric rehabilitation program ( prp ) . in total , these settings serve 2000 individuals annually . although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005 , it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh . behavioral health home referrals were made to patients who were considered to require the most assistance . this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division . individuals in these intensive services were referred to the bhh staff by their mental health provider , and for those who consented , an intake was conducted by bhh initiate services . once enrolled , the bhh provided a minimum of 2 bhh services per month , coordinated by a nurse case manager . a wide variety of services qualified for bhh services , including care coordination , comprehensive transitional care , and health promotion . one service per month was also allowed to be delivered in a group format . given this allowance , a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi ( eg , diabetes and hypertension ) . while initially designed to alternate the somatic condition being tracked every month ( eg , may diabetes , june hypertension ) , it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment . it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness , nutritional guidelines , and value of regular exercise . patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics . changes in , as well as severity of , hba1c levels ( eg , normal , prediabetes , or type 2 diabetes ) were assessed during monthly chart reviews . this bhh was embedded in the division of community psychiatry at the university of maryland school of medicine . the division consists of 2 large outpatient mental health clinics , adult and child assertive community treatment ( act ) team , and an adult psychiatric rehabilitation program ( prp ) . in total , these settings serve 2000 individuals annually . although efforts to enhance somatic care for patients with smi had been a top priority of the division since 2005 , it was in 2013 that the state of maryland approved funding that allowed for the creation of the bhh . behavioral health home referrals were made to patients who were considered to require the most assistance . this was determined by selecting persons with smi who were enrolled in the more intensive act or prp services of the division . individuals in these intensive services were referred to the bhh staff by their mental health provider , and for those who consented , an intake was conducted by bhh initiate services . once enrolled , the bhh provided a minimum of 2 bhh services per month , coordinated by a nurse case manager . a wide variety of services qualified for bhh services , including care coordination , comprehensive transitional care , and health promotion . one service per month was also allowed to be delivered in a group format . given this allowance , a population - based health management program was created to target highly prevalent somatic conditions for individuals with smi ( eg , diabetes and hypertension ) . while initially designed to alternate the somatic condition being tracked every month ( eg , may diabetes , june hypertension ) , it was quickly realized that the high prevalence of these conditions required more monitoring time for a more comprehensive assessment . it was determined that monitoring a specific health condition for several consecutive months provided a better understanding of the population s care needs and impact of interventions that included additional education about the illness , nutritional guidelines , and value of regular exercise . patients who did not have their hba1c level drawn within 6 months of the monthly chart review were identified to be not in compliance with clinic recommendations for individuals prescribed atypical antipsychotics . changes in , as well as severity of , hba1c levels ( eg , normal , prediabetes , or type 2 diabetes ) were assessed during monthly chart reviews . a total of 120 ( 73 males and 47 females ) patients with smi were enrolled and receiving population - based health management services within 10 months of starting the bhh . the average age of the population was 50 years ( standard deviation = 12.75 years ) . of the 120 patients , 54 ( 45% ) were identified who did not have hba1c levels drawn per the clinic s recommended guideline of having hba1c drawn every 6 months for those prescribed antipsychotic medications . thirty - three patients had a diagnosis of type 2 diabetes , 4 of which were newly diagnosed as a result of the population - based health management ( phm ) initiative . another 39 patients were identified as having prediabetes ( hba1c levels between 5.7 and 6.4 ; table 1 ) . abbreviations : bhh , behavioral health home ; hba1c , hemoglobin a1c . of the 33 patients identified as having type 2 diabetes , of the 16 patients , 4 within their target goal achieved this range after initiating in bhh services . two patients who were originally in the prediabetes range when starting bhh services had higher hba1c levels and were in the type 2 diabetes after 10 months . another 7 patients had improvements in hba1c but remained above their target goal , and 4 patients had elevated hba1c since initiating bhh services . behavioral health home services were able to identify 54 individuals who did not have their hba1c levels checked according to the program s recommended guidelines for individuals being prescribed antipsychotic medications . this screening is particularly important for individuals with smi because , consistent with the literature , approximately 27% of this sample had a diagnosis of diabetes . also , another 4 patients were identified to be within diabetic range but did not have any diagnosis of diabetes . preliminary data indicate that the use of bhh services may be particularly useful in identifying gaps in care such as laboratory monitoring or missed diagnoses and allow for the delivery of preventative and individualized proactive care . in regard to health improvements , the data for the 33 patients who were within the type 2 diabetes range are mixed . slightly over 12% ( n = 4 ) of patients improved to be within the recommended guidelines for hba1c levels . another 21% ( n = 7 ) showed improvements in their hba1c levels , albeit still above the target goal . however , 6% ( n = 2 ) of individuals who were originally considered to be in the prediabetes range moved within the diabetic range , and another 12% ( n = 7 ) had higher hba1c levels in comparison to when they started receiving services . given identified problems with traditional primary care settings for this population , new models of care that can deliver patient - centered primary care within the mental health setting are needed to decrease the high rates of mortality and morbidity . while more empirical data are needed , preliminary results indicate that the use of a bhh may be particularly useful in understanding the unique characteristics of a specific population . this allows for earlier recognition of common medical disorders through more careful monitoring according to program guidelines and promotes proactive preventative care to prevent advancement of disease . as a result of these findings , a center for disease control and prevention diabetes prevention program group has been started within the mental health clinic and closer monitoring of clinician practice with regard to monitoring and referral to specific services is being done . it is important to note that the current report is limited by the fact that not all patients received equal amounts of phm services , given that enrollment occurred throughout the entirety of the 10 months . also , given the lack of a comparison group , meaningful statistical tests could not be conducted . future research examining the utility of bhh services would benefit from investigating both a clinical and a control sample . furthermore , given that these services are designed to provide more patient - centered care for individuals with smi , an examination of patient satisfaction with bhh and phm services is warranted . while the current data are mixed , the bhh may provide a cost - effective way to provide patient - centered care that helps identify gaps in somatic care for individuals with smi and provide this population with much need interventions .

objective : individuals with serious mental illness ( smi ) have higher rates of preventable diseases such as diabetes in comparison to the general population . while multifaceted , these high rates of preventable diseases in the population with smi may be partially attributed to limited access to primary care . a new program , the behavioral health home ( bhh ) , which allows for the delivery of somatic care coordination and population - based care , may provide this population with the much needed somatic coordination and education it requires.methods:the impact of the population - based health management program of the bhh identification and severity rating of glucose metabolism disorders was assessed during the initial 10 months of the bhh.results:multiple patients were identified who either were not having hemoglobin a1c ( hba1c ) levels drawn per recommended guidelines for individuals prescribed antipsychotic medications or were within diabetic range but did not have a diagnosis of diabetes . mixed results occurred in regard to patients hba1c levels while engaging in the bhh.conclusion:this case study provides some initial evidence for the utility of the bhh in regard to identifying patients who need preventive care .

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the american demographer and sociologist valerie oppenheimer wrote a series of influential articles in which she emphasized the role of men s socioeconomic position in demographic change , in particular in the declining rates of marriage and the underlying tendency to increasingly postpone and perhaps even forego marriage ( oppenheimer 1988 , 2000 , 2003 ; oppenheimer et al . i review oppenheimer s original theoretical study , i discuss how her study was held up in empirical research in europe , and i provide a new test of the theory for the european setting . in doing so , i try to resolve some remaining gaps in the empirical literature , and i evaluate whether the theory is equally valid in different countries that make up the european context . given the recent economic crisis in the united states and in europe , and the growing concerns about economic inequality , the influence of men s economic position on marriage and family formation remains a vital concern . at the time oppenheimer began writing her articles on how men s economic position influenced marriage formation in the late 1980s and early 1990s this was generally not a popular idea . the declining rates of marriage and increasing rates of divorce were typically conceptualized in terms of an " erosion of marriage . " one theory looked for the culprit in the growing economic role of women in society . this theory was voiced by demographers and economists working from a micro - economic perspective ( becker 1981 ; espenshade 1985 ; farley 1988 ) , although , as oppenheimer noted ( 1988 , p. 575 ) , it bore a strong resemblance to classic sociological theories formulated by functionalists like talcot parsons ( parsons 1949 ) . the explanation basically argued that more symmetrical economic roles of men and women would lead to a decline in the gains to marriage , or to put it in parsonian terms , would undermine marital solidarity . the second explanation argued that the decline of marriage was related to value change , and in particular to the increasing need for individual autonomy on the one hand , and the ideological condemnation of traditional institutions like marriage on the other . this second perspective was expressed more strongly by european demographers like lesthaeghe and van de kaa although it was also used by the influential american demographers at the time ( bumpass 1990 ; rindfuss and van den heuvel 1990 ) . in their second demographic transition theory , lesthaeghe and van de kaa argued that ideological change in combination with secularization was driving not only the postponement of marriage , but also the increase in cohabitation , the rise in divorce , and the decline of fertility ( lesthaeghe 1983 ; lesthaeghe and meekers 1986 ; lesthaeghe and surkuyn 1988 ; van de kaa 1987 ) . while the first explanation saw the engine of the demographic transition in economic change , the second emphasized the primacy of cultural change . both theories , however , were pessimistic about the future of marriage : the economic perspective saw marriage as incompatible with symmetrical gender roles , the second saw it as incompatible with individualistic values . while there was a considerable debate between the proponents of economic and cultural explanations , oppenheimer criticized both perspectives . for example , she noted that there were no signs of a so - called independence effect . women with attractive economic resources were not less likely to enter marriage , as would be predicted from the micro - economic perspective ( oppenheimer and lew 1995 ) . although women s employment and education had an effect on fertility and divorce , this did not appear to be the case for marriage timing ( oppenheimer 1997 ) . oppenheimer also had empirical critique on the cultural perspective . when looking at simple descriptive statistics on what people want for themselves on people s hopes and desires she noted that the majority of both single men and women still wanted to be married ( oppenheimer 1994 ) . the anti - marriage ideology may have existed in feminist circles or in the pop culture of the sixties , but it had not spread to a larger audience in the way that , for example , egalitarian gender norms had done . oppenheimer also had theoretical criticisms of the two explanations ( oppenheimer 1994 , 1997 ) . first , she believed that the theories were basically about nonmarriage and not about delays in marriage . as other demographers also had observed , the declining marriage rate was primarily driven by increases in the age at marriage , and not so much by a decline in the proportion of persons who marry eventually , although the latter could of course not yet be observed in the late 1980s . this seems by and large correct now , although the proportion of the marrying persons among the lower educated in the united states did appear to decline ( goldstein and kenney 2001 ) . a second part of her theoretical critique was against the micro - economic model of specialization . quoting historical demographic work , oppenheimer noted that wives in the past had always worked for pay when circumstances required this . wives worked to make ends meet when the husband was not making enough money , when he was unemployed , or when household costs were temporarily pressing ( oppenheimer 1982 ) . oppenheimer argued that specialization in marriage is an inflexible and risky strategy in many different societal contexts . if marriage was not based on a model of full specialization in the more distant past , oppenheimer argued , why would it then cease to exist in the modern era in which wives began to work ? oppenheimer not only criticized the then dominant perspectives on demographic change , she also presented an alternative . her explanation can be placed in the economic rather than the cultural camp , but it was different in that it focused on men rather than women . during the 1980s and 1990s , young men s economic position in the united states had deteriorated quickly , especially for those with little schooling . in the poor and uncertain economic prospects of young men , oppenheimer saw an important potential for understanding the decline of marriage . because the earlier explanation had focused more on women especially through arguments about women s economic independence one could say that oppenheimer was in fact " bringing men back into the debate . " first , she reinstated older malthusian ideas about the economic costs of marriage ( hajnal 1965 ; easterlin 1980 ) . as setting up and running a household costs money , men who are unable to fulfill oppenheimer recognized that this traditional male - breadwinner hypothesis may have lost some of its force when gender roles become more symmetrical . nonetheless , she argued that it would also be naive to expect men s economic resources to become unimportant in influencing marriage prospects : this would be " throwing out the baby with the bathwater . " the second way in which she brought men back in the debate was through her uncertainty hypothesis ( oppenheimer 1988 ) . the argument is that unstable careers , as indicated by low - status jobs , nonemployment , and irregular and temporary employment , signal uncertainty . this uncertainty applies not only to whether the husband will be able to provide in the future , but also to the type of life he will lead . work structures the lifestyle a person will develop , and when men have not yet settled in their career it is difficult to predict what married life will be like . in this way . an important difference between the breadwinner and the uncertainty hypotheses is that the former focuses primarily on the financial aspects of employment whereas the latter is also concerned with its social consequences . an implication is that the neo - malthusian argument would be fully covered by effects of income , whereas the uncertainty argument would also be reflected in indicators like irregular attachment to the labor market , the amount of work experience , career trajectories , and temporary employment . in a separate article , oppenheimer also developed and operationalized the concept of a stopgap job , i.e. , a job that is not a reflection of an employee s educational credentials and that is meant as temporary by both employer and employee ( oppenheimer and kalmijn 1995 ) . men in such stopgap jobs would postpone marriage because they are not settled in their career and therefore can not yet make a suitable match in the marriage market . compared to the other two perspectives , oppenheimer s theory has a more optimistic implication for the future of marriage . after all , female labor force participation was unlikely to decline in the future and individualism did not appear to be receding . in oppenheimer s theory , the economic position of young men largely depends on macro - economic conditions . because unemployment rates tend to have cyclical rather than linear trend patterns , the economic position of young men could improve and this would then have positive repercussions for marriage . moreover , the theory only implies the postponement of marriage until men accumulate more work experience and become settled in their career , and not an erosion of the institution of marriage , as the other theories seem to imply . oppenheimer 's explanation had a second attractive feature : it could also explain another important demographic trend , namely the rise in cohabitation ( oppenheimer 2003 ) . oppenheimer argued that a man s failure to provide economically would be less of a problem for cohabitation than for marriage . for many couples , cohabitation is a trial stage before marriage , and it may be that uncertainty about a young man s position is more tolerable during the cohabiting stage than it would be for a long - term commitment to marriage . assuming that the costs of breaking up a cohabiting union are lower , cohabitation can therefore provide a way for couples to reduce uncertainty about future career prospects . in a sense , oppenheimer argued that a cultural innovation like cohabitation before marriage ( on a massive scale ) was the outcome of economic needs rather than the result of ideological change . in line with this , other authors even argued that cohabitation is a rational response to uncertainty : a flexible partnership well - suited for a flexible labor market ( mills et al . american research generally supports the view that poor economic prospects for men are associated with a delay in marriage . this has been demonstrated for a range of indicators , including employment per se , unstable employment , low earnings , and other indicators of career " immaturity " ( clarkberg 1999 ; lichter et al . 1992 ; lloyd and south 1996 ; mare and winship 1991 ; oppenheimer 2003 ; oppenheimer et al . 1997 ; sassler and schoen 1999 ; smock and manning 1997 ; sweeney 2002 ; xie et al . there is also evidence in the united states that cohabitation is less strongly influenced by men s economic position than marriage , although there is no clear reverse income effect , i.e. , that the poor are being selected into cohabiting unions . furthermore , in the united states , the income effect on marriage timing appears to be stable over time . sweeney ( 2002 ) compared two cohorts in the united states and found that in the cohort marrying during the 1980s and 1990s , men s income had an equally strong positive effect on the entry into marriage as in the cohort marrying during the 1960s and 1970s ( sweeney 2002 ) . in this article , i develop a new test of oppenheimer s theory for the european context . first , the demographic trends that occurred in europe were similar , although sometimes less dramatic and sometimes occurring later . the age at marriage has risen , the rate of marriage has declined , and cohabitation has increased as well ( kiernan 2002 ; lesthaeghe 1983 ; van de kaa 1987 ) . second , many european countries experienced economic problems that were similar to those in the united states . they were especially salient for outsiders on the labor market , such as young adults , ethnic minorities , and women . several authors argued that partly in response to economic globalization , young european men ( and women ) faced increasing levels of economic uncertainty in their transition from school to work ( blossfeld et al . 2005 ) . in many european countries , especially in southern europe , levels of youth unemployment were even higher than in the united states , a phenomenon which has often been linked to the higher degrees of employment protection in europe ( mller and gangl 2003 ; nickell 1997 ) . there are also reasons to believe that the theory would be less applicable to europe . one counter argument lies in the role of the welfare state . in several european societies , and particularly in social - democratic welfare states like sweden , denmark , and the netherlands , social security is more generous and more universally provided than it is in the united states ( arts and gelissen 2002 ; esping - andersen 1993 ) . this means that in many european countries , young men receive unemployment benefits when they are out of work . moreover , for those who have never worked , basic welfare is provided , albeit at a minimum level . as a result following the neo - malthusian argument , it could thus be argued that employment problems do not per se lead to marriage postponement in europe . a rebuttal of this point is that oppenheimer s argument about uncertainty and assortative mating , which is not only about money , but also about stability and predictability , could still apply to europe . a young man who is on unemployment benefits remains an uncertain candidate on the marriage market even if he has the financial means to support a household at that point in time . another important difference between the american and the european case lies in the degree of heterogeneity . although the united states is certainly not a homogeneous country there are important ethnic , racial , and regional differences it is fair to say that europe is more heterogeneous ( at least regionally ) than the united states . in comparative studies , it has often been argued that european countries can be rated on a continuum from more traditional societies such as spain , greece , and italy on the one hand , to more modern and more economically developed societies such as sweden and the netherlands on the other ( hagenaars et al . these differences are expressed in a number of social and cultural domains , including differences in marriage and family living . for example , in more traditional european societies , cohabitation and divorce are less common and less accepted , marriage has a higher social status , gender roles in marriage are more unequal , female labor force participation is lower , and extended family ties are stronger ( hans - peter blossfeld and hakim 1997 ; gelissen 2003 ; kalmijn 2003 ; knudsen and waerness 2008 ; reher 1998 ) . these indicators are strongly correlated , both with each other and with the level of economic development in a country ( gdp ) . this degree of heterogeneity suggests that oppenheimer s theory may not apply equally to all european countries . for example , in contexts where gender roles are more egalitarian , men s economic situation could be less important for the entry into marriage and cohabitation . in these settings , men are not the only breadwinners and women s economic resources should be of growing importance , making men s economic resources less important . what has the evidence in europe shown so far ? perhaps the most important source of evidence comes from a large multi - nation project initiated by the german sociologist blossfeld and his colleagues ( blossfeld et al . in this project , blossfeld brought together a number of demographers and sociologists from different parts of the world ( with an overrepresentation of european countries ) , with the aim of examining the effect of men and women s individual economic resources on the timing of marriage and parenthood . while the authors used their own country - specific longitudinal data , they used similar methods and variables , leading to a reasonably uniform and comparable set of outcomes . the project s goal was to test the exact same set of hypotheses in each country . the hypotheses were borrowed in part from oppenheimer s work but they were translated by mills and blossfeld to make them fit for a broader societal setting ( mills and blossfeld 2005 ) . the articles were combined in a volume for which oppenheimer wrote the foreword ( blossfeld et al . the articles in blossfeld s volume provide generally positive evidence for the theory in the european countries studied ( germany , the netherlands , france , sweden , hungary , great britain , italy , and spain ) . in virtually all countries , school enrollment one of the indicators of uncertainty negatively affected the entry into marriage . more importantly , men s unemployment appeared to lower the chances of entering marriage in most countries ( bernardi and nazio 2005 ; kieffer et al . 2005 ; liefbroer 2005 ; noguera et al . 2005 ; robert and bukodi 2005 ) . in britain , an effect of unemployment was observed only on the transition from cohabitation to marriage , and not on the transition from being single to living together ( francesconi and golsch 2005 ) . in sweden , only unemployment after leaving school appeared to delay marriage formation , not unemployment after a period of employment ( bygren et al . 2005 ) . some evidence was also found for the effect of temporary contracts . in italy , spain , france , and the netherlands , it was shown that men who were employed temporarily were less likely to enter marriage than men who had permanent employment . in germany and hungary , there was no effect of temporary work , however , and in several other countries , the effect could not be studied . a recent analyses of fertility in europe has also pointed to the delaying effect of temporary contracts ( adsera 2011 ) . outside the blossfeld project , there were a number of important individual articles in which aspects of oppenheimer s theory were tested . for example , in sweden , it was found that men s employment increased the chances of union formation while it did not affect the chances of marriage after cohabitation ( bracher and santow 1998 ) . in norway , men s employment increased the chances of marrying after being single and the chance of marrying after living together ( kravdal 1999 ) . in the netherlands , men s employment had a stronger effect on direct marriage than on cohabitation but there was no effect of employment on marriage after cohabitation ( kalmijn and luijkx 2005 ; cf . the evidence outside europe ( i.e. , israel ) has been supportive as well ( raz - yurovich 2010 ) , as has been the evidence in central and eastern europe , a region not included in the present article ( gerber and berman 2010 ) . while the role of employment has often been studied in europe , less is known about how men s income and earnings affect union formation . many of the studies discussed above were based on retrospective rather than prospective longitudinal data . the panel data that exist have been collected by economists and labor market researchers and do not always have the desirable demographic indicators . because income can not be measured well in a retrospective fashion , this has also meant that we know little about the income effects on marriage and divorce in europe . this is unfortunate because employment and income need to be examined simultaneously , especially if one wants to make a distinction between the neo - malthusian breadwinner hypothesis on the one hand , and oppenheimer s uncertainty hypothesis on the other . because in many european welfare states nonemployment does not , per se , another drawback of the prevailing evidence is that most studies are based on single countries . blossfeld s multi - nation project is clearly a major step forward in trying to summarize the evidence for europe as a whole , but the analyses are not pooled so the results can only be summarized verbally . moreover , possible differences that exist between countries can be described but they can not be compared or tested in a more rigorous fashion . for these reasons , in answering this question , i not only look at employment , but also i look at men s income , work experience , and type of labor contract . by looking at income and employment patterns simultaneously , i obtain more direct evidence on the underlying mechanisms . the period for which i answer unemployment rates increased substantially in the early 1990s before declining again in the mid to late 1990s ( oecd 2009 ) . in italy , greece , and belgium , unemployment remained high in the late 1990s but began to decline later , in the early 2000s . in other words , for most countries , the period that i examine covers a recovery stage of the economy , a stage which should have been positive for marriage and family formation . second , are the effects of men s economic position similar or different for the chances of entering marriage and the chances of entering a cohabiting union ? with this part of the study , i replicate the last influential study of oppenheimer ( 2003 ) , in which she studied this issue for the united states . we would expect effects to be weaker for cohabitation than for marriage : marriage would require a stronger economic underpinning than cohabitation ( kravdal 1999 ; oppenheimer 2003 ) . in addition , i examine the chances that cohabiting unions turn into marriage . here too , oppenheimer expects men s economic position to have an effect , but because those who cohabit already have an independent household , the effects of income will probably be weaker . third , to what extent are the effects of men s economic position on union formation different across societal contexts ? in this part , i focus on differences between traditional and egalitarian societies . the expectation is that men s economic characteristics remain important in traditional societies but are less important in more modern , egalitarian ones . by looking at differences among societal contexts , i try to generalize the cross - cohort comparison that sweeney ( 2002 ) made for the united states . answering this question is also of more general importance because if we find conditions under which the theory is ( not ) true , this could in principle lead to theoretical progress in the field . i use panel data that are collected in the same format for a number of european countries , i.e. , the european community household panel ( echp ) . the echp was an annual panel survey held between 1994 and 2001 ( behr et al . samples are large and representative , and ( almost ) the same questionnaire was used each year . for the analyses in this article , i use data from 13 countries : denmark , finland , germany , austria , the netherlands , belgium , france , the united kingdom , ireland , portugal , spain , italy , and greece . my sample is limited to men who were never married in the first wave of the panel . although i am able to exclude previously married men , i can not exclude men who ended a cohabiting union before the panel began . never married men who were cohabiting in the first wave are also included because these men can make the transition from cohabitation to marriage . the first wave of data from the netherlands is excluded because no information is available on cohabitation status there have been previous demographic analyses of the echp most notably by adsera ( 2011 ) who analyzes the effect of individual and aggregate labor market characteristics on fertility . although adsera focused more on women than on men , her general conclusion is that labor market uncertainty is very influential in delaying fertility , in line with the perspective suggested above . in the current article , we go back one step by analyzing how labor market uncertainty affects union formation , a transition which probably remains the most important necessary condition for family formation . i use discrete time event - history analysis by estimating logistic regression models on a person - year file . the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data , such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) . the first logit model is estimated for person - years in which men are single and never married . the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 ( union formation ) . in the second logit model , i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred . this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) . the sequential approach does not need the assumption implicit in a competing risk model that the two choices this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al . if a person is single in year t , missing in year t + 1 , and married or cohabiting in year t + 2 , i also regarded this as an event . duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) . blossfeld used log ( age a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample . there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage . the second logit model is estimated for person - years in which men were living with a partner without being married . the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 ( marriage after cohabitation ) . respondents who were living alone in wave t + 1 are truncated . hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t + 1 , and married in year t + 2 , i also regarded this as an event . duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative age is obviously less ideal than duration since persons enter a cohabiting union at different ages . it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave . hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave . all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1 , the independent variables precede the dependent variable in time . the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week . the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15 h. given that a man is employed , i also considered the type of contract he has . temporary contracts include fixed - term or short - term contracts , casual work with no contract , and " some other working arrangement " that is not a permanent contract ( all included within the 15 + hours category ) . the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract . i also include whether a person is enrolled in full - time schooling in the interview week . data on work history are obtained from a monthly calendar that all respondents had to fill out . for the year t 1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called " active " ) . i checked whether the effects of this variable were different when excluding the netherlands but this was not the case . i consider personal income and not only income from work since social security income may also provide a stable source of income . ideally , we would like to measure the income a man had in the interview month or in the 12 months before the interview . unfortunately , incomes are measured for ( full ) calendar years only . to solve this , i took a weighted average of the income in calendar year t 1 and the income in calendar year t , using information on the month of interview . for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 . it is noted that some of the income in year t may be earned after a man marries . marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight . i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries i first calculated income quintiles in each country using data from all men in the first wave of the echp . next , i used these quintiles to categorize the men in the person - year file . i also use a linear income variable which is coded 15 for the five quintiles . three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) . health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al . descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany ( n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark ( n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total ( n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level . first , i considered a measure of the division of household labor in marriage in a society . this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) . the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) . . countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r = .74 ) . the most traditional societies are southern european countries , the most egalitarian are northern european countries . i construct a single macro - level indicator , which is the means of the two standardized items . i use discrete time event - history analysis by estimating logistic regression models on a person - year file . the person - year file begins at the first wave and ends in the last wave or when a transition is made . as is the case with all event - history analyses of panel data , such left truncation problems can be solved in principle , but not without losing our time - varying independent variables ( guo 1993 ) . the first logit model is estimated for person - years in which men are single and never married . the dependent variable is whether a man is living without a partner in wave t and living with a partner in wave t + 1 ( union formation ) . in the second logit model , i estimated which choice was made , cohabitation or marriage , using only the person - years in which the event occurred . this sequential approach is slightly different from the approach taken by oppenheimer , who estimated multinomial logit ( i.e. , competing risk ) models ( 2003 ) . the sequential approach does not need the assumption implicit in a competing risk model that the two choices this assumption is problematic because it is plausible that unmeasured factors like personality , wealth , attractiveness , and the like , influence marriage and cohabitation to the same extent ( hill et al . if a person is single in year t , missing in year t + 1 , and married or cohabiting in year t + 2 , i also regarded this as an event . duration dependency is modeled with two age effects , according to the approach developed by blossfeld and huinink ( 1991 ) . blossfeld used log ( age a1 ) and log ( a2age ) to capture the nonlinear age - dependency of union formation , where a1 is the lowest and a2 the highest age in the sample . there were 4,492 transitions to a first union of which 2,499 were to cohabitation and 1,993 to marriage . the second logit model is estimated for person - years in which men were living with a partner without being married . the dependent variable is defined as living with a partner unmarried in wave t and being married in wave t + 1 ( marriage after cohabitation ) . respondents who were living alone in wave t + 1 are truncated . hence , separation is treated as a competing risk . if a person is cohabiting in year t , not in the panel in year t + 1 , and married in year t + 2 , i also regarded this as an event . duration dependency could not be modeled directly because for those who are in a cohabitating union in the first wave of the panel , no data on the start of the union is available . as an alternative , age is obviously less ideal than duration since persons enter a cohabiting union at different ages . it is noted that there was no question in the interview about whether the partner in one wave was the same partner as in the previous wave . hence , i could not check if a man changed ( cohabiting ) partners between the subsequent waves , nor could i check if the married partner was the same person as the cohabiting partner in the previous wave . all independent variables , except where noted , refer to time t. as the dependent variables refer to whether or not a transition was made between the time t and t + 1 , the independent variables precede the dependent variable in time . the first indicates a man is working on a paid job or is self - employed at the time of the interview for at least 15 h a week . the second variable indicates other less common forms of employment , such as military service , apprenticeships , unpaid family work , and working less than 15 h. given that a man is employed , i also considered the type of contract he has . temporary contracts include fixed - term or short - term contracts , casual work with no contract , and " some other working arrangement " that is not a permanent contract ( all included within the 15 + hours category ) . the coding is cumulative so that the effect of temporary work refers to the difference between the men with a temporary contract and men with a permanent contract . i also include whether a person is enrolled in full - time schooling in the interview week . data on work history are obtained from a monthly calendar that all respondents had to fill out . for the year t 1 , i counted the number of months that a man was employed , self - employed , or in school ( hereafter called " active " ) . i checked whether the effects of this variable were different when excluding the netherlands but this was not the case . i consider personal income and not only income from work since social security income may also provide a stable source of income . ideally , we would like to measure the income a man had in the interview month or in the 12 months before the interview . unfortunately , incomes are measured for ( full ) calendar years only . to solve this , i took a weighted average of the income in calendar year t 1 and the income in calendar year t , using information on the month of interview . for example , if the interview was in september 1994 , income for that person - year is 9/12 of the income earned in 1994 plus 3/12 of the income earned in 1993 . it is noted that some of the income in year t may be earned after a man marries . marrying in the year of the interview is more likely when the interview took place early in the year , but then , this income receives a lower weight . i corrected all incomes for changes in the consumer price index and converted them to pounds sterling . to estimate effects of income in a comparable way across countries , i first calculated income quintiles in each country using data from all men in the first wave of the echp . next , i used these quintiles to categorize the men in the person - year file . i also use a linear income variable which is coded 15 for the five quintiles . three control variables are used which may affect union formation : general health ( time varying ) , the highest level of educational attainment , and the year of the interview ( using dummies ) . health is controlled because having a poor health may be detrimental for finding a partner and is also related to job and income opportunities ( waldron et al . descriptive findings for the independent variables for each country are presented in table 1.table 1means of independent variables in person - year file by countryyear ( 18)agegeneral health ( 15)secondary educationtertiary educationworking 15 + hoursother worktemporary contractenrolled in schoolmonths active ( 012)relative income ( 15)income in poundsuk ( n = 7,585)4.0731.883.910.080.680.750.030.050.059.943.0410546ireland ( n = 7,741)3.4032.314.370.440.230.620.050.080.129.522.707184germany ( n = 9,581)4.0630.123.790.640.260.680.090.070.1210.412.559536austria ( n = 5,782)4.4829.124.400.790.080.700.080.060.1310.672.508995france ( n = 11,385)3.9031.993.870.420.320.600.060.090.149.492.528537belgium ( n = 3,842)3.8132.254.170.440.440.630.010.080.199.962.469182netherlands ( n = 5,248)4.5232.664.110.540.220.730.030.100.149.002.449841italy ( n = 18,316)4.0229.884.110.520.140.530.060.070.178.552.494696greece ( n = 7,942)3.8929.644.690.470.280.610.110.120.119.572.574220spain ( n = 12,764)3.8330.834.080.250.390.550.040.200.168.682.544553portugal ( n = 10,097)4.0929.423.690.220.110.680.040.150.1210.062.883062finland ( n = 5,360)5.0132.954.060.560.260.610.040.090.179.452.5511526denmark ( n = 5,381)3.8833.384.420.450.400.710.060.080.1110.202.9912814total ( n = 111,024)4.0330.974.090.430.280.630.060.100.139.502.627227source : echp ( own calculations ) means of independent variables in person - year file by country source : echp ( own calculations ) to explore differences across societies , i constructed two measures at the macro level . first , i considered a measure of the division of household labor in marriage in a society . this measure was obtained from a article by knudsen and waerness ( 2008 ) and measures the extent to which husbands and wives share four household tasks ( i.e. , laundry , grocery shopping , meal preparation , caring for sick family members ) . the second measure is the female labor force participation rate in the 1990s , which is defined as the percentage of women aged 2049 who are active in the labor force ( obtained from ilo , geneva ) . . countries in which wives participate more often in the labor force are also countries in which the household tasks are divided more equally ( r = .74 ) . the most traditional societies are southern european countries , the most egalitarian are northern european countries . i construct a single macro - level indicator , which is the means of the two standardized items figure 1 shows that there are small differences within europe in the age at first union entry . in most countries , male age at entry is low in portugal and austria , and high in the netherlands and italy . for example , the age at union formation is quite high in italy and greece where the family is a strong institution . europe , youth unemployment is high . when we look at the type of first union chosen by men , presented in the left side of fig . 1 even more in northern europe , direct marriage is a minority experience . in many countries , more than 80% of men cohabit first . in southern europe , the proportions are almost reversed : 6080% of the men in these countries marry directly . the united kingdom resembles western europe but ireland resembles the south , with most men choosing marriage as their first way to enter a union . 1men s first union formation in 13 countries men s first union formation in 13 countries the effects of men s economic characteristics on union entry are presented in table 2 . the odds of entering a union are 58% higher for employed men than for men who are not employed and not in school ( e ) . the type of labor contract also matters for the choice between marriage and cohabitation . compared to men with a permanent job , men with a temporary job who enter a union are 23% less likely to choose marriage than cohabitation . we also note that school enrollment has a negative effect on union entry while it does not affect the type of union.table 2discrete time event history models of union formation : logit regression coefficients and p - values in parentheses union formationmarriage vs. cohabitationmarriage after cohabitationmodel amodel bmodel cmodel dmodel emodel fmodel gmodel hmodel iln ( age15)0.841 * * * ( 0.000)0.661 * * * ( 0.000)0.676 * * * ( 0.000)0.746 * * * ( 0.000)0.745 * * * ( 0.000)0.741 * * * ( 0.000)0.230 * ( 0.040)0.317 * * ( 0.006)0.316 * * ( 0.006)ln ( 65age)1.872 * * * ( 0.000)1.816 * * * ( 0.000)1.847 * * * ( 0.000)0.810 * * * ( 0.000)0.813 * * * ( 0.000)0.809 * * * ( 0.000)0.296 * ( 0.038)0.264 ( 0.061)0.268 ( 0.058)general health0.106 * * * ( 0.000)0.100 * * * ( 0.000)0.098 * * * ( 0.000)0.135 * ( 0.012)0.135 * ( 0.013)0.135 * ( 0.012)0.107 * * ( 0.008)0.104 * ( 0.010)0.103 * ( 0.011)education secondary versus primary0.029 ( 0.491)0.002 ( 0.955)0.007 ( 0.867)0.046 ( 0.655)0.035 ( 0.731)0.047 ( 0.648)0.005 ( 0.951)0.012 ( 0.890)0.014 ( 0.873)education tertiary versus primary0.225 * * * ( 0.000)0.141 * * ( 0.003)0.122 * * ( 0.010)0.113 ( 0.311)0.149 ( 0.194)0.111 ( 0.326)0.408 * * * ( 0.000)0.361 * * * ( 0.000)0.349 * * * ( 0.000)working 15 + hours versus no work / school0.457 * * * ( 0.000)0.193 * * ( 0.003)0.220 * * * ( 0.001)0.450 * * ( 0.003)0.419 * * ( 0.007)0.445 * * ( 0.004)0.178 ( 0.166)0.090 ( 0.495)0.102 ( 0.434)other employed versus no work / school0.144 ( 0.088)0.204 * ( 0.016)0.188 * ( 0.026)0.279 ( 0.176)0.350 ( 0.096)0.282 ( 0.173)0.237 ( 0.277)0.250 ( 0.254)0.245 ( 0.263)temporary versus fixed contract0.067 ( 0.182)0.026 ( 0.609)0.031 ( 0.544)0.253 * ( 0.033)0.262 * ( 0.028)0.251 * ( 0.035)0.120 ( 0.249)0.086 ( 0.410)0.090 ( 0.386)in school versus no versus no work / school0.623 * * * ( 0.000)0.431***(0.000)0.468 * * * ( 0.000)0.180 ( 0.388)0.089 ( 0.679)0.173 ( 0.412)0.515 * ( 0.015)0.410 ( 0.059)0.424 * ( 0.046)months worked last 2 years0.022 * * * ( 0.000)0.006 ( 0.243)0.007 ( 0.178)0.008 ( 0.518)0.011 ( 0.405)0.009 ( 0.504)0.031 * ( 0.010)0.021 ( 0.089)0.023 ( 0.066)first income quintile versus third quintile0.604 * * * ( 0.000)0.272 ( 0.054)0.256 * ( 0.044)second income quintile versus third quintile0.253 * * * ( 0.000)0.039 ( 0.735)0.182 * ( 0.037)fourth income quintile versus third quintile0.232 * * * ( 0.000)0.136 ( 0.210)0.094 ( 0.224)fifth income quintile versus third quintile0.366 * * * ( 0.000)0.127 ( 0.328)0.109 ( 0.233)linear income ( 15)0.239 * * * ( 0.000)0.007 ( 0.861)0.098 * * * ( 0.001)constant11.814 * * * ( 0.000)10.677 * * * ( 0.000)11.595 * * * ( 0.000)7.903 * * * ( 0.000)7.789 * * * ( 0.000)7.895 * * * ( 0.000)3.346 * * * ( 0.000)2.771 * * * ( 0.000)3.138 * * * ( 0.000)n776217762177621447244724472116781167811678224024602451162516301625335348346note : controlled for country and year dummies*p < .05 , * * p < .01 , * * * p < .001 discrete time event history models of union formation : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies * p < .05 , * * p < .01 , * * * p < .001 next to the effects of current employment characteristics , we see effects of men s work history . the more months men were employed or otherwise active in the past calendar year , the more likely they are to enter a union in the next two calendar years . men who were active for an entire year were e = 30% more likely to enter a union in the next 2 years than men who were inactive the entire year . this effect comes on top of the effect of a man s current employment situation . there is no effect of work history on the choice between marriage and cohabitation . in sum , the findings from model a and model d confirm that men who are not yet settled in their career postpone union formation . the findings also confirm that cohabitation is less sensitive to economic insecurity than marriage , although this applies more to employment per se and the type of employment than to a man s work history . in model the poorest quintile of men are 45% less likely to enter a union than the middle quintile , while the richest quintile are 44% more likely to do so . the income effects increase monotonically but the differences between the quintiles become smaller at higher income levels . a formal test shows that the effect is not strictly linear : the nonlinear model for union formation ( model b ) has a better fit than the linear model ( model c , = 9.3 , p = .03 ) . when we look at the income effect on choice between the marriage and cohabitation , we only see one marginally significant effect . men in the lowest quintile who enter a union are 31% more likely than the middle quintile to choose cohabitation rather than marriage ( p = .054 ) . this result seems to confirm the notion of cohabitation as the poor man s marriage , although the poor are still more likely not to enter a union in the first place . when we compare the two models for union entry ( model a and b ) , the employment effects that we observed in model a are reduced by more than half once income is added to the model . in other words , the effects of employment and work history run in part via income . nevertheless , the employment effect remains statistically significant even when income is included , which means that the influence of employment on union formation also has a non - financial element . it is also interesting to observe that the effects of employment and temporary jobs on the choice between the marriage and cohabitation are not affected by whether or not income is added ( compare model d and e ) . hence , the non - financial aspects of work are more important for the type of union than for the chance of entering a union in the first place . in this sense , oppenheimer s uncertainty theory seems to work better for the type of union than for union formation per se . this confirms theories about selection into marriage and suggests that screening or selection may be less strong for cohabitation . education , finally , has a positive effect on union entry but these effects are reduced when income is controlled for . in the last columns of table 2 , we focus on marriage formation after cohabitation ( model g , h , and i ) . we see that it is not affected by employment , but being enrolled in school does reduce the chance of marrying . we also find that men who worked more months in the past are more likely to marry . the higher the men s income during cohabitation , the more likely they are to change from cohabitation to marriage . when comparing the linear income effect on marriage after cohabitation with the linear effect on initial union formation , it appears that income is less important for marriage after cohabitation ( b = .10 ) than it is for union formation after being single ( b = .21 ) . this is in line with the neo - malthusian hypothesis , which suggests that it is primarily for initial union entry that the costs of setting up a household play a role ( although the wedding is a cost factor which applies specifically to marriage ) . men in good health are more likely to experience a transition from cohabitation to marriage , suggesting that the selection effects also play a role in the decision to marry , not only in the decision to live together . we also see that higher educated cohabiting men are more likely to marry , hence , for the higher educated , cohabitation is less often seen as a long - term option . do the effects we found vary across societal contexts ? to assess this , i present interaction effects of key independent variables with the macro - level indicator of traditional versus egalitarian societies . the p - values are based on standard errors that are corrected for the clustering of cases within countries . this yields a more conservative test for macro - level effects and is a good alternative to multilevel models when the number of units at the macro level is limited . to check for outliers at the country level , i calculated dfbetas for the interaction effects of modernization and employment , and of modernization and income , and re - estimated the models while leaving out countries for which dfbetas exceeded the critical value ( kalmijn 2010 ; ruiter and de graaf 2006 ) . dfbeta is calculated as the difference in an effect with and without the outlier divided by the standard error of the effect . these outlier analyses are presented in the third and fourth columns of table 3.table 3discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parenthesesmodel a all casesmodel b all casesmodel a w / o outliersmodel b w / o outliersln ( age15)0.953 * * * ( 0.000)0.781 * * ( 0.002)0.943 * * * ( 0.001)0.686 * ( 0.036)ln ( 65age)1.970 * * * ( 0.000)1.956 * * * ( 0.000)1.934 * * * ( 0.001)2.040 * ( 0.010)general health0.121 * * ( 0.001)0.107 * * ( 0.002)0.102 * * ( 0.006)0.119 * ( 0.016)education secondary versus primary0.071 ( 0.287)0.023 ( 0.732)0.031 ( 0.638)0.115 ( 0.079)education tertiary versus primary0.271 * * * ( 0.000)0.152 * ( 0.014)0.248 * * * ( 0.000)0.224 * * * ( 0.001)working 15 + hours versus no work / school0.501 * * * ( 0.000)0.194 * * * ( 0.000)0.549 * * * ( 0.000)0.175 * * * ( 0.000)in school versus no versus no work / school0.672 * * * ( 0.000)0.604 * * * ( 0.000)0.614 * * * ( 0.000)0.673 * * * ( 0.000)income linear0.242 * * * ( 0.000)0.243 * * * ( 0.000)index ( traditional egalitarian)1.916 ( 0.177)2.075 ( 0.144)2.092 ( 0.262)2.039 ( 0.327)index * ln ( age15)0.278 ( 0.058)0.314 * ( 0.037)0.311 ( 0.110)0.217 ( 0.272)index * ln ( 65age)0.233 ( 0.442)0.262 ( 0.386)0.288 ( 0.471)0.309 ( 0.485)index * health0.003 ( 0.914)0.004 ( 0.872)0.007 ( 0.718)0.005 ( 0.897)index * secondary education0.079 ( 0.333)0.087 ( 0.285)0.086 ( 0.368)0.121 * ( 0.041)index * tertiary education0.119 ( 0.106)0.130 ( 0.079)0.139 ( 0.127)0.205 * * * ( 0.000)index * working0.158 * * ( 0.001)0.122 * * ( 0.001)0.152 * * * ( 0.000)0.098 * ( 0.043)index * enrolled0.236 * * ( 0.001)0.210 * * ( 0.002)0.215 * * ( 0.004)0.288 * * * ( 0.000)index * income0.002 ( 0.935)0.037 * * ( 0.008)constant13.107 * * * ( 0.000)12.953 * * * ( 0.000)12.853***(0.000)13.112 * * * ( 0.000)n77621776216883758354note : controlled for country and year dummies . outliers for model a are denmark and greece , for model b these are italy and the united kingdom * p < .05 , * * p < .01 , * * * p < .001 discrete time event history models of union formation with interactions : logit regression coefficients and p - values in parentheses note : controlled for country and year dummies . outliers for model a are denmark and greece , for model b these are italy and the united kingdom * p < .05 , * * p < .01 , * * * p < .001 in table 3 , we see a negative interaction of the societal index with men s employment ( b = .16 , p < .01 ) . hence , the average effect of employment ( b = .50 ) is reduced by 32% for each standard deviation increase in the egalitarian context ( .16/.50 ) . this shows that the effect of men s employment on the entry into a union is considerably weaker in more egalitarian countries than in more traditional countries . i also present the effects for each country separately in a graph ( fig . , the effect of selected independent variables is plotted against the macro - level index . although the graph does not provide a test , like the interaction effect , it does give a good intuitive feel of the importance of the interaction effect . in line with expectations , the graph shows that in more traditional societies like spain , italy , and ireland , the effect of men s employment is quite strong . in more egalitarian countries like the netherlands , denmark , and finland , men s enrollment in school deters union formation but this effect is weaker in more egalitarian societies . s economic characteristics on union entry effects of men s economic characteristics on union entry table 3 further shows no negative interaction of the societal index with men s income effect on union formation . after deleting two outliers ( italy and the united kingdom ) , the interaction becomes negative and significant ( b = .037 , p = .01 ) . hence , for these 11 countries , the effect of men s income on union formation is weaker in more egalitarian countries than in more traditional countries . the magnitude of the effect is a 15% reduction in the income effect per standard deviation increase in the egalitarian context ( .037/.234 ) . when we look at the graph , the pattern appears to be weaker than it was for employment . spain and portugal reveal strong effects of men s income and denmark and the netherlands have weak effects . however , there are also outliers like the united kingdom ( stronger effect than expected ) and italy and belgium ( weaker effect than expected ) . so far , these results apply to union formation regardless of the type of union . traditional and egalitarian societies also differ in the extent to which cohabitation occurs . the more egalitarian countries like denmark , finland , and the netherlands also have the highest levels of cohabitation ( soons and kalmijn 2009 ) . because the effects of men s economic position differ depending on whether marriage or cohabitation is the outcome , the results could in part be due to such compositional differences . for this reason , i also look at the entry into marriage only ( either after being single or after cohabitation ) . the interaction with employment is .066 ( p = .11 ) , the interaction with enrollment is .326 ( p < .01 ) , and the interaction with income is .073 ( p < .01 ) . hence , the employment interaction effect is weaker in this model , although still negative , while the other interaction effects are not affected . we therefore conclude that part of the reason why the employment effects depend on the context lies in the fact that unions are more often unmarried unions in more egalitarian societies . even apart from that , however , there is evidence that men s economic status matters more for marriage in more traditional societies . this article has re - examined the importance of oppenheimer s theory on marriage timing in the european context . by and large unemployment , little work experience , low income , and temporary employment on the part of men deter union formation . by analyzing income and employment effects simultaneously in a panel perspective , it was possible to obtain more direct evidence for the two contrasting hypotheses suggested by oppenheimer , i.e. , the neo - malthusian male breadwinner hypothesis and the career uncertainty hypothesis ( sometimes also called the career instability or immaturity hypothesis ) . many previous european analyses have not been able to take income into account and have therefore not been able to separate these two mechanisms empirically . my analyses first show that income effects are strong and significant , which supports the male breadwinner hypothesis . second , the income effects explain about half of the effects of employment and work experience , suggesting that employment effects by themselves are not sufficient evidence for the uncertainty hypothesis . this suggests that employment effects on union formation are more than just a matter of financial resources , in line with the uncertainty hypothesis . by analyzing the choice between cohabitation and marriage , marriage in europe appears to be more sensitive to men s economic position than cohabitation . men who are not employed or who have temporary jobs are more likely to choose cohabitation rather than marriage . this finding provides additional evidence for the uncertainty hypothesis since cohabitation is more like a trial marriage and hence , more compatible with uncertainty in other life domains ( i.e. , employment ) . we do not , however , see a clear income effect on the choice between marriage and cohabitation . hence , the choice between marriage and cohabitation has more to do with employment uncertainty than with income . there is a small effect of the lowest income group , however . among men who enter a union , hence , there is some evidence that cohabitation is the " poor man s marriage . " following oppenheimer s last study , i also examined the transition from cohabitation to marriage . we would expect economic uncertainty to also reduce the chances of a transition from cohabitation to marriage , but in general , the effects we find are weaker for this transition . the weaker income effect could be due to the fact that the cost of setting up a household plays no role for this transition . while this is in line with the neo - malthusian argument , the finding that the employment effects on the transition from cohabitation to marriage are also weaker is unexpected . other authors have found this too ( e.g. , bracher and santow 1998 ; liefbroer 2005 ) . if couples buy a house or have a child , they may decide to marry . although such transitions may be partly governed by economic uncertainty , they may also be exogenous and hence reduce the effects of other determinants like employment . another speculation has to do with the lack of information on the ( female ) partner . the partner may have become economically more secure during the cohabitation stage , thereby promoting the transition to marriage , but we do not observe such changes . european countries differ considerably in terms of their economic , social , and cultural characteristics , and so , it is important to also examine country variation in the degree to which the theory applies . we hypothesized that men s economic position would be less influential in more developed countries where gender roles are more symmetrical . the effects of men s employment and school enrollment on union formation are stronger in more traditional societies than in more egalitarian ones . income effects are also weaker in egalitarian societies but the evidence for this pattern is weaker . our interpretation is that in egalitarian settings , the costs of setting up a household are more often shared between men and women . hence , men can afford to experiment with their career if they have a partner who has a ( more ) stable career . similarly , women may attach less weight to the career options of men than they did in more traditional circumstances and for instance , pay more attention to other traits , such as men s willingness to participate in child rearing . my finding is in contrast to a previous cohort comparison for the united states which suggested that men s economic effects on marriage timing did not change over time ( sweeney 2002 ) . perhaps this difference is due to the limited time period that was examined in sweeney s trend study , a design which may have reduced the variance in contextual gender roles . at the same time , however , it needs to be investigated whether the patterns that i found still hold when a larger number of countries is considered . the income interaction effects , for example , are sensitive to outliers and therefore less convincing . more units at the macro - level will be needed to confirm this finding . in closing , it is important to re - emphasize on the role of women . although oppenheimer was justifiably " bringing men back " into the debate at a time when there was too little attention on the changing economic fate of young ( american ) men , the growing egalitarian model that is now supported by many couples in europe and the united states , suggests that men and women should be examined simultaneously for a better understanding of trends and differentials in marriage and cohabitation . traditionally , studies of women were largely done from a beckerian perspective in which it was argued that women s status would lead to a decline of marriage ( and fertility ) . currently , we can speculate that a strong economic position and career stability on the part of women might foster marriage . it could be that certainity on just one side either the man or the woman alternatively , it could be that both the man s and the woman s career need to have been settled before couples enter a more committed union . in this respect , it is somewhat unfortunate that analyses of marriage ( timing or formation ) have often been one - sided , focusing either on men or on women . this design is unavoidable given that the partner of a respondent is typically observed too late , i.e. , when the respondent is married or living with the partner . empirically , this problem does not exist when we observe cohabiting couples chances of marrying . some authors in the past have regarded the transition from cohabitation to marriage as a two - sided problem and have analyzed economic characteristics of both partners in one model ( lichter et al . these studies have not been able to examine the more important entry into a first union , however . for this transition , the problem can , in principle , be solved by examining dating couples in a more systematic fashion . for example , prospective surveys could collect data on the school and work careers of both members of a dating couple and subsequently analyze whether or not ( and when ) they begin to live together , while using the dissolution of the dating relationship as a competing risk . in this way , the analysis of marriage and cohabitation can become truly two - sided and the economic characteristics of men and women can be included in one model . there are some innovative sociological studies of transitions from dating to cohabitation and marriage , but so far , they have not focused on the partners economic characteristics ( joyner and kao 2005 ) .

this article discusses oppenheimer s theory on marriage timing , reviews the way this theory was received in european demography and family sociology , and develops a new test of the theory using annual panel data from 13 european countries for the period 19942001 . several indicators of men s economic status are used , including school enrollment , employment , type of labor contract , work experience , income , and education . effects of these indicators are estimated for the transition to marriage and cohabitation , as well as for the transition from cohabitation to marriage . country differences in these effects are examined as well . the evidence provides strong support for the male breadwinner hypothesis on the one hand , and for oppenheimer s career uncertainty hypothesis on the other . however , the relevance of these hypotheses also depends on the national context , and especially on the way gender roles are divided in a society .

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osteoporosis is a condition of impaired bone strength which leads to increased risk of fracture . the enhanced bone fragility reflects the integration of the amount of bone ( bone mass ) and bone quality . bone quality depends on its macro- and microarchitecture and on the intrinsic properties of the materials that comprise it ( e.g. , matrix mineralization , microdamage accumulation , or collagen quality ) . bone is continually adapting to changes in its mechanical and hormonal environment via the process of bone remodeling . bone remodeling maintains bone structure and its mechanical competence by removing damaged bone and replacing it with new bone and thus restores bone 's material composition , micro- , and macroarchitecture . this process depends on the normal production , work and lifespan of osteoclasts , osteoblasts , and osteocytes . thus , diseases and drugs that have an impact on bone cells and bone remodeling will influence bone 's structure and its resistance to fracture . multiple sclerosis ( ms ) is a chronic progressive disease affecting the myelin sheath covering of nerve fibers in the brain and spinal cord , leading to functional impairments such as visual impairment , abnormal walking mechanics , poor balance , muscle weakness , fatigue , and progressive immobilization . the disease affects mainly young adults ( 20 to 40 years ) and its incidence is more frequent in women ( approximately 2 : 1 ) . impaired mobility or lack of weight - bearing physical activity reduced mechanical stress on bone , which causes a marked imbalance in bone remodeling with a disruption of osteocytes network . management of ms requires long - term disease - modifying therapy , such as glucocorticoids ( gcs ) with a further negative effect on bone remodeling and bone strength . secondary osteoporosis may develop and low - trauma fractures occurring in patients with ms more frequently than in healthy controls [ 915 ] . fractures and their sequelae can have important personal as well as ( economic ) implications for society . therefore , the attention on the issue of bone health among patients with ms is warranted . this paper examines the underlying pathogenic mechanisms of osteoporosis in patients with ms as well as its management . understanding the causes associated with decreased bone strength in patients with ms will help in the optimal therapeutic intervention . the analysis of a registry of 9029 patients with ms in the usa found that 27.2% responders reported low bone mass , and more than 15% of responders reported a history of fracture . most studies in patients with ms evaluated bmd in comparison with the control group of healthy subjects and showed significantly lower bmd in patients with ms than in controls [ 1116 ] . several of these studies were shown that vertebral bmd is affected to a lesser degree than femoral bmd [ 11 , 12 , 16 ] . interestingly , one study in men with ms reported low bmd ( osteoporosis ) in 37.5% ( 15 out of 40 ) and 21% ( 8 out of 38 patients ) had vertebral , rib , or extremities fractures . patients with progressive forms of ms showed a more severe loss of bmd than those with relapsing - remitting ms . cosman group found fracture rates of 22% in patients with ms compared with 2% in controls . it remains unexplained whether all patients with ms are more susceptible to osteoporosis and fractures ; for example , there is evidence that patients with a low expanded disability status scale ( edss ) score did not show any significant difference in bmd with comparison with healthy control subjects [ 17 , 18 ] . therefore , further elucidation is needed to qualify which risk factors are most responsible for a bone loss in patients with ms . bone remodeling is under way throughout life and maintains bone strength by removing damaged bone and replacing it with new bone and thus restores bone 's micro- and macroarchitecture . this process depends on the normal production , work , and lifespan of osteoclasts , osteoblasts , and osteocytes . chronic diseases , such as ms , may significantly disturb the process of bone modeling and remodeling with resulting bone loss , deterioration of bone 's quality and increased frequency of fractures [ 20 , 21 ] . secondary osteoporosis and low - trauma fractures occur in patients with ms more frequently than in healthy controls [ 9 , 11 ] . the underlying pathogenic mechanisms of the osteoporosis in patients with ms are probably based on the progressive immobilization , long - term gcs treatment , vitamin d deficiency , skeletal muscle atrophy and possibly on the presence of various cytokines involved in the pathogenesis of ms . in addition , chronic use of other drugs , such as antidepressants may contribute to the development of osteoporosis and fractures . the functional impairments also leads to an increased risk of falling that , combined with bone loss and impaired quality of bone mass , can increase the frequency of bone fracture in individuals with ms . increased bone loss in immobilized subjects is well - recognized complication in patients after spinal cord injury with tetraplegia [ 23 , 24 ] , in bedridden patients , or in astronauts , whereas localized bone loss is well documented in patients with regional disuse , for example , after fracture itself . immobilization causes an overall progressive bone loss at a similar rate to osteoporosis caused by estrogen deficiency , but at the same amount of induced bone loss , disuse led to more deteriorated bone structure and mechanical properties than estrogen deficiency . the available studies showed that cortical thinning and substantial decline of trabecular bone density account for increased bone fragility [ 2729 ] . the duration and degree of motor disability appears to be a major contributor to the pathogenesis of secondary osteoporosis in patients with ms . the degree of disability measured by the kurtzke edss score significantly correlated with bmd in patients with ms . specifically , site - specific effects of motor disability were documented in ms patients , and edss correlated mainly with bmd in the hip but not in the lumbar spine . in wheelchair - bound patients , an atrophy of hip muscles affects proximal femur , while bmd of lumbar spine is not decreased because of its adequate mechanical stimulation by the trunk and back muscles in the upright position . also , hemiplegic patients showed a significant loss of bmd in both trabecular and cortical bone at the forearm and at the neck and great trochanter on the paretic hip . higher total body bone mineral content was documented in ambulatory patients ( edss score 6.5 ) compared with nonambulatory patients ( edss score 7.0 ) [ 12 , 13 ] . also , higher prevalence of osteoporosis was found in nonambulatory patients . in male patients , a positive correlation has been observed between bmd and both edss score ( correlation with femoral and also vertebral bmd ) and bmi ( correlation with femoral bmd only ) . there was shown that also edss score and bmi two years prior to the study could be used as future indicators of low bmd . a reduced mechanical stress on bone causes a marked imbalance in bone remodeling with a transient increase in bone resorption ( which occurs initially ) and a decrease in bone formation ( which is sustained for a longer duration ) [ 25 , 33 , 34 ] ( table 1 ) . the mechanism causing this decrease in bone formation probably lies in the reduction of mechanical stress during immobilization which results in a marked disruption of osteocytes network due to increase of osteocyte apoptosis . osteocytes represent 95% of all bone cells and form a mechanosensory system which is based on a three - dimensional network of tightly interconnected osteocytes entombed in mineralized bone matrix . disruption of this system affects probably several aspects of bone homeostatic system , such as mechanosensitivity , mechanotransduction , and basic multicellular units responsible for bone remodeling . while molecular mechanisms of disuse osteoporosis are not well understood , recent evidence found that mechanical unloading caused upregulation of sost gene in osteocytes and increased levels of sclerostin ( product of sost gene ) . sclerostin is responsible for the inhibition of wnt / beta - catenin signaling in vivo and for the suppressed viability of osteoblasts and osteocytes . interestingly , sclerostin - deficient mice ( sost / ) were resistant to mechanical unloading - induced bone loss . importantly , the administration of sclerostin neutralizing antibody in experimental model of immobilization resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass . osteocytes are also necessary for targeted bone remodeling to avoid microdamage accumulation , which could lead to whole - bone failure . recently , waldorff et al . showed that osteocyte apoptosis may be insufficient for repair of microdamage without the stimulation provided through physiologic loading . ms affects a wide range of neurological function and most of patients with ms have abnormal muscle strength , impaired balance , and gait control which leads to frequent falls [ 5 , 41 ] that combined with a bone loss increase the frequency of bone fractures . it was demonstrated that changes in postural control in most patients with ms are probably the result of slowed afferent proprioceptive conduction in the spinal cord . disuse , inflammatory changes , as well as gcs treatment or vitamin d deficiency , may also contribute to weakness and loss of muscle strength and thus to frequent falling . gcs are frequently used to control ms relapses . oral gcs treatment in patients with ms may increase the risk of osteoporosis . epidemiological studies showed that fracture risk is increased rapidly after starting oral gcs treatment and is related to the dose and duration of gcs exposure . doses as low as 2.55 mg of prednisolone equivalents per day can be associated with a 2.5-fold increase in vertebral fractures , and the risk is greater with higher doses used for prolonged periods . bone loss due to gcs treatment is steep during the first 12 months and more gradual but continuous in subsequent years . however , the fracture risk returns towards baseline levels after discontinuation of oral gcs treatment . the mechanism of osteoporosis in patients on gc treatment is complex ( table 2 ) . however , the contribution of other risk factors , such as vitamin d insufficiency and physical disability confounds the assessment of gcs effects on bone in patients with ms . repeated pulses of high - dose methyprednisolone in ms patients did not result in a subsequent decrease in bmd ; however , the risk of osteoporotic fractures remains slightly increased in patients undergoing cyclic gcs treatment at high doses . high - dose , short - term intravenous gc regimens cause an immediate and persistent decrease in bone formation and a rapid and transient increase of bone resorption . in fact , gcs may increase proresorptive il-6 signaling as well as increase the expression of receptor activator of nf-b ligand ( rankl ) and decrease the expression of its soluble decoy receptor , osteoprotegerin ( opg ) , in stromal and osteoblastic cells . however , discontinuation of such regimens is followed by a high bone turnover phase . in physically active patients with ms treated with low - dose steroids , the bone turnover markers were not different from controls . addressing the question of whether duration of low - dose gcs use in combination with other immunomodulators in patients with ms increases risk of osteoporosis requires further prospective study by taking into account other risk factors , particularly the level of disability . although no harm effect of low - dose methotrexate was observed in patients with ms , several case reports have described associations between pathological nonvertebral fractures and low - dose methotrexate ( mtx ) in rheumatoid arthritis ( ra ) patients . in addition , methotrexate osteopathy , characterized by pain , osteoporosis , and microfractures , has been very rarely observed in patients with low - dose mtx treatment . other immune - modifying drugs , such as interferon - beta or azathioprine , which are used in conjunction with gcs have not been shown to promote bone loss experimentally or clinically . on the contrary , interferon - beta may have favorable effect on bone metabolism in patients with ms , probably due to the inhibitory effect of interferon - beta on osteoclasts development . experimentally , also treatment with the s1p(1 ) agonist fty720 , a new and promising drug for the treatment of ms , relieved ovariectomy - induced osteoporosis in mice by reducing the number of mature osteoclasts attached to the bone surface . however , further investigation with regard to their effects on bone health is needed . the role of vitamin d in bone homeostasis is well understood , and the use of vitamin d to prevent and treat osteoporosis was recently reviewed . there is also evidence from both observational studies and clinical trials that hypovitaminosis d are predisposing conditions for various common chronic diseases . in addition to skeletal disorders , vitamin d deficiency is associated with increase the risk of malignancies , particularly of colon , breast , and prostate gland cancer , of chronic inflammatory and autoimmune diseases ( e.g. , insulin - dependent diabetes mellitus , inflammatory bowel disease , or multiple sclerosis ) , as well as of metabolic disorders ( metabolic syndrome and hypertension ) . vitamin d intake , decreasing latitude , increased sun exposure , and high serum vitamin d levels have all been shown to be associated with a decreased risk of ms . patients with ms have more often vitamin d deficiency due to its low intake as well as limited sunlight exposure . mean 25-hydroxyvitamin d3 ( 25ohd ) levels in patients with ms are more often lower ( below the level of 20 ng / ml ) than in age - matched controls [ 11 , 14 ] . there was no significant correlation between 25(oh)d and bmd in patients with ms [ 11 , 14 ] . thus , while patients with ms are susceptible to low 25ohd levels , the evidence implicating linking levels to reduced bmd and osteoporosis in patients with ms is unclear . only a few studies have investigated this link [ 11 , 12 , 14 ] . a low vitamin d state , from inadequate diet intake and decreased exposure to sunlight , contributes to malabsorption of calcium and vitamin d insufficiency in ms patients . secondary hyperparathyroidism may develop , which can contribute to bone remodeling imbalance and bone loss in patients with ms . moreover , patients with ms treated with gcs will be at greater risk for an imbalance between bone formation and bone resorption and , therefore , more susceptible to development of osteoporosis due to vitamin d insufficiency / deficiency . gcs treatment is associated with reduced calcium absorption from the gastrointestinal tract by opposing vitamin d action . in addition , gcs may affect pth secretory dynamic , with a decrease in the tonic release of pth and an increase in pulsatile burst of the hormone . ms is an inflammatory disease of the central nervous system ( cns ) with a prominent role of immune cells and cytokines in degradation of the myelin sheaths . recent evidence has indicated that a number of additional cell types , such as t cells , play a key role in bone loss . in inflammatory or autoimmune disease states , activated t - cells produce receptor activator of nuclear factor kappab ligand ( rankl ) and proinflammatory cytokines , such as tnf- , il-1 , or il-11 , all of which can induce rankl expression in osteoblasts and bone marrow stromal cells . the systemic or local activation of t - cells may , therefore , trigger bone loss via the expression of rankl . osteoprotegerin ( opg ) , a protein member of the tumor necrosis factor ( tnf ) receptor family and its ligand rankl were identified as a key cytokines that regulate osteoclastogenesis . significantly , higher levels of rankl and opg were found in the patients with ms with low mean edss as compared to the age - matched controls . among other cytokines , osteopontin ( opn ) has been studied in the shared pathogenesis of ms and osteoporosis . opn is a member of the sibling ( small integrin - binding ligand n - binding glycoprotein ) family of noncollagenous matricellular proteins . opn was identified as the most abundantly expressed cytokine in ms lesions , and opn levels were found to be increased in cerebrospinal fluid of ms patients [ 64 , 65 ] and in the plasma in patients with relapsing - remitting ms . however , other studies found that opn circulating levels are low in patients with ms . it seems likely that further future studies experiments will uncover the role of opn and additional molecules mediating bone loss in inflammatory diseases , such as ms . meta - analyses have revealed that barbiturate , antidepressant , antipsychotic , and benzodiazepine treatment increases patient 's risk of osteoporosis . more recently , current use of antidepressant drugs with a high affinity for the 5-hydroxytryptamine reuptake transporter ( 5-htt ) was associated with a higher risk of osteoporotic fractures compared to use of antidepressants with a medium or low affinity . bmd was lower among those reporting current selective serotonin reuptake inhibitors ( ssri ) use but not among users of other antidepressants [ 72 , 73 ] . in vivo studies have found that 5-ht could alter bone architecture and could reduce bone mass and density . the 5-htt has been located in osteoclasts , osteoblasts , and osteocytes , and the the inhibition of 5-htt using a ssri ( fluoxetine hydrochloride ) had antianabolic skeletal effects in rats . further research is needed to confirm this finding in light of widespread ssri use and potentially important clinical implications . despite the fact that patients with ms can develop osteoporosis and fractures more often than their age - matched healthy controls , many patients with ms are not evaluated for their bone status , and there are no clinical guidelines for prevention and treatment of osteoporosis in patients with ms . patients with ms are also at a higher risk of falls that can increase the frequency of bone fracture combined with bone loss and impaired bone 's quality . clinical evaluation in all patients with ms should include the assessment of the clinical risk factors for osteoporosis and fractures , such as the hereditary disposition of osteoporosis , previous low trauma fractures , and smoking or alcohol habits . the specific risk factors of the osteoporosis in patients with ms are the level of disability ( specifically motor disability ) and possibly a long - term gcs treatment , vitamin d deficiency , skeletal muscle atrophy , and increased risk of falling . the examination of the motor function using the edss score could provide a useful indicator for further evaluation . cutoff edss 6 represents reasonable end of motor performance of the patient ; 6.5 means only several meters with bilateral support , and 7 is only the ability of transfer to wheelchair from the bed . the edss scores of 6 or greater has been found to correlate well with decreased bmd [ 12 , 15 ] , and bmd should be routinely measured in these patients . on the other hand , patients with a good physical activity and low edss score ( < 5 ) may have normal bmd as well as markers of bone turnover . bmd measurement should be also performed in all patients who are receiving 5 mg of prednisone equivalents daily for more than 3 months . bmd testing using dual - energy x - ray absorptiometry ( dxa ) should be conducted at the lumbar spine and hip . this measure provides an assessment of fracture risk prior to the occurrence of a fragility fracture as well as monitors the course of the disease and response to therapy . no consensus exists as to how frequently patients at risk osteoporosis should have followup scans . however , bmd should be remeasured after 1 or 2 years to ascertain that it is stable or to identify the patient with ongoing bone loss , especially in patients treated with long - term gcs treatment . in the presence of clinical risk factors therefore , combination of bmd with clinical risk factors is recommended to identify a risk patient and to target pharmacologic therapy . in postmenopausal women and men ( between 40 and 90 ) , the assessment of individualized 10-year absolute fracture risk ( frax , fracture prediction algorithm ) the identification of previous low - trauma fractures , especially vertebral fractures is important for the decision - making process as a previous vertebral fracture is a particularly strong risk factor . importantly , vertebral fractures may occur in 3050% of patients receiving chronic gcs therapy and up to 50% of vertebral fractures are asymptomatic and , therefore , do not come to the attention of physicians . spinal x - rays should be performed in those with localized back pain or a loss of more than 3 cm in height in order to detect prevalent vertebral fractures . alternatively , the vertebral fracture assessment tool of the bone densitometer , which is associated with low radiation , may be useful screening test for vertebral fractures assessment . laboratory tests are indicated to exclude other secondary causes of osteoporosis , such as vitamin d deficiency , renal insufficiency , malabsorption , and hypogonadism . useful biochemical tests include routine standard tests to exclude renal or hepatic impairment , blood count , serum calcium , 24-hour urinary calcium , 25-hydroxyvitamin d3 ( to exclude vitamin d deficiency ) , and gonadal hormones ( to exclude hypogonadism ) . nonpharmacological therapies should be considered for prevention of skeletal fragility , including adequate weight - bearing exercise , nutrition ( protein , calcium , vitamin d ) , and lifestyle modifications . as reviewed above , disability is the most often cause of bone loss in patients with ms , and mechanical loading and exercise interventions can prevent osteocyte apoptosis and bone loss [ 77 , 78 ] . exercises have beneficial effects on strength , physical endurance , mobility - related activities ( transfer , balance , and walking ) , and on mood , without any evidence of detrimental effects ; however , there was no evidence that any particular exercise programs were more effective in improving or maintaining function . whole - body vibration is a new approach to improve neuromuscular functions and bone strength , but there is limited evidence that whole body vibration provides any additional improvements . further experimental studies are necessary to identify optimal physical activities for the prevention of osteocyte apoptosis and bone loss . recurrent falls may be an important risk factor for fracture in disabled patients with ms . in patients with ms , falls are related to the level of disability , and possibly other factors may contribute to muscle weakness and imbalance , such as vitamin d deficiency or gcs treatment . calcium and vitamin d supplementation has been routinely provided in most clinical trials of bone protective therapy for both primary and secondary osteoporosis , for example , in glucocorticoid - induced osteoporosis ( gio ) . the effect of calcium and vitamin d supplementation is maximized in patients whom baseline intake is low . as patients with ms are at a higher risk of calcium and vitamin d deficiency should have their calcium and vitamin d status checked and intake must be individualized . those with a personal or family history of nephrolithiasis an increase in serum calcium is provoked by bed rest alone and additional calcium intake would not be helpful and might be harmful and provoke an increased risk of kidney stone formation . however , calcium and vitamin d should be used as an adjunct treatment , because a low calcium intake may exacerbate calcium loss during low mechanical loading . in general , the amount of vitamin d supplementation should aim at achieving serum 25ohd levels above 50 nmol / a daily dose of 8001000 iu of vitamin d3 should be able to obtain this minimal 25ohd target . due to vitamin d resistance in patients receiving gcs , those patients may require amounts of 10002000 iu of vitamin d3 daily . measurement of serum 25oh vitamin d is recommended , especially in gcs - treated patients . although some evidence suggests that daily supplemental intake of 20004000 iu colecalciferol is required to obtain at least 75 nmol / l 25ohd , which may be optimum for many health outcomes , prospective trials showing that higher 25ohd levels ( > 7580 although a number of drugs have been evaluated for the prevention and treatment of postmenopausal osteoporosis and gio , the evidence of their efficacy in patients with ms , especially in premenopausal women and younger men is less strong . as osteoporosis in ms patients have multiple pathogenesis , medical interventions used in women with postmenopausal osteoporosis may not be similarly efficient . patients requiring long - term gcs treatment and those being immobilized may require pharmacological therapy to prevent excessive bone loss and fractures . options for treatment include antiresorptive drugs , such as estrogen , or aminobisphosphonates , or anabolic agents such as teriparatide . although the use of bps may be appropriate , the etiology of osteoporosis in patients with ms is fundamentally different from the osteoporosis commonly found in the postmenopausal women for whom these drugs were originally developed . as immobilization in patients with ms can cause substantial bone loss and increase in the risk of fractures , bps may be option for treatment for those patients . although bps have not been systematically evaluated in the therapy of these conditions , some studies support the potential benefit of bps in the management of bone loss associated with immobilization [ 24 , 85 , 86 ] . in immobilized patients , bps is known to reduce immobilization - induced hypercalcaemia by inhibiting bone resorption of calcium . an immobilization - related elevated serum calcium level may inhibit parathyroid hormone ( pth ) secretion , and hence renal 1 , 25(oh)2d3 production , in disabled long - standing ms patients . if oral therapy of bps can not be tolerated or excluded due to gastroesophageal disease , intravenous route of administration of ibandronate or zolendronate may be applied . however , acute phase reaction with fever , particularly after the first application of bp , may occur . bps ( alendronate , risendronate , or zolendronate ) were also approved for the treatment of gio . these drugs were shown to improve bmd , whereas the data on fractures were scanty in gio , particularly in premenopausal or younger men . the mechanism by which bps reduce the adverse skeletal effects of gcs have not been elucidated . the disadvantage of long - term bps treatment is that it may lead to a reduction in bone turnover to a level inadequate to support normal bone remodeling . although experimental data showed that bps also prevents osteocyte apoptosis , there is also experimental evidence of increased accumulation of microdamage with long - term bps therapy . also , as bps accumulate in the skeleton ( with a long - term residual time ) , they cross the placenta , accumulate in fetal skeleton , and cause toxic effects in pregnant rats . therefore , bps should be used with caution in women who may become pregnant . drugs , such as bps , that suppress bone resorption have been proposed as interventions for prevention of gio as well as disuse osteoporosis . the disadvantage of this approach is that it may lead to a reduction in bone turnover to a level inadequate to support normal bone remodeling . an alternative approach is to maintain a normal level of bone formation using a bone anabolic agent such as pth . the human recombinant n - terminal parathyroid hormone ( pth 134 or teriparatide ) is a potent osteoanabolic agent , which decreases osteoblast and osteocyte apoptosis and increases bone formation and bone strength . because of gcs - induced decrease in the number of osteoblasts and rate of bone remodeling , anabolic , and antiapoptotic treatment with teriparatide may directly counteracts the key pathogenetic mechanisms of gcs excess on bone , thus , it may be a more effective treatment than bps . the same rationale applies to immobilization - induced osteoporosis , as progressive immobilization as well as long - term gcs exposure results in osteocyte apoptosis and reduced bone formation . as sclerostin augments osteocyte apoptosis , the antibody - mediated blockade of sclerostin represents a promising new therapeutic approach for the anabolic treatment of immobilization - induced osteoporosis and probably also for gcs - induced osteoporosis . indeed , more recently , experimental data showed that administration of sclerostin neutralizing antibody in rat model of right hindlimb immobilization resulted in a dramatic increase in bone formation and a decrease in bone resorption that led to increased trabecular and cortical bone mass . we have described a spectrum of pathogenetic factors which may contribute to the development of osteoporosis and low - trauma fractures in patients with ms . whilst there is evidence to support an important role for many of the risk factors , the most significant etiology of bone loss in patients with ms seems to be the level of motor disability and reduced bone load within individual patients . other risk factors , such as long - term gcs treatment , hypovitaminosis d , or inflammation , may also play an important part in subset of patients with ms ; however , further examinations in prospective studies are required . with regard to diagnostic as well as therapeutic interventions , there are currently no specific recommendations in patients with ms ; however , identification and treatment of underlying cause should be the goal of therapeutic management . optimally , the patients in a higher risk of osteoporosis should be early identified and preventively promptly treated to avoid the bone loss and fractures . because the long - term disability and long - term gcs are probably two most significant etiologic risk factors for osteoporosis development in the majority of the patients with ms , the interventions which can counteract the osteocyte apoptosis as well as loss of muscle mass and muscle weakness will be promising .

multiple sclerosis ( ms ) is a gait disorder characterized by acute episodes of neurological defects leading to progressive disability . patients with ms have multiple risk factors for osteoporotic fractures , such as progressive immobilization , long - term glucocorticoids ( gcs ) treatment or vitamin d deficiency . the duration of motor disability appears to be a major contributor to the reduction of bone strength . the long term immobilization causes a marked imbalance between bone formation and resorption with depressed bone formation and a marked disruption of mechanosensory network of tightly connected osteocytes due to increase of osteocyte apoptosis . patients with higher level of disability have also higher risk of falls that combined with a bone loss increases the frequency of bone fractures . there are currently no recommendations how to best prevent and treat osteoporosis in patients with ms . however , devastating effect of immobilization on the skeleton in patients with ms underscores the importance of adequate mechanical stimuli for maintaining the bone structure and its mechanical competence . the physical as well as pharmacological interventions which can counteract the bone remodeling imbalance , particularly osteocyte apoptosis , will be promising for prevention and treatment of osteoporosis in patients with ms .

pubmed

malaria is one of the main scourges of the world , affecting half a million people a year . it is caused by parasites belonging to the phylum apicomplexa , family plasmodiidae , and genus plasmodium . there are five known species that parasitize man , the most prevalent being plasmodium falciparum , p. vivax , and p. malariae in tropical areas such as latin america , the indian subcontinent , southeast asia , and parts of africa . p. ovale has been reported in africa , middle east , and the indian subcontinent , while p. knowlesi has been recently described as responsible for large foci of infections in southeast asia . the natural transmission occurs through the bite of infected female anopheles mosquito , anopheles darlingi being the most prevalent species in the country . the ministry of health directs therapy and provides free antimalarial medicines used throughout the national territory , in units of the unified health system ( sus ) , through a national policy on malaria treatment . malaria caused by p. vivax ( or p. ovale ) is treated with chloroquine in brazil at a dose of 30 mg / kg divided into three days and primaquine in the 30 mg dose / day for 7 days or 15 mg / day for 14 days . also , low doses of chloroquine can be used for the prevention of vivax malaria . pregnant women and children under 6 months of age with vivax malaria receive only chloroquine in different dosage schedules . primaquine is the only antimalarial drug with effective activity against all the species gametocytes of plasmodium which cause malaria in humans , as well as against hypnozoites , latent forms of p. vivax and p. ovale responsible for relapses . however , it has hemolytic effects , especially in people with glucose-6-phosphate dehydrogenase deficiency ( g6pd ) , in which primaquine as a stressor to the erythrocyte induces hemolysis . hemolysis induced by primaquine can result in discoloration of urine and faeces and it is dose related [ 8 , 9 ] . gastrointestinal disorders , such as nausea , dizziness , and vomiting , are common but usually not present as severe complications , especially when primaquine is administered with food . with respect to chloroquine , there are relatively few adverse effects with use in usual doses , of which the most serious are retinopathy , cardiomyopathy , myopathy , and neuromyopathy . prolonged treatment or use of high level doses may cause retinal toxicity , long subtle symptoms of decreased visual acuity , diplopia , and bilateral loss of vision . adverse reactions of the gastrointestinal tract are the most common side effects , usually controlled by reducing drug dose . there are also reports of ototoxicity , tingling , itching , and change in skin color , in addition to seizures , insomnia , and paresthesia [ 12 , 13 ] . the occurrence of symptoms specifically related to the intake of antimalarials and the overlap of symptoms of malaria with the side effects of primaquine and chloroquine may decrease adherence to treatment . therefore , it becomes important to assess the main side effects of antimalarial drugs in patients from endemic areas , where treatment is aimed not only at improving the clinical status but also at reducing the transmission of plasmodium . this study evaluated the frequency of the symptoms that commonly occur in vivax malaria before and after treatment and the frequency of side effects reported during the term of antimalarial treatment with chloroquine and primaquine in standardized doses according to the protocol of the brazilian ministry of health . the study was conducted in the urban area of the municipality of mncio lima , located in the western part of the brazilian amazon , in the state of acre . this municipality , with 5000 km , is bordered by the cities of cruzeiro do sul and rodrigues alves and the republic of peru . mncio lima has 14,884 inhabitants living in urban areas ( 57.3% ) , rural or riparian areas ( 37.9% ) , and indian villages ( 4.8% ) . the county is located 38 km from cruzeiro do sul and 650 km from rio branco . patients with microscopic diagnosis of vivax malaria were identified in microscopy stations of the city of mncio lima before the medication and invited to participate in the study . after signing the informed consent or having it signed by parents or legal guardians , patients answered a questionnaire on malaria symptoms that occurred up to the time of diagnosis ( before antimalarial treatment ) , to verify the occurrence of the following symptoms : nausea , vomiting , diarrhea , pale stools , loss of appetite , mesogastric and/or hypogastric pain , pain in hypochondrium , headache , myalgia , arthralgia , back pain , retroocular pain , fever , chills , sweating , malaise or weakness , itchy throat , jaundice , choluria , dyspnea , and bitter taste sensation , covering up thereby symptoms commonly occurring in malaria and overlapping with possible side effects of chloroquine and primaquine . between 3 and 5 days after the start of medication , a second interview was conducted asking about the occurrence of the same symptoms plus some symptoms related to the use of chloroquine that are infrequent in malaria : blurred vision , itching , stinging sensation of the skin , convulsions , numbness , and insomnia . the distributions of relative and absolute frequencies , median , mean , and standard deviations of the variables of interest were calculated . as primaquine and chloroquine may lead to the onset of symptoms and also intensify or reduce some of the symptoms of malaria , the occurrence of symptoms before and after drug treatment was analyzed with the mcnemar 's test for paired samples . some of the symptoms considered to be side effects of chloroquine and primaquine and that are not usual in malaria were assessed only after drug treatment was started . the research protocol was submitted to the ethics committee for experimentation with human beings of universidade federal do acre and approved , cae number 22876013.9.0000.5010 ( 2013 ) . informed consent was obtained from each adult participant or legal guardian in case of minors , before the start of the study . the study included 50 people with vivax malaria , 28 men and 22 women , aged between 7 and 68 years ( average age 28.38 years ) . the average length of stay in an endemic area was 24.79 years , and about one - third of the study population ( 30% ) lived in an endemic area for malaria between 11 and 20 years . of the 50 patients evaluated , only one had a primary infection , and all the others had had between 1 and 41 episodes of previous malaria , and 36% report having had more than 10 episodes of malaria . about 52% had an episode in the 12 months leading up to the current episode , ranging from one to 10 , the number of malaria events in 2012 . most of the patients had low parasitemia ( less than 200 parasites / mm in 66% of cases ) , 12% of them had between 301 and 500 parasites / mm , and 12% were found to have 50110000 parasites / mm ( table 1 ) . the main symptoms observed in untreated malaria were fever ( 72% ) , chills ( 54% ) , sweating ( 54% ) , and weakness ( 42% ) , as well as pain symptoms including headache ( 76% ) , arthralgia ( 48% ) , and lower back pain ( 52% ) . less commonly reported symptoms were retroocular pain ( 36% ) and myalgia ( 30% ) . gastrointestinal symptoms occurred in low frequency , such as bitter taste ( 30% ) , decreased appetite ( 32% ) , nausea ( 22% ) , vomiting ( 16% ) , and pain in hypogastric or mesogastric region ( 20% ) . before treatment , only 20% of patients reported dark urine , and no patient reported having had pale stools . after initiation of treatment , a significant reduction in the prevalence of fever ( 38% ) , arthralgia ( 26% ) , back pain ( 32% ) , and retroocular pain ( 18% ) ( p < 0.05 , table 2 ) was noted . however , there was significant increase in the frequency of nausea ( 46% ) , diarrhea ( 26% ) , pale stools ( 12% ) , abdominal pain ( 38% ) , bitter taste in the mouth ( 60% ) , weakness ( 36% ) , and hypochondrial pain ( 32% ) ( p < 0.05 , table 2 ) . the frequency of choluria increased to 56% after initiation of antimalarial treatment ( p = 0.01 , table 2 ) . other symptoms such as vomiting , loss of appetite , headache , myalgia , chills , and sweating , as well as respiratory symptoms and upper respiratory tract symptoms , suffered no major change in frequency ( table 2 ) . among the adverse effects of chloroquine described in the literature and which are not often reported in malaria patients without treatment , there were reported changes in visual acuity ( 54% ) , insomnia ( 46% ) , pruritus ( 22% ) , the feeling of stings into the skin ( 22% ) , and paresthesias ( 6% ) . most gastrointestinal symptoms appeared after the patient started the antimalarial treatment ( table 3 ) . the symptoms most commonly seen in patients with malaria reported in the literature are fever , chills , sweating , headache , myalgia , and arthralgia . da silva - nunes and ferreira evaluated 326 cases of untreated malaria , reporting high frequency of headache ( 84.8% ) , fever ( 80.9% ) , and myalgia ( 68.7% ) , while gastrointestinal symptoms were infrequent ( < 20% ) . in pregnant women with vivax or falciparum malaria , the most frequent symptoms were also fever ( 97.9% ) , chills ( 71.7% ) , and headache ( 62.4% ) . the treatment of vivax malaria with chloroquine , a blood schizonticide , and primaquine ( active against latent tissue forms of p. vivax or so - called hypnozoites ) is very effective in reducing parasitemia and some of the symptoms caused by the plasmodium infection , such as fever and algic symptoms . fever tends to disappear between 24 and 48 hours after the beginning of treatment ; parasitemia decreases significantly at about 48 to 72 hours , and pain symptoms decrease rapidly . reported the disappearance of fever in 91.2% of patients , chills in 86% of patients , and headaches in 65.6% of patients at the end of the third day of treatment with chloroquine and primaquine and significant reduction of peripheral parasitemia between the second and third days of treatment . however , chloroquine can cause side effects or intensify symptoms already present , such as abdominal discomfort , nausea , vomiting , and diarrhea . these side effects can also occur during the use of primaquine being suitable to ingest the medication with food to avoid such side effects . chloroquine can rarely cause neurological symptoms , such as mental confusion , seizures , and coma , and cardiovascular symptoms such as hypotension , vasodilation , suppression of myocardial function , and cardiac arrhythmias , usually associated with rapid infusion of the drug parenterally . in usual doses for treatment of malaria , headache , blurred vision , and lichenoid rash can occur . chloroquine may block the entrance of potassium into the cells and primaquine also has an effect on blocking sodium channels . both may also have an effect on the chloride channels present in cardiac myocytes , which may explain the occurrence of cardiac and gastrointestinal side effects . in studies conducted in belm ( par ) , silva et al . found , as major side effects of chloroquine , the following symptoms : itching ( 5.55% ) , epigastric pain ( 1.28% ) , diarrhea ( 0.85% ) , and choluria ( 3.42% ) . the itching and stinging sensation are explained by formation of haptens during the metabolism of chloroquine , which bind to degraded products erythrocytes or phospholipids involved in allergic reactions , stimulating the production of ige - like antibodies and degranulation of mast cells and basophils . also , the binding of chloroquine at the dermoepidermal junction may possibly result in stimulation of nerve fibers , causing these symptoms . regarding primaquine use , the most prevalent side effect is hemolytic anemia , resulting in jaundice and choluria . data on primaquine use worldwide indicates that , among 200 million people , there were 14 deaths in six decades related to its use , from which 12 were caused by severe hemolysis . hemolytic anemia can also occur in people without g6pd deficiency when using primaquine , although it is a very scarce event . the gene encoding the g6pd is located on the x chromosome ( locus xq28 ) , with a recessive pattern of inheritance linked to sex . in heterozygous females normal red blood cells can coexist with red blood cells deficient in g6pd , in variable proportion , and this can cause a milder hemolytic anemia when exposed to primaquine . at the same time , there are 180 different genetic variants of g6pd , resulting in different levels of hemolysis . the mediterranean variant , which predominates in europe , western and central asia , and north of india , is more severe , while the african variant ( common in sub - saharan africa , amazon , and afro - americans ) is a milder variant . in these milder variants , hemolysis will be clinically detected only after one or two days of exposure to primaquine . the prevalence of g6pd deficiency is between 3 and 30% in endemic countries for malaria , corresponding to 350 million people . in the americas , it has a lower prevalence , varying between 5 and 10% in the amazon region and higher levels in tropical africa , middle east , and the mediterranean basin , such as sudan and congo , where it is present in more than 20% of the population . in brazil , the frequency of g6pd deficiency varies between 1.7% and 6.0% , prevailing the african variant . in manaus , a city with high levels of transmission of malaria in the brazilian amazon , the prevalence of g6pd was 2.5% . this difference in prevalence may explain why , in brazil , primaquine is administered to all nonpregnant patients older than 6 months with an unknown g6pd deficiency status , while in other countries with higher prevalence of this enzymatic deficiency ( or in which more severe variants are common ) primaquine use is very much debated and controversial . these events are dependent on the extent of hemolysis , which is related to the severity of g6pd deficiency and the dose of primaquine . recommended primaquine doses used to block falciparum malaria transmission ( 0.25 mg of base per kilogram ) are usually considered to have low toxicity , because it is a single - dose treatment . however , primaquine use in vivax malaria , aimed at preventing relapsing events , is a longer course ( between 7 and 14 days ) , with varying dosage among malaria control policies , and therefore hemolytic events are more prone to happen . in a systematic revision , monteiro et al . detected 47 cases of hemolytic anemia in patients using primaquine in latin america and caribbean , and 23 occurred in brazil . however , brazil has the largest population at risk for malaria ; thus it is expected to have more cases reported . a few cases of primaquine - induced hemolysis have also been reported in other countries , such as cuba , el salvador , puerto rico , and trinidad and tobago , between 1963 and 2013 . in our study , the frequency of choluria ( dark urine ) increased significantly from 6% to 56% in malaria patients after primaquine was started . since it is an event referred by the patient , we can not be sure how many of the patients really presented with choluria , or how severe was the choluria , but it is known that g6pd deficiency occurs among amazonian residents . it is possible that the hemolytic anemia is the cause of weakness and malaise , reported by these patients . however , none of the patients studied suffered from severe hemolysis or severe anemia ( data not shown ) or required hospitalization during or after malaria treatment . although severe events related to the use of primaquine were infrequent in our and other studies in the amazon , it is important to bear in mind the possible adverse side effects of primaquine and also mild anemia occurring in patients with the less severe forms of g6pd deficiency , which can be associated or not with weakness . reported that subjects with no g6pd deficiency showed reduction of 1 to 2 g / dl of hemoglobin after primaquine use , while in g6pd deficient patients hemoglobin levels decreased between 3 and 5 g / dl , with fast normalization after treatment was over . reported abdominal pain as a side effect for primaquine in falciparum malaria patients from myanmar , occurring in 16% of patients receiving a single dose . there is no clear evidence why primaquine can cause abdominal pain ; it can be related to the direct effect of the drug on an empty stomach or related to the release of pharmacological substances during hemolysis . despite its cause , it is noteworthy that , in our study , abdominal pain increased significantly from 6% to 32% after primaquine was started . although the present study has a limitation in the number of subjects studied , it was possible to detect significant association of symptoms and malaria treatment . thus , while the treatment of vivax malaria with a combination of chloroquine and primaquine leads to decrease in fever and pain symptoms , it can lead to the onset of gastrointestinal symptoms and hemolysis or enhances these symptoms when already present in the patient with malaria , contributing to reducing treatment adherence , especially during the use of primaquine , which has a longer course of treatment . moreover , the sensation of itching and stinging , although they do not occur in all patients , can be quite intense , hindering treatment with chloroquine . it is important to warn the patient of these possible side effects and seek measures to minimize them , either with concomitant use of antihistamines or antiemetics , according to the individual needs of each patient . the occurrence of severe hemolysis after primaquine use , although not common in brazil , can still occur , and since brazilian control programs currently do not have a policy for g6pd deficiency screening before malaria treatment is offered , health care personnel must be aware of it to interrupt primaquine use and manage its side effects properly .

side effects of antimalarial drug can overlap with malaria symptoms . we evaluated 50 patients with vivax malaria in mncio lima , acre , treated with chloroquine and primaquine . patients were evaluated for the presence of 21 symptoms before and after treatment and for reported side effects of these drugs after treatment was started . the most frequent symptoms before medication were headache , fever , chills , sweating , arthralgia , back pain , and weakness , which were present in between 40% and 76% of respondents . the treatment reduced the occurrence of these symptoms and reduced the lack of appetite , but gastrointestinal symptoms and choluria increased in frequency . there were no reports of pale stools before medication , but 12% reported the occurrence of this symptom after treatment started . other symptoms such as blurred vision ( 54% ) , pruritus ( 22% ) , paresthesia ( 6% ) , insomnia ( 46% ) , and stings into the skin ( 22% ) were reported after chloroquine was taken . the antimalarial drugs used to treat p. vivax malaria reduce much of the systemic and algic symptoms but cause mainly gastrointestinal side effects that may lead to lack of adherence to drug treatment . it is important to guide the patient for the appearance and the transience of such side effects in order to avoid abandoning treatment .

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from a worldwide perspective , the life span of humans has been prolonged , and the proportion of elderly people in the total population has increased . various changes occur in the human body with age . in elderly men , lower urinary tract symptoms ( luts ) , erectile dysfunction ( ed ) , and decreased libido often appear concurrently . several community - based studies have shown a strong correlation among sexual dysfunction , increasing age , and the severity of luts . this coexistence of sexual problems with luts and benign prostatic hyperplasia ( bph ) further affects quality of life ( qol ) . luts and ed share increased prevalence as age increases , and in many cases , the two are synchronously present . thus , continuous efforts have been made to disclose the common pathophysiology between these two conditions . no definite explanations have been given to clarify the pathophysiological mechanism of the correlation between luts and ed , but several hypotheses have been proposed [ 2 - 5 ] . one possible cause is overactivation of the autonomic nervous system and increased tension of the sympathetic nervous system . according to this hypothesis , luts increases sympathetic nervous system activity , which induces the occurrence of urinary storage symptoms due to the contraction of smooth muscles in the prostate gland and urinary bladder . ed occurs as the result of smooth muscle contractions in the corpus spongiosum , and the myosin light chain is phosphorylated because of increased rho - kinase activity , which can cause smooth muscle layer contraction . it has been proposed that endothelial dysfunction in the prostate gland and corpus spongiosum and hormonal imbalance are physiological causes for the correlation between luts and erectile dysfunction . changes in lifestyle due to nocturia and rapid eye movement sleep disorder may affect the occurrence of erectile dysfunction . also , the result of our preliminary study showed that age , international prostate symptom score ( ipss ) , nocturia , and the uroflow rate correlated significantly with the 5-item version of the international index of erectile function ( iief-5 ) . even in cases in which age was controlled for , qol was found to have a significant correlation with sexual function . prostate gland volume was not significantly correlated with sexual function , implying that erectile function is not affected by subjective luts even with a relatively greater prostate size . irwin et al reported that overactive bladder ( oab ) was significantly associated with increased prevalence of ed and that sexual activity , sexual enjoyment , and sexual satisfaction were reduced as the result of urinary symptoms . in recent years , many studies have reported that administration of alpha - blockers improves luts due to bph and sexual dysfunction . these findings suggest that rudimentary treatments of luts , a risk factor for erectile dysfunction , would be mandatory to effectively improve sexual function in patients with bph who concurrently have luts and ed . the primary treatment regimen for bph is to administer alpha - blockers , for which the main mode of action is based on smooth muscle relaxation in the prostate and the resulting improvement in the urinary flow rate and voiding symptoms . however , a synchronous improvement of irritative and voiding symptoms may be the most effective treatment regimen for luts / bph . it is expected that the concomitant administration of alpha - blockers and anticholinergics would improve luts more effectively in patients with luts / bph . these effects are also expected to have a positive effect in secondarily improving sexual function . thus , the aim of this study was to examine the effects on erectile function of a combination of the alpha - blocker tamsulosin to improve luts and the antimuscarinic agent solifenacin to improve irritative symptoms of the bladder . male patients aged 40 years or older who had concurrent luts / bph and ed were enrolled in this study . inclusion criteria were an ipss total score > 12 , an ipss - qol score > 3 , and an iief-5 score < 20 . exclusion criteria were as follows : 1 ) antiandrogens administered in the 4 weeks previously for the management of bph or luts ; 2 ) sex hormone agents or pde-5 inhibitors administered in the 4 weeks previously for the management of male sexual dysfunction ; 3 ) surgical treatment of the prostate gland or urethra ; 4 ) diagnosis of urethral stricture , urinary tract infection , prostatitis , prostate cancer , or bladder cancer ; 5 ) psa > 4 mg / dl ; 6 ) severe renal dysfunction or hepatic dysfunction ; 7 ) residual urine > 100 ml ; and 8) ineligibility for the current study as judged by the investigators . sixty male patients between the ages of 41 and 76 years with luts / bph and ed were divided into two groups by using a table of random sampling numbers : group 1 ( the alpha - blocker treatment group ; n=30 ) and group 2 ( the alpha - blocker+solifenacin treatment group ; n=30 ) . in group 1 , an alpha - blocker ( tamsulosin 0.2 mg q.d . ) was solely administered for 3 months . in group 2 , an alpha - blocker and an antimuscarinic drug ( tamsulosin 0.2 mg and solifenacin 5 mg q.d ) were concomitantly administered during the same period . in patients who were suspected of having a significant bladder outlet obstruction ( postvoiding residual urine > 100 ml or maximum flow rate < 5 ml / s ) , solifenacin was not administered . to examine the correlation between luts and the degree of erectile function , ipss and iief-5 then , prostate - specific antigen ( psa ) , transrectal ultrasonography ( trus ) , urine flowmetry ( ufm ) , and residual urine ( ru ) were measured . ipss was categorized into total scores , storage symptoms ( st ) , voiding symptoms ( vd ) , and quality of life ( qol ) . in group 1 , in which tamsulosin was solely administered ( n=30 ) for 3 months , and group 2 , in which tamsulosin and solifenacin were concomitantly administered ( n=30 ) for 3 months , comparative analyses were also done to examine the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . the t - test ( t ) and mann - whitney u - test were used to compare parameters indicating the degree of luts and sexual function between the two groups , such as trus , psa , ipss ( total , vd , and st ) , ipss - qol , iief-5 total , amount of urination , amount of ru , maximal urinary flow rate ( qmax ) , and mean urinary flow rate ( qave ) . by use of the paired t - test ( t ) and wilcoxon signed rank test ( z ) , a comparative analysis was performed on each group for ipss ( vd , st , total ) , ipss - qol , iief-5 total , the amount of urination , the amount of ru , qmax , and qave before and after treatment . regression analysis was used to inspect the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . the t - test ( t ) and mann - whitney u - test were used to compare parameters indicating the degree of luts and sexual function between the two groups , such as trus , psa , ipss ( total , vd , and st ) , ipss - qol , iief-5 total , amount of urination , amount of ru , maximal urinary flow rate ( qmax ) , and mean urinary flow rate ( qave ) . by use of the paired t - test ( t ) and wilcoxon signed rank test ( z ) , a comparative analysis was performed on each group for ipss ( vd , st , total ) , ipss - qol , iief-5 total , the amount of urination , the amount of ru , qmax , and qave before and after treatment . regression analysis was used to inspect the linear relationship between ipss scores and iief-5 scores before and after treatment in the two groups . of a total of 60 study patients , 1 patient in group 1 and 3 patients in group 2 dropped out of the 3-month follow - up study . therefore , analyses were performed on 29 patients in group 1 and 27 patients in group 2 . the incidences of hypertension and diabetes were 11 and 3 in group 1 and 9 and 5 in group 2 , respectively . before treatment , no significant differences ( p<0.05 ) were found between groups 1 and 2 , respectively , in age ( 59.176.73 yr vs. 56.410.80 yr ) , prostate gland size ( 27.18.0 g vs. 28.438.18 g ) , psa levels ( 1.090.97 mg / dl vs. 1.180.80 mg / dl ) , amount of urination ( 245.86160.97 ml vs. 223.36125.76 ml ) , amount of ru ( 2434.50 ml vs. 32.239.79 ml ) , qmax ( 14.417.16 ml / s vs. 14.806.1 ml / s ) , or qave ( 7.333.62 ml / s vs. 8.063.53 ml / s ) . no significant differences were found in iief-5 ( 12.456.85 vs. 12.047.65 ) or ipss - qol ( 4.280.88 vs. 3.820.88 ) scores . however , the ipss total score was significantly higher ( p<0.05 ) in group 1 than in group 2 ( 20.287.39 vs. 15.225.55 ) . this was due to the exclusion of patients in group 2 who were suspected of having significant bladder outlet obstruction ( postvoiding residual urine > 100 ml or maximum flow rate < 5 ml / s ) . in both groups 1 and 2 , the ipss total , ipss - vd , ipss - st , and ipss - qol scores were significantly improved after the 3-month treatment ( p<0.05 ) . however , no significant differences in the iief-5 total score , amount of urination , amount of ru , qmax , or qave were found before or after treatment ( table 1 ) . a comparison of the change ( ) in luts and sexual function after treatment between group 1 and group 2 revealed no significant differences in ipss total ( 5.726.90 vs. 4.155.72 ) , ipss - vd ( 3.694.77 vs. 2.044.71 ) , ipss - st ( 2.243.50 vs. 2.112.33 ) , ipss - qol ( 0.831.47 vs. 0.821.04 ) , or iief-5 a comparison of the degree of improved sexual function ( iief-5 ) associated with improved luts in each patient ( ipss total , ipss - vd , ipss - st , and ipss - qol ) showed significant improvement in the iief-5 score associated with the degree of improvement in the ipss - st domain ( ipss - st ) in group 1 , but no significant associations were found in group 2 ( table 3 , fig . 1 ) . in cases in which tamsulosin was administered , the iief-5 score significantly improved as the ipss - st domain score improved . in the group in which tamsulosin and solifenacin were concomitantly administered , improvement of the ipss - st domain score had no significant effect on the iief-5 score . luts / bph is the most common disease of lower urinary tract obstruction that occurs in men aged 60 years or older . in most cases , it develops slowly , but in some patients , it progresses rapidly . the incidence of both bph and ed is increased in elderly people , and the synchronous occurrence of luts and ed is considered to happen as the result of aging . even in cases in which age and underlying disease such as cardiovascular disease , diabetes mellitus , and hypertension were statistically controlled , epidemiologic studies have shown a definite correlation between bph and ed . these two disease entities are not due to aging , but they share a common pathophysiology . thus , a synergistic effect whereby the treatment of one disease would lead to improvement in the other disease is expected . conversely , khler and mcvary reported that the improved sexual function resulting from treatment with pde-5 inhibitors led to improved luts . a questionnaire study of 12,815 men aged 50 years or older found a significant correlation between bph and ed . furthermore , age and luts were found to be greater risk factors for ed than were organic factors such as diabetes mellitus , hypertension , and hyperlipidemia . in our series , age had the highest degree of correlation with sexual function . voiding symptoms of the ipss , storage symptoms , qol , nocturia symptoms , residual urine , maximal urinary flow rate , and mean urinary flow rate were significantly correlated with the degree of sexual satisfaction as well as with erectile function . luts accompanied by bph can be divided mainly into voiding ( obstructive ) symptoms and storage ( irritative ) symptoms . however , little is known about which of the various symptoms are more closely associated with ed . it remains unclear which luts might effectively improve the concurrent presence of ed , and a substantial number of studies in recent years have aimed to resolve this issue . this study was also performed to investigate which symptoms of bph , obstructive or irritative symptoms , are closely related to erectile function . morant et al reported that both irritative and voiding symptoms are significantly correlated with sexual function . tsai et al reported that irritative symptoms are more highly correlated with ed than are obstructive symptoms . a preliminary study that we conducted in patients with bph found a significant correlation between sexual function and various irritative and voiding symptoms , qol , nocturia , residual urine , and urine flow rates . following a 3-month treatment of luts with alpha - blockers , the improvement in voiding symptoms , qol , and average flow rate was significantly correlated with improved sexual function . to date , however , few studies have reported similar results , and controversy remains . storage symptoms due to oab may cause a higher degree of pain than do voiding symptoms . in cases of bph accompanied by oab , for this reason , the use of anticholinergic agents ( or antimuscarinic agents ) has been attempted in patients with oab accompanied by bph . several previous studies have confirmed the efficacy and safety of concomitant administration of alpha - blockers and anticholinergic agents . even so , in some cases , residual urine is increased or voiding symptoms are aggravated following the use of anticholinergic agents , and in patients with luts / bph , anticholinergic agents are used selectively . in our series , we excluded patients who likely had a significant bladder outlet obstruction ( pvr > 100 ml or qmax < 5 ml / s ) and who had luts / bph ; we therefore found significantly improved luts with no complications such as acute urinary retention . common side effects following the use of anticholinergic ( or antimuscarinic ) agents are dry mouth , constipation , blurred vision , headache , and dry eye , but almost no reports have described the effects on sexual function . some concern exists that the use of anticholinergic ( or antimuscarinic ) agents in patients with bph may lead to aggravation of voiding difficulty or the development of urinary retention . to date , urologists have not actively used anticholinergic ( or antimuscarinic ) agents , but many types of antimuscarinic agents have been used in recent years to treat oab in a clinical setting . as related experiences have accumulated , these drugs have been selectively used for patients with luts / bph . almost no reports have addressed the effects of antimuscarinic agents on sexual function in luts / bph patients . in a study of 39 men with bph and luts in whom previous alpha - blocker therapy had failed , kaplan et al found normal sexual function in 27 ( 63% ) and 29 ( 67% ) patients before and after administration , respectively . no reports have indicated that the administration of anticholinergic ( or antimuscarinic ) agents significantly impairs sexual function . in our series , a comparison of iief-5 scores before and after concomitant drug treatment with solifenacin and tamsulosin revealed no statistically significant changes . thus , it can be inferred that administration of anticholinergic ( or antimuscarinic ) agents had no significant effect on sexual function . in patients with luts / bph , concomitant treatment with alpha - blockers and antimuscarinics may lead to improvement in luts , but this treatment can not be expected to improve sexual function . in patients with luts / bph and ed , concomitant use of tamsulosin and solifenacin was effective for improving luts . however , sexual function was not significantly improved by the concomitant administration of alpha - blockers and antimuscarinics .

purposeto examine the effects on erectile function of concomitant treatment with an alpha - blocker ( tamsulosin ) and an antimuscarinic agent ( solifenacin ) in patients with lower urinary tract symptoms ( luts)/benign prostatic hyperplasia ( bph).materials and methodsfifty - seven male patients with luts / bph were assessed for the degree of luts and erectile function . in group 1 ( tamsulosin ) and group 2 ( tamsulosin and solifenacin ) , changes in the international prostate symptom score [ ipss : total scores , storage symptoms ( st ) , voiding symptoms ( vd ) , and quality of life ( qol ) ] , prostate - specific antigen , trans - rectal ultrasonography , urine flowmetry , residual urine , and a 5-item version of the international index of erectile function ( iief-5 ) were assessed after a 3-month treatment period . in both groups , it was determined whether treatment was associated with changes in luts and erectile function and whether improvement in the ipss was correlated with the iief-5 . comparative analysis was also done to examine the linear relationship between improved ipss scores and iief-5 scores.resultsa comparison of the degree of improvement in all the parameters indicated that both groups showed significant improvement in total ipss , ipss - st , ipss - vd , and ipss - qol ( p<0.05 ) . a comparison of the degree of improved sexual function associated with improved luts in each patient showed significant improvement in the iief-5 score associated with the degree of improvement in the ipss - st domain in group 1 , but no significant associations were found in group 2 . in cases in which tamsulosin was administered , the iief-5 score significantly improved as the ipss - st domain score improved . in the group in which tamsulosin and solifenacin were concomitantly administered , improvement of the ipss - st domain score had no significant effect on the iief-5 score.conclusionsin patients with luts / bph , tamsulosin and solifenacin combination therapy was effective for luts , but erectile function was not significantly improved . therefore , although effective for improving luts , combination therapy with an alpha - blocker and an antimuscarinic agent was not effective for improving erectile function .

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specific mirnas have been found to be expressed in cell type - specific manner , at specific developmental stages , and in disease states including cancer [ 1 , 2 ] . during the initiation and progression of cancer , mirnas have been observed to act as oncogenes or tumor suppressors [ 3 , 4 ] . while some mirnas are overexpressed in cancers , the majority appear to be lost and often localize to fragile sites . differences in the mirna expression profiles of normal compared to cancerous tissue of the endometrium , breast and ovary have been documented [ 611 ] . mirnas can affect the expression of a large number of proteins , including those involved in pathways relevant to cancer , such as apoptosis , migration and metastatis . thus , mirnas hold promise as biomarkers for several types of cancer [ 12 , 13 ] . epithelial to mesenchymal transition ( emt ) is a normal process that occurs during development in which individual cells or groups of cells become motile . the same process is thought to be used by cancer cells during tumor progression to enable them to become more motile and thus more metastatic . emt involves reprogramming of the cells by transcription factors such as zeb1 , sip1 ( zeb2 ) , twist , snail , and slug . a hallmark of emt is loss of e - cadherin expression , loss of polarity , acquisition of mesenchymal markers , and increased motility [ 16 , 17 ] . both zeb1 and the closely related zeb2 bind e - box like sequences in the e - cadherin promoter , recruit the corepressor ctbp and thereby repress e - cadherin . zeb1 expression is confined to cells of mesenchymal origin , while normal epithelial cells and low grade carcinomas do not express zeb1 . however , we and others have shown that in high grade , aggressive carcinomas that have undergone emt , zeb1 can be expressed , leading to loss of e - cadherin [ 1922 ] . several mirnas have been implicated in the process of emt , among them are the members mir-200 family [ 2325 ] . this family contains five members ( mir-200a , -200b , -200c , -141 and , -429 ) which are highly homologous . originally , mir-200c was reported to directly bind zeb1 and cause degradation of the mrna , resulting in an upregulation of e - cadherin . subsequently , other reports have shown that all members of the mir-200 family , since they share a high degree of homology especially in their seed sequence , are capable of repressing both zeb1 and zeb2 [ 2729 ] . we have demonstrated that mir-200c represses not only zeb1/2 , but a program of transcripts normally expressed only in cells of mesenchymal origin . since members of the mir-200 family are responsible for repressing zeb1 and zeb2 as well as other mesenchymal genes , these mirnas are considered mir-200 family members are therefore thought to be expressed in an epithelial cell - specific manner in normal tissues . recently , the ability of zeb1 to transcriptionally repress expression of mir-200 family members has been documented [ 31 , 32 ] . this double negative feedback loop between mir-200 family members and zeb1 allows for plasticity between the epithelial and mesenchymal states . in this paper , we focus on the role of mir-200c in breast , ovarian , and endometrial cancers . the mutual repression between zeb1 and mir-200c is functional in some , but not all cells that we have tested . we demonstrate that the decrease in migration and invasion observed when mir-200c is reintroduced to cancer cells that lack it is independent of restoration of e - cadherin . lastly , we have previously demonstrated that class iii beta tubulin ( tubb3 ) is directly controlled by mir-200c . expression of tubb3 is known to be a common mechanism of resistance to microtubule - targeting agents in many types of cancer . here , we present conclusive data that repression of tubb3 is the mechanism whereby mir-200c restores sensitivity to paclitaxel . taken together , these data demonstrate that loss of mir-200c is a marker for chemoresistance and aggressiveness in breast , ovarian , and endometrial cancers . hec50 cells , representing the more aggressive type ii endometrial cancers , were cultured in dmem with 10% fbs , and 2 mm l - glutamine . mda - mb-231 are a triple negative breast cancer cell line and were grown in media containing 5% fbs , hepes , nonessential amino acids , l - glutamine , penicillin , streptomycin , and insulin . the identity of all the cell lines was confirmed by dna profiling using the identifiler kit ( applied biosystems ) . lipofectamine 2000 ( invitrogen ) was combined with pre-200c ( mirna mimic ) or scrambled negative control ( ambion ) at a concentration of 60 nm and incubated in serum free rpmi for 20 minutes prior to addition to hey cells . cells were incubated at 37c for 4 hours before replacement of fbs to 10% . protein and rna were harvested 48 hours posttransfection . tubb3 ( from fernando cabral , university of texas - houston medical school ) was cloned into pci - neo . transient transfection of 3.3 g of tubb3 plasmid or empty vector ( pci - neo , promega ) per well in a 6-well plate was performed using lipofectamine 2000 . transduction of cells was performed using smartvectortm shrna lentivral particles ( thermo scientific dharmacon ) . each cell line was transduced with 3 separate lentiviral constructs targeting zeb1 as well as two controls : smartvector empty vector particles and smartvector firefly luciferase control particles . the former is a negative control and does not correlate with gene silencing and the latter is a positive control targeting firefly luciferase plasmids pgl2 and pgl3 . all vectors are packaged and contain a turbogfp and an scmv promoter , as well as a puromycin - resistance selectable marker . mda231 and hec50 cells were plated at 3000 cells / well and 1500 cells / well , respectively , in triplicate using 96 well plates . the following day , media was replaced with 80 l of fresh media containing 10 g / ml polybrene ( sigma ) . the amount of viral particles / well was determined using the following calculation : ( moi cn)/vt , where moi ( multiplicity of infection ) = 10 tu / cell , cn = number of cells / well , and vt = stock viral titer of 10 tu/l . viral particles were added in a total volume of 20 l to each well . the following day , transduction media was removed and wells were rinsed with pbs and replaced with regular media . once confluent , cells were trypsinized and replated in 48 well plates . at this point , antibiotic selection was initiated and cells were ultimately expanded and maintained using 1 g / ml of puromycin ( sigma ) . whole cell protein extracts were denatured and 50 g separated on 8% sds page gels and transferred to pvdf membranes . the membranes were blocked in 5% milk in tbs - t , and then probed overnight at 4c . doug darling , university of kentucky , 1 : 1500 dilution ) , e - cadherin ( clone nch-38 from dako , 1 g / ml ) , tubb3 ( rabbit polyclonal prb-435p from covance , 1 : 5000 dilution ) , and -tubulin ( clone b-5 - 1 - 2 from sigma , 1 : 20000 dilution ) . after incubation with appropriate hrp - conjugated secondary antibody , bands were detected using western lightning chemiluminescence reagent plus ( perkin elmer ) . rna was harvested from cells using trizol ( invitrogen ) as per the manufacturer 's instructions . prior to generating cdna , mrna was treated with dnase1 ( invitrogen ) for 15 minutes at room temperature . rna was reverse transcribed into cdna in a reaction containing reaction buffer , dntps , rnase inhibitor ( applied biosystems ) , random hexamers , and 200 u of mulv - rt ( applied biosystems ) . the reaction proceeded at 22c for 10 minutes , then at 37c for one hour . for normalization , real time rt - pcr was performed on the cdna using eukaryotic 18s rrna endogenous control primers and fam - mgb probe ( applied biosystems ) . taqman microrna reverse transcription kit was used to generate cdna for real time rt - pcr reaction in conjunction with a mir-200c specific primer and probe ( abi , assay i d 002300 ) . the reverse transcription primer for mir-200c is a hairpin primer which is specific for the mature mirna and will not bind to the precursor molecules . reported values are the means and standard errors of 3 biological replicates . the relative mrna or mirna levels were calculated using the comparative ct method ( ct ) . briefly , the cycle threshold ( ct ) values for the rrna were subtracted from ct values of the target gene to achieve the ct value . the 2 was calculated for each sample and then each of the values was then divided by a control sample to achieve the relative mirna levels ( ct ) . the assays were performed on mda - mb-231 stable empty vector or shzeb1 # 2 , or hey cells transiently transfected with the mir-200c mimic for 48 hours . bd biocoat control insert chambers 24-well plate with 8 micron pore size and bd biocoat matrigel invasion chambers were used for migration and invasion assays , respectively . after starvation , cells were removed from the plate and 50000 hey cells or 250000 mda - mb-231 cells were plated in 0.5 ml media with 0.5% fbs in the upper chamber . in the lower chamber 0.8 ml of 50% conditioned media plus 50% complete media containing an additional 10% fbs hey cells were incubated for 24 hours and mda - mb-231 cells for 48 hours at 37c . migrating or invading cells on the lower surface of the membranes were stained with diff - quik stain ( fisher ) and counted manually using imagepro plus software ( mediacybernetics inc . ) . adhesion assays were performed using innocyte ecm cell adhesion assays ( calbiochem ) for collagen iv , fibronectin , basement membrane complex and laminin . to each well 50000 cells the plates were incubated for 1 hour at 37c , and fluorescence was read with an excitation wavelength of 485 nm and an emission wavelength of 528 nm . the relative fluorescent units were plotted , and the error bars represent standard error of the mean over four replicates . hey cells were plated into 6-well plates at a density of 2000 cells per well . twenty - four hours after plating , the cells were treated with 0 , 1 , 2 , 3 , 4 , or 5 nm paclitaxel ( sigma ) in triplicate . the cells were incubated at 37c for 8 days before fixing and staining with crystal violet . photos were taken of the plates and the images analyzed using imagej software ( nih ) . the average number of colonies and the average total area was plotted , with error bars representing the standard error of the mean over the three replicates . hec50 cells were transfected with the mir-200c mimic as described previously for the hey cells . twenty - four hours after transfection , cells were treated with 0 , 15 , 20 or 25 nm paclitaxel ( sigma ) . twenty - four hours after treatment , the cell death elisa ( roche ) which recognizes mono- and oligonucleosomes in the cytoplasm of dying cells was performed as per manufacturer 's instructions . cells were plated into 6-well plates ( 4000 hey cells / well and 6000 mda - mb-231 or hec50 cells / well ) . at time points indicated , cells were trypsinized and counted using the vi - cell cell viability counter ( beckman coulter ) . just as members of the mir-200 family can repress zeb1 by degradation of its transcript , zeb1 can repress expression of the mir-200 family members by binding to e - boxes within their promoter regions [ 31 , 32 ] ; see figure 1(a ) . directly increasing mir-200c levels in hey cells ( aggressive serous ovarian cell line ) by transfection of a mir-200c mimic ( pre-200c ) results in repression of zeb1 expression ( figure 1(b ) ) . although zeb1 is a repressor of e - cadherin , we did not observe e - cadherin expression induced by the repression of zeb1 in these cells ( data not shown ) . however there are several other mechanisms through which e - cadherin can be lost including methylation of the promoter [ 34 , 35 ] and chromosomal deletion [ 36 , 37 ] . in contrast , we have previously shown that transient transfection of the mir-200c mimic into mda - mb-231 ( an aggressive triple negative breast cancer cell line ) and hec50 ( an aggressive type 1 endometrial cancer cell line ) causes a marked repression of zeb1 and a restoration of e - cadherin expression . presently , we stably transduced lentiviral shrnas targeting zeb1 into these two cell lines ( hec50 and mda - mb-231 ) . while two of the shrnas did not decrease zeb1 protein , shrna # 2 caused an almost complete repression of zeb1 expression resulting in re - expression of e - cadherin in both cell lines ( figures 2(c ) and 2(d ) ) . intriguingly , while knock down of zeb1 in mda - mb-231 cells causes the expected increase in mir-200c levels ( indicative of the reciprocal regulation ) , no such increase is observed in hec50 cells . this suggests that while reciprocal repression of mir-200c and zeb1 occurs in some cell lines , it does not occur in all . it has been previously shown that the mir-200 family members cause a decrease in cell migration and invasion [ 27 , 29 , 30 ] . we observe a decrease in migration and invasion in the mda - mb-231 cells in which zeb1 has been knocked down , resulting in an increase in mir-200c levels . in the mda - mb-231 cells there is 52% decrease in migration and a 50% decrease in invasion in the shzeb1 # 2 containing cells in which zeb1 is completely knocked down versus luciferase control ( figures 2(a ) and 2(b ) ) . we show here that the same holds true in the aggressive ovarian cancer hey cell line . this cell line is highly migratory and invasive , and reintroduction of mir-200c to these cells results in an 83% decrease in migration and a 7986% decrease in invasion compared to negative controls ( figures 2(c ) and 2(d ) ) . however , it is interesting to note that the effect on migration and invasion caused by mir-200c is independent of the e - cadherin status of the cells , since unlike the mda - mb-231 cells , hey cells do not regain e - cadherin expression in response to decreased zeb1 levels . while e - cadherin protein affects epithelial cell - cell contact , we also wished to determine if mir-200c affects adhesion to substrates as measured by fluorescent adhesion assays . hey cells transiently transfected with the mir-200c mimic showed a small but statistically significant decrease in adhesion to basement membrane complex ( bmc ) and laminin ( figures 3(a ) and 3(b ) ) . there is also a trend towards decreased adhesion to collagen iv ( figure 3(c ) ) ; however , this did not reach statistical significance . no difference in adhesion to fibronectin was observed ( figure 3(d ) ) . since there was an affect on adhesion to bmc and laminin in ovarian cancer cells with high mir-200c levels , we performed the adhesion assay with the hec50 and mda - mb-231 cells in which zeb1 had been stably knocked down . we again see a decrease in adhesion to bmc and laminin in the mda - mb-231 cells ; however , only the decrease in bmc binding is statistically significant ( figures 3(e ) and 3(f ) ) . in contrast to the hey and mda - mb-231 cells , there was no decrease in adhesion to either substrate in the hec50 cells in which zeb1 is knocked down ( data not shown ) , but there is not a concomitant increase in mir-200c , as shown in figure 1(d ) . we have previously demonstrated that mir-200c expression causes increased chemosensitivity to microtubule targeting agents such as paclitaxel . while the elisa cell death assay that we have used previously to demonstrate this property of mir-200c is a short - term assay , we confirm here , in a relatively long - term clonogenic assay , that there is increased sensitivity of hey cells to paclitaxel when transfected with pre-200c ( figure 4(a ) ) . we observe a 4955% decrease in total area and a 6770% decrease in the number of colonies in the pre-200c treated cells versus the negative control with 5 nm paclitaxel treatment ( figures 4(b ) and 4(c ) ) . as the assay is conducted over a relatively long period of time , the maximum dose of paclitaxel used is relatively small compared to what is used in the assays that look at acute toxicity ( i.e. , 24 hours ) . at doses of paclitaxel of 10 nm and higher , we have previously implicated the ability of mir-200c to directly target tubb3 ( class iii beta tubulin ) as being the mechanism responsible for the increased chemosensitivity to microtubule targeting agents . tubb3 is normally only expressed in neuronal cells ; however aberrant expression of tubb3 in several different types of cancers has been shown to cause resistance to paclitaxel [ 3843 ] . to definitively test whether tubb3 is responsible for the mir-200c - mediated increase in chemosensitivity to paclitaxel , we transfected cells with a tubb3 construct lacking its 3 utr ( containing the mir-200c binding site ) which is therefore not able to be targeted by mir-200c . transfection of this exogenous tubb3 construct does not affect the transfection of the mir-200c mimic , nor its ability to downregulate zeb1 and upregulate e - cadherin ( figures 5(a ) and 5(b ) ) in hec50 cells . when the hec50 cells are transfected with an empty vector ( no exogenous tubb3 ) in addition to mir-200c mimic , there is a statistically significant increase in sensitivity to paclitaxel as measured in a cell death elisa ; see figure 5(c ) . however , when the cells are transfected with the tubb3 expression vector lacking its 3 utr , the enhanced sensitivity to paclitaxel is lost ; see figure 5(d ) . therefore , expression of exogenous tubb3 lacking the mir-200c target site reverses the chemosensitivity to paclitaxel caused by increased mir-200c expression . it can be argued that cells with increased proliferation would be more sensitive to microtubule poisons and that could be an alternative explanation for the observed chemosensitivity upon restoration of mir-200c . we therefore performed proliferation assays in the three cell types and found decreased proliferation in all three ( figures 6(a ) , 6(b ) , and 6(c ) ) . since the decrease in proliferation is observed in all three cell types , including the hec50s where there was no increase in mir-200c levels , it is likely that the effects on proliferation occur via zeb1 and not by mir-200c . the fact that the increase in chemosensitivity is found in cells that are proliferating more slowly than the negative controls demonstrates that increased proliferation is not the mechanism behind the increase in chemosensitivity . in this paper we build on our previous work to further characterize the role that loss of mir-200c plays in generating an aggressive cancer phenotype . zeb1 is normally only expressed in cells of mesenchymal origin ; however , its aberrant expression is observed in cancers that have undergone emt . zeb1 ( and the closely related zeb2 ) transcripts are targeted by mir-200c and the other mir-200 family members . interaction of any of the mir-200 family members with the zeb transcripts results in degradation and inhibition of translation . therefore the maintenance of mir-200c expression in normal epithelial cells serves to prevent zeb1 and zeb2 from being expressed . since both zeb1 and zeb2 repress genes involved in polarity , repression of these proteins serves to maintain polarity , an important epithelial cell characteristic . we have recently shown that in addition to repressing zeb1 and 2 , mir-200c represses a program of transcripts normally only expressed in cells of mesenchymal and neuronal origin , such as fibronectin ( fn1 ) , neurotrophic tyrosine kinase ( ntrk2 ) , quaking 1 ( qki ) , and tubb3 . thus , mir-200c maintains epithelial cell characteristics not only by maintaining polarity via repression of zeb1 and zeb2 , but also by repressing additional non - epithelial genes . it has been recently demonstrated that mir-200c and zeb1 regulate each other in a double - negative feedback loop [ 31 , 32 ] . the mir-200 family of mirnas is expressed in two clusters , one on choromosome 1p36.33 and the other on chromosome 12p12.31 . therefore , in cells that have undergone emt , zeb1 and 2 not only serve to repress genes involved in polarity , but also repress the mir-200 family and thereby release the repression of many genes characteristic of the mesenchymal phenotype . central to the double feedback loop between mir-200c and zeb1 is tgf-. during tgf- -induced emt , there is an increase in ets1 which binds to and activates the promoter of zeb1 . therefore , in a tumor microenvironment , increased tgf- levels are thought to result in an increase of zeb1 transcription to a point where it can overcome the repression caused by mir-200c . as zeb1 protein begins to be made , it can then repress the mir-200 family members , resulting in progression through emt . we have previously shown that restoration of mir-200c in hec50 endometrial cells and mda - mb-231 breast cancer cells causes repression of zeb1 and re - expression of e - cadherin protein . here we show that transfection of mir-200c mimic into hey cells , an aggressive serous ovarian cell line , also causes a dramatic repression of zeb1 ; however no expression of e - cadherin was observed . to test whether the double - negative feedback loop is intact in the hec50 and mda - mb-231 cells , these cells were infected with lentivirus expressing an shrna against zeb1 . in both cell lines , efficient knock down of zeb1 was achieved , as was re - expression of e - cadherin . in mda - mb-231 cells , zeb1 knock down resulted in an increase in mir-200c levels , as would be expected from the negative feedback loop . however , this was not the case in hec50 cells , where there was no increase in mir-200c . whether the break in the negative feedback loop is an anomaly of this particular cell line remains to be tested . the mechanism behind the phenomenon is also unknown ; however , it does offer an opportunity to dissect the contribution of zeb1 versus that of mir-200c to the phenotype of the cells . for example , significantly decreased proliferation was observed in the hey cells transiently transfected with the mir-200c mimic as well as in the mda - mb-231 and hec50 cells that have zeb1 stably knocked down , although mir-200c levels did not rise in the hec50s . therefore it is likely that the decrease in proliferation is due to the lack of zeb1 , not an increase in mir-200c . conversely , the decrease in adhesion to the basement membrane complex and laminin was only observed in the hey and mda - mb-231 cells , and not the hec50s , suggesting that this phenotype is a function of mir-200c expression rather than zeb1 . we and others have previously shown that restoration of mir-200c to cancer cells that do not express it causes a decrease in invasion and migration [ 27 , 30 ] . here we show that knock down of zeb1 in mda - mb-231 cells , which causes an increase in mir-200c , negatively affects migration and invasion . furthermore , we show that restoration of mir-200c in hey ovarian cancer cells results in a dramatic decrease in migration and invasion even though e - cadherin is not restored in these cells , despite complete repression of zeb1 . loss of e - cadherin expression can result from mechanisms other than zeb1 transcription repression , including chromosomal deletion and promoter hypermethylation [ 3437 ] . possibilities for its continued absence in these cells include promoter methylation such that even when repression by zeb1 is relieved , e - cadherin will not be expressed , or perhaps levels of another transcriptional repressor such as snail or twist remain high and repress e - cadherin . regardless of the mechanism , the effects of mir-200c on invasion and migration appear to be independent of e - cadherin status . while e - cadherin is involved in epithelial cell - cell adhesion and its expression has been shown to negatively affect migration and invasion [ 45 , 46 ] , increased mir-200c is able to decrease migration and invasion on its own . we have previously observed that restoration of mir-200c affects genes involved in cell motility and invasion such as arhgdib , ntrk2 , ephb1 , and fn1 . we demonstrate that mir-200c causes a decrease in adhesion to basement membrane complex , laminin , and perhaps collagen type iv . this observation is particularly relevant to ovarian cancer because the cancerous cells adhere to sites within the peritoneal cavity . during the progression of cancer there is switching of the expression patterns of the cell surface adhesion molecules , such as the cadherins and integrins [ 4749 ] . although the change in the number of adherent cells appears modest , this might play a significant role in developing a potential treatment for ovarian cancer . the ability of ovarian cancer cells to spread and adhere to the peritoneal cavity is one of the major phenotypes of this disease . a small change in ability of the cells to adhere might reflect a great decrease in the tumor burden and/or increase the ability to debulk the tumor . these results are independent of e - cadherin expression since the decrease in adhesion is observed in both the mda - mb-231 cells ( where e - cadherin expression is regained with increased mir-200c ) and in hey cells , where it is not . clonogenic assays reveal that there is an increase in chemosensitivity to paclitaxel with increased mir-200c levels . indeed , acquired resistance to paclitaxel in ovarian cancer cells has been shown to be associated with emt , resulting in an aggressive phenotype . clinically , aberrant expression of tubb3 ( not normally expressed in epithelial cells ) has been found to be associated with resistance to taxanes [ 3841 ] . we have previously shown that tubb3 is a direct target of mir-200c and suggested that its repression by mir-200c is the mechanism behind the ability of mir-200c to increase chemosensitivity to microtubule targeting agents . here , we perform the definitive experiment to prove that mir-200c - mediated tubb3 downregulation is indeed the cause of the enhanced chemosensitivity . we utilized exogenous tubb3 lacking its 3utr such that it can not be targeted by mir-200c and show that resistance to paclitaxel is maintained even in the presence of mir-200c . in contrast , endogenous tubb3 is reduced when mir-200c is added , resulting in enhanced chemosensitivity to paclitaxel . microtubule targeting agents such as paclitaxel work more efficiently in cells that are rapidly dividing . consequently , it could be argued that the increase in chemosensitivity caused by mir-200c is due to increased proliferation . however , we show that increase of mir-200c or direct knockdown of zeb1 results in decreased proliferation in three different types of cancer cells . it is therefore the downregulation of tubb3 , not an increase in proliferation that is responsible for the enhanced chemosensitivity to taxanes observed with restoration of mir-200c to resistant cancer cells . mir-200c expression serves to maintain the epithelial phenotype in well - differentiated , low - grade , breast , ovarian , and endometrial cancer cells . furthermore we find that not all of mir-200c 's actions can be attributed to the restoration of e - cadherin via targeting of zeb1 . we further prove that mir-200c - mediated repression of tubb3 is the cause of enhanced chemosensitivity to microtubule targeting agents . lastly we demonstrate that not all cells exhibit the double negative feedback loop between mir-200c and zeb1 and that this can be exploited to identify the distinct roles of mir-200c as compared to zeb1 .

we focus on unique roles of mir-200c in breast , ovarian , and endometrial cancers . members of the mir-200 family target zeb1 , a transcription factor which represses e - cadherin and other genes involved in polarity . we demonstrate that the double negative feedback loop between mir-200c and zeb1 is functional in some , but not all cell lines . restoration of mir-200c to aggressive cancer cells causes a decrease in migration and invasion . these effects are independent of e - cadherin status . additionally , we observe that restoration of mir-200c to ovarian cancer cells causes a decrease in adhesion to laminin . we have previously reported that reintroduction of mir-200c to aggressive cells that lack mir-200c expression restores sensitivity to paclitaxel . we now prove that this ability is a result of direct targeting of class iii beta - tubulin ( tubb3 ) . introduction of a tubb3 expression construct lacking the mir-200c target site into cells transfected with mir-200c mimic results in no change in sensitivity to paclitaxel . lastly , we observe a decrease in proliferation in cells transfected with mir-200c mimic , and cells where zeb1 is knocked down stably , demonstrating that the ability of mir-200c to enhance sensitivity to paclitaxel is not due to an increased proliferation rate .

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the transobturator approach to treat female stress urinary incontinence by means of a midurethral sling ( mus ) placement was first described by delorme to potentially prevent complications associated with the retropubic mus placement originally described by ulmsten ( delorme , 2001 ; ulmsten et al . , 1996 ) . a similar transobturator passage utilizing an inside - out approach , as opposed to delorme s outside - in approach , was later described by de leval , aiming to further reduce the potential injury to the urethra and bladder ( de leval , 2003 ) . a recent meta - analysis found similar cure rates between the 3 most commonly used surgical approaches for treating stress urinary incontinence , namely the retropubic , outside - in transobturator , and inside - out transobturator mus procedures ( latthe et al . , 2010 ) . multiple studies have discussed the anatomical trajectories of both obturator approaches and confirmed their anatomical safety from a neurovascular perspective ( bonnet et al . , 2005 ; achtari et al . , 2006 ; hinoul et al . , 2007 ; zahn et al . , 2007 ; reisenauer et al . , 2006 ) . the large body of clinical evidence supports these findings . to date , anatomical studies have focused on the mus s trajectory in relation to the obturator foramen , the muscles and especially the neuro - vascular structures . traditional anatomical dissections do not allow the study of the mus s configuration in relation to the urethra , as a progressive dissection of the entire tape s trajectory would be required , inherently leading to a distortion of the relevant anatomy . the aim of this cadaveric study was to compare the specific relationship between the tape and the urethra in both the inside - out and outside - in approach . to allow such a comparison , ct - scan image analysis was performed after tantalum wire marked mesh slings were implanted in a series of cadavers . all 10 cadavers were operated upon , by independent surgeons , one experienced with the inside - out ( in - out ) and one with cadavers were positioned in the dorsal supine lithotomy position with the legs in abduction and at a 100 flexion position ( at the level of the hips ) on the in - outside and at 90 flexion on the out - in side . the surgical technique for the transobturator mus procedures were performed according to the instructions for use as provided by the respective manufacturers ( out - in : monarc , american medical systems , minnetonka , mn and in - out : tvt - obturator system , ethicon inc . , all cadavers were catheterized using a foley catheter with its lumen filled with a radio - opaque solution ( hypaquemeglumine 60% , amersham health , princeton , nj ) to allow subsequent identification of the urethral position . both the in - out and the out - in transobturator procedures were performed on each cadaver , alternating the type of procedure and tape between the right and left sides in the consecutively operated cadavers . to be able to do this with a single tape in each cadaver , a device was conceived for this study in which the tape on one side consisted of an in - out mus device , while an out - in mus device was attached to the other side at the midpoint . to allow radiographic visualization of these tapes , a double tantalum wire was threaded through each side of both midurethral tapes as shown in figure 1 . this design allowed for an intra - cadaveric comparison between 10 independent in - out transobturator mus trajectories and 10 out - in transobturator mus trajectories . care was taken to have a flat positioning of both tape ends at the end of the procedures . the vaginal incision sites were closed and the mus tapes were cut 2 cm from the level of the skin . to exclude the possible influence of the surgeon s right handedness , an equal number of both procedures was performed on the right and left sides ; bmi of the cadavers ranged between 15 and 33 kg / m2 ( mean : 25.3 ; median : 26 ) . post capture image analysis was performed using the vitrea core 5.1.1618.1808 image analysis software ( vital , 5850 opus parkway , suite 300 , minnetonka , mn 55343 - 4414 ) . plane that included the urethra at the point where it crossed the urethra and the two arms of the sling extending from the urethra to the point where it crossed the obturator membrane . this was done to prevent parallax effects that might alter the measured angle ( hoyte and ratiu , 2001 ) . on the mips view of the images , the relative angulations between a line through the urethral axis and the slope drawn through each mus tape s mean upward pathway ( forming the hammock part of its trajectory ) were measured ( table i ) . the angle inside the upward trajectory of the tape constituted the angle under consideration for this study and is referred to as the tape s hemi inner angle , as only half a procedure was performed on each cadaver ( fig . for the study s purposes the assumption was made that a symmetric , bilateral placement of a full length tape would yield the full inner angle of the mus hammock corresponding to double the hemi - inner angle measurement . 2a . ventro - dorsal view ( frontal view ; l = left , r = right ) . note : dotted lines schematically demonstrates the hemi - inner angle but do not correspond to the mips view used to measure the actual angles . statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples . three cadavers were noted to have stage 2 prolapse and 1 had a notably high vaginal sulcus . the 3-dimensional ct images revealed that the mus tapes lie as a band posterior / dorsal to the urethra rather than inferior . the out - in muss were more closely wrapped around the inferior ischio - pubic ramus than the in - out muss . qualitative assessment of the images demonstrated a safe distance from the tapes to the obturator canal for both techniques . distances of the tapes trajectories to the individual obturator nerve branches could not be determined . the mean full inner angle in a strict antero - posterior view formed by the in - out mus measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in mus ( p = 0.02 ) . the ( paired ) differences between the tapes inner angles were significantly different ; with a mean difference of 19.8 ( median 19.0 ) , ( wilcoxon signed rank test : p = 0.008 ) . correlation analysis only showed a trend between the out - in and the in - out mus angle ( correlation coefficient : 0.6 , p = 0.08 ) . a similar trend was observed between the patient s bmi and the in - out mus angle ( correlation coefficient : 0.6 , p = 0.08 ) , but not for bmi and the out - in mus angle ( correlation coefficient : 0.08 , p = 0.8 ) . there was no correlation between the infra - pubic angle measurement and the out - in mus angle nor the in - out mus angle . the use of tantalum wires threaded through the polypropylene-/ prolene - tape in combination with three - dimensional ct - scanning yielded a unique method to visualize midurethral tapes throughout the entirety of their trajectory . this technique can be applied to different types of non radio - opaque implants , allowing for a better spatial understanding of the anatomical position within the pelvis . this technique circumvents the technical problems encountered by ultrasound where visualization is partially obstructed by the bony pelvis and the fact that traditional anatomical techniques rely on a progressive dissection intrinsically only allowing visualization at a single level each time . this is the first controlled anatomical study to compare the spatial relationships between the in - out and out - in mus tapes and the female urethra . the difference could be objectified by measuring the angle formed between the two legs of the mus tape . the in - out approach angulated around the urethra in a more acute fashion than the out - in approach , which seemed to follow a more horizontal , flatter , trajectory ( 122 versus 142 ) . these findings were counter - intuitive as the exit - points of both devices at the level of the skin would suggest the opposite . the three dimensional radiographic visualization of the tape s trajectory also allowed for an appreciation of the mus tapes spatial configuration in relation to the bony pelvis and the urethra . contrary to the belief that the urethra is suspended in a cranially directed u - shaped band , both obturator mus approaches were demonstrated to be u - shaped bands that head backwards or dorsally . the tape is not positioned caudally but lies as a band behind the urethra , suggestive of its passive ( non obstructive ) role in achieving continence . as observed in the different measurements of the tapes angles , there seemed to be a higher variability in the in - out s than the out - in trajectory ( 58 versus 40 variation respectively ) , as previously described by hinoul et al . in a traditional anatomic study , underscoring the need for strict adherence to the prescribed operative technique ( hinoul et al . , 2007 ) . the importance of this becomes clear when comparing it to two ultrasound studies investigating the angles formed by the two arms of mus tapes . lin et al described an angle at rest for the in - out tape to be 115 ( sd13 ) while chene et al , measured the same angle at rest also using ultrasound to be 138(sd7 ) ( lin et al . the differences in outcomes obtained in these studies demonstrate that ( inter- and intra - individual ) comparisons may prove to be difficult to interpret . the cadaveric study design was also the study s limitation as no clinical findings could be correlated to the static anatomic findings . a direct meta - analysis comparing both routes of transobturator tapes by madhuvrata et al . ( 2012 ) reported no significant differences in the objective and subjective efficacy but showed that the inside - out route was associated with significantly fewer vaginal angle injuries but with a non - significanttrend towards higher risk of postoperative groin pain . in a small retrospective study , out - in mus tapes were more frequently associated with a finding of superficial placement at the level of the lateral vaginal sulcus , described as paraurethral banding ( cholhan et al . , 2010 ) . coincidentally , the only cadaver with a notably high vaginal sulcus was found to have a very palpable out - in mus tape post placement . increased rates of dyspareunia and tape exposures after the out - in compared to the in - out approach ( 2012 ) , but these finding were not confirmed in an rct by abdel - fattah et al . the more horizontal positioning of the out - in tape s trajectory , taking the mesh closer to the vaginal wall , as described in this study could explain these clinical observations . the radiographic images explain the increased risk of button - holing during an out - in procedure because the more acute in - out mus trajectory buries the tape deeper inside the internal adductor muscles . on the other hand , this study also confirmed that the out - in mus trajectory stayed closer to the ischiopubic ramus . the slightly more lateral in - out mus trajectory causes the tape to be seated in more muscular tissue . the corresponding inflammatory reaction might explain the trend to a slightly higher risk of transient thigh pain . the clinical implications from this cadaveric imaging study are that surgeons must pay more attention to the 2nd half of the helical pass , whichever way they chose to go . for the in - out passage , one must rotate hard and carefully around the ischiopubic ramus and not get lazy with the rotation ; while for the out - in pass , care must be taken that dissection of the peri - urethral tunnel provides for a full thickness vaginal epithelial layer . the advent of three - dimensional ultrasonography will allow for similar measurements of the different tapes trajectories in vivo in a dynamic setting , possibly allowing correlation of these findings to nuances in clinical outcomes . ultrasound will not be able to provide the same holistic visualization within the bony pelvis , but mesh - tape beyond the obturator membrane probably does not contribute to the mus functional effect or possible adverse events at the level of the vagina . recently , a new approach , incorporating iron particles into polymer - based implants , allowed in vivo visualization of mesh by magnetic resonance imaging ( mri ) following inguinal hernia surgery ( hansen et al . , 2013 ) . approval of these types of products for human use in the future will bring new insights into the action mechanism of mesh in the pelvic floor as well as a better understanding of the mesh- tissue interaction .

the three - dimensional configuration of mid - urethral sling tapes is difficult to demonstrate in traditional anatomical dissections or imaging studies . the aim of this study was to test the utility of a novel technique using mesh tapes to assess spatial differences between the in - out and out - in transobturator mid - urethral slings . two independent surgeons performed their usual transobturator mid - urethral sling placement on 10 fresh thawed cadavers , alternating sides in the consecutive cadavers . tantalum wires threaded through the polypropylene - tapes rendered them radio - opaque . following placement , ct scans were obtained to generate 3-d and mips images for analysis . results showed that the mean angle formed by the in - out sling measured 122 ( 95%ci : 107-136 ) ; versus 144 ( 95%ci : 131-151 ) for the out - in sling ( p = 0.02 ) . the paired differences between the tapes inner angles were significantly different ; with a mean difference of 20 ( median 19.0 ) , ( p = 0.008 ) . there was no significant correlation between either approach and bmi or angle of the pubic arch . the images revealed that the tapes lie as a band posterior / dorsal to the urethra rather than inferior . in conclusion : marking mesh with tantalum wire , in combination with 3-d and mips ct - scan reconstruction images , provided a unique method to visualize the entire sling trajectory . the clinical implications of the more horizontal positioning after the out - in approach remain to be determined .

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