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+{"text": "E. coli and B. subtilis both regulate the same task, namely, excitation and adaptation to environmental signals. Both pathways employ many orthologous genes. Yet how these orthologs contribute to network function in each organism is different. To investigate this problem, we propose what is to our knowledge the first computational model for B. subtilis chemotaxis and compare it to previously published models for chemotaxis in E. coli. The models reveal that the core control strategy for signal processing is the same in both organisms, though in B. subtilis there are two additional feedback loops that provide an additional layer of regulation and robustness. Furthermore, the network structures are different despite the similarity of the proteins in each organism. These results demonstrate the limitations of pathway inferences based solely on homology and suggest that the control strategy is an evolutionarily conserved property.Comparable processes in different species often involve homologous genes. One question is whether the network structure, in particular the feedback control structure, is also conserved. The bacterial chemotaxis pathways in  E. coli and B. subtilis, differences that are hard to predict on the basis of sequence homology aloneComputational modeling reveals some important differences in the networks that regulate chemotaxis in  Escherichia coli and Bacillus subtilis, swimming alternates between smooth runs and reorientating tumbles. Smooth runs require that the flagellar motors spin counterclockwise, whereas tumbles result from clockwise spins. Bacteria follow a random walk that is biased in the presence of gradients of attractants and repellents by alternating the frequency of runs and tumble. Owing to their small size, most bacteria are unable to sense chemical gradients across the length of their body. Rather, they respond only to temporal changes. In particular, their stimulated response always returns to prestimulus levels despite the sustained presence of attractants or repellents. Sensory adaptation involves a rudimentary form of memory that allows bacteria to compare their current and past environments. Bacteria regulate chemotaxis using a network of interacting proteins. The basic mechanism in flagellated bacteria involves receptor-mediated phosphorylation of a cytoplasmic protein (CheY) that binds to the flagellar motor and changes the spin direction , E. coli does not adapt  indicate that CheY interacts with the receptors. This model provides one possible feedback mechanism for methylation-independent chemotaxis. The other possibility is CheV. While either CheY or CheV is sufficient for methylation-independent chemotaxis, the model predicts that both feedback loops are necessary to generate the oscillations that are observed in the cheBCDR strains . However in the mutant, there are no complementary changes at residue E637, as it cannot be methylated.When thylated . Cast in removed . These r of CheY  or when  of CheY , we propB. subtilis. While in E. coli CheB phopshorylation is not necessary for adaptation . Likewise, when the majority of CheY is unphosphorylated, CheA is not repressed and residue E630 is preferentially methylated (activating residue). This feedback loop provides a regulatory mechanism for adaptation otherwise absent in aptation , it formaptation . This feaptation . HowevercheC and cheD, chemotaxis genes present in B. subtilis and missing in E. coli, are not treated explicitly in the model. Mutations to either gene are modeled implicitly by perturbing the kinetic parameters governing CheA activation and selective methylation. CheC is homologous to the P2 domain of CheA and the N-terminal domain of FliM .receptor . Adaptatnhibited . The cueB. subtilis. On the one hand, we expect that the rates and concentrations are comparable to their E. coli counterparts, given that many B. subtilis chemotaxis proteins complement in E. coli. On the other hand, the additional feedback loops involving CheV and CheY could mask differences in the rates and concentrations between the two species. Unlike E. coli, many properties of the B. subtilis model, such as the steady-state bias and adaptation time, are insensitive to the kinetic parameters, suggesting that perhaps chemotaxis is more robust in B. subtilis than in E. coli. For lack of a better alternative, we used E. coli parameters for the B. subtilis model when available, as they produce results in the B. subtilis model consistent with experimental measurements.Few kinetic measurements have been made for B. subtilis model were inferred from mutants and lack explicit experimental confirmation. There are a number of experiments that could test the predictions made by the model, and we describe just a few. One experiment is to correlate receptor methylation with CheA activity in vitro using purified components .Many regulatory interactions proposed in mponents . This inmponents . The in E. coli  binds only inactive receptors (IT) and that phosphorylated CheB (PB) binds active receptors (AT). In a simplified version of the model  and that CheB demethylates active receptors (AT) at residue 630 and inactive receptors (IT) at residue 637. If we simplify the model, the concentrations of receptors with residues methylated at 630 (m630)and 637 (m637) are described by the following two differential equations:The YK is the Michaelis constant for phosphorylated CheY and the receptor. Subtracting m = m630 \u2013 m637. We assume the concentration PY is proportional to the concentration of active receptors. The relative rate of methyation at residue 630 in E. coli model, E. coli forms memory using the absolute level of receptor methylation m, and B. subtilis forms memory using the differential level of receptor methylation \u0394m. The structure of Equations The difference between the two species is how receptor methylation forms memory. m and \u0394m\u2014are equivalent.Both mechanisms are robust; adaptation does not depend on the values of the kinetic parameters. Robust adaptation requires feedback with integral memory . The samE. coli chemotaxis; changes in the relative level of CheR expression did not alter the ability of E. coli to adapt to attractants  and, as a result, are subject to different selective pressures, so it is difficult to explain their differences without further investigating the role of their environment. There is also the issue of sensitivity; E. coli is able to sense gradients in concentrations spanning five orders of magnitude. As formulated, both models fail to capture this observed behavior. Other mechanisms, such as receptor clustering . The results are similar when either gene is deleted. A subpopulation (60%) of B. subtilis cheBCDR cells oscillates when stimulated with chemoattractants . The remaining cells partially adapt to the addition of attractants . Matlab m-files are available from T exist in either an active (AT) or inactive (IT) state. Let iT denote the concentration of receptor complexes with i residues methylated and \u03b1i(L) denote the probability that the receptor complex iT is active when the concentration of chemoattractant is L. The concentration of active receptors isThe chemotaxis model combines the two-state model proposed for adaptation by i(L) are given by the expression0 = 0; \u03b11 = 0.1; \u03b12 = 0.5; \u03b13 = 0.75; \u03b14 = 1; \u03b1L0 = 0; \u03b1L1 = 0; \u03b1L2 = 0.1; \u03b1L3 = 0.5; \u03b1L4 = 1; LK = 10 \u03bcM iT and the rate of methylation is Br(1 \u2013 \u03b1i(L))iT, whereWe modeled receptor methylation using the mechanism proposed by i(L))iT and the rate of demethylation is proportional to the concentration of active receptors \u03b1i(L)iT. A simple mass balance yields the following set of differential equations for the receptors:rk = 0.255 s\u20131; RK = 0.251 nM; Bk = 0.5 s\u20131; BK = 5.5 nM; A + pA = 5 nM; B + pB = 2 nM; Y + pY + [pMY] = 17.9 nM; M + [pMY] = 5.8 nM; and T0 + T1 + T2 + T3 + T4 = 5 nM  active receptors is given by the expressionThe te model . Let Tijiji is the probability that the receptor complex ijT adopts an inactive conformation. The concentration of weakly active receptors is given by the expressionL) is the probability that a weakly active receptor adopts an active conformation. The concentration of weakly inactive receptors is given by the expressionE. coli model, there are two boundary conditions: fully methylated receptors and unmethylated receptors. Furthermore, methylated receptors are active (\u03b1 = 1) and unmethylated receptors are inactive (\u03b1 = 0). In the B. subtilis model, there are four boundary conditions: T20, T02, T11, and T00. Furthermore, the methylated receptors T11 and unmethylated receptors T00 are partially active. We needed, therefore, to distinguish additional states to construct a robust model involving activity-dependent methylation.E. coli model, we assume that the kinetics for ligand binding are fast and employ the quasi-steady-state assumption for simplicity. The probabilities \u03b1ij(L) and iji(L) are given by the expressionsIn a similar manner to the 20 = 1; \u03b110 = 0.4, \u03b111 = 0.2; \u03b100 = \u03b101 = \u03b102 = 0; \u03b1020 = 1; \u03b1010 = 0.99, \u03b1011 = 0.8; \u03b1000 = \u03b1001 = \u03b1002 = 0; i02 = 1; i01 = 0.99, i11 = 0.8; i00 = i10 = i20 = 0; i002 = 1; i001 = 0.4, i011 = 0.2; i000 = i010 = i020 = 0; \u03b2 = 0.2; \u03b20 = 0.8; LK = 10 \u03bcM. The parameters were inferred from tethering experiments, where the attractant asparagine is added and then removed in a flowcell containing wild-type cells and the rotation of the flagellar motor is observed (observed pY) binds receptors. We model receptor binding with the following two differential equations:The model assumes that CheY negatively regulates CheA activity. The model assumes that only phosphorylated CheY  and Ik = 0.5 [pTY](2 + 10IT + 0.1WIT). The term C denotes the concentration of inactive receptor complexes. Evident from the expressions for A kand Ik, weakly active and inactive receptors contribute less to the state of the receptor complex.B. subtilis is an extension of the model proposed for E. coli. The key differences are the addition of CheV and the loss of CheZ. We used a Michaelis\u2013Menten-type expression to model inhibition of the CheA kinase by unphosphorylated CheV (V). There is no dedicated phosphatase for CheY in B. subtilis. However, the motor switch appears to enhance the CheY dephosphorylation when phosphorylated CheY is bound to the motor ijT and the rate of demethylation for residue 637 is ijB ir(L)ijT, whereFor simplicity, we used Michaelis\u2013Menten kinetics to model the methylation reactions. Similar results were obtained using mass action kinetics. For the receptor 1ij R Trand the rate for residue 637 is 2ijR Tr, wherepY and vice versa. A simple mass balance yields the following differential equation for the receptors:E. coli model: rk = 0.255 s\u20131; RK = 0.251 nM; Bk = 0.5 s\u20131; BK = 5.5 nM; A + pA = 5 nM; B + pB = 2 nM; Y + pY + [pMY] + [pTY] = 17.9 nM; M + [pMY] = 5.8 nM; T20 + T10 + T00 + T01 + T02 + T11 = 5 nM; [T] + [pTY] = 5 nM. The model assumes that the concentration of CheV is 8 nM: V + pV = 8 nM.cheBCDR strain described in ACTo model oscillations for the Ak = 0.001T (1 + 0.1WAT) and Ik = 0.001[pTY] (2 + 0.1WIT) with the initial condition T00 = 5 nM. The concentrations of CheB and CheR were set to 0 to account for their deletion. The subpopulation that partially adapts was modeled by setting the concentration of CheV = 4 nM. In this formulation, receptors adopt either a weakly active or weakly inactive conformation. We also induced a timescale separation necessary for a relaxation oscillator by decreasing the transition rate between active and inactive receptor complexes by a factor of 500. This change produced oscillations with a period of 100 s.AC:To model precise adaptation with simple negative feedback by CheY as described in Ak = 0.1[T]WAT and Ik = 0.1[pTY]WIT with the initial condition T00 = 5 nM. The concentrations of CheB and CheR were set to 0. We also needed to change the model for receptor binding:Ak = 0.01/(10 + L) + 0.036L/(10 + L).http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the genes and gene products discussed in this paper are E. coli CheA (AAC74958) and B. subtilis CheA (CAB13516), E. coli CheB (AAC74953) and B. subtilis CheB (CAB13506), B. subtilis CheC (CAB13518), B. subtilis CheD (CAB13519), E. coli CheR (AAC74954) and B. subtilis CheR (CAB14188), B. subtilis CheV (CAB13274), E. coli CheW (AAC74957) and B. subtilis CheW (CAB13517), E. coli CheY (AAC74952) and B. subtilis CheY (CAB13506), E. coli CheZ (AAC74951), and B. subtilis FliY (CAB13505).The GenBank ("}
+{"text": "Possible methods for distinguishing receptor binding models and analysing their parameters are considered.The conjugate gradients method is shown to be optimal for solving problems of the kind considered. Convergence with experimental data is rapidly achieved with the appropriate model but not with alternative models.Lack of convergence using the conjugate gradients method can be taken to indicate inconsistency between the receptor binding model and the experimental data. Thus, the conjugate gradients method can be used to distinguish among receptor binding models. Most medicinal preparations and biologically active substances do not penetrate into cells and must therefore exert their influence on intracellular processes by interaction with specific protein molecules at the cell surface -3, for wNon-cooperative interaction between ligand and receptor:R is the receptor molecule, L is the ligand molecule, RL is the ligand-receptor complex, and k+1 and k-1 are respectively the kinetic constants of formation and dissociation of the complex.where Cooperative interaction between ligand and receptorInteraction of one ligand with N types of binding sitesRL] are determined. The investigator has to solve two basic interrelated problems  , and = [R0] - [RL], [L] = [L0] - [RL].But [So equation (4) can be rewritten:Rikkatty equations. It can be solved analytically with the help of a special substitution [R] = [R0] - [RL], [L] = [L0] - [RL] do not generate analytically soluble equations. Therefore, all equations of this form were solved numerically using the Runge-Kutta method u. Random error assuming the normal distribution law was superimposed on the magnitude of [RL]u, and was calculated at 5, 10, 20 or 100 points.Numerical solution of equations (5\u20137) was carried out to determine [RT]m was calculated using parameters other than [RL]u from models (1\u20133). These parameters were applied to the determination of [RL]u by the following functional minimization:The magnitude [RL]u - [RL]m)2. \u00a0\u00a0\u00a0 (8)\u03a6 = , Newton's method and its variants (the conjugate gradients method and coordinate descent method in various modifications) were used -17. The R0] and Kd cannot be <10-15 M or >10-5 M. Hence the iteration procedure could be improved by re-scaling these parameters logarithmically, making 10-15 M equivalent to -1 on the new scale and 10-5 M equivalent to 1.It is clear from the literature  that [R0R0] Kd can be seen. Therefore the magnification of the random error in evaluating the magnitude of [RL]u displaces the functional (8) global maximum from its true values. In a sufficiently large neighbourhood of the global maximum, the functional magnitude (8) is practically invariant. However, this modification becomes more essential for evaluating the ratio of the functional (8) to basis vector of values [RL]u. Therefore this ratio was used with the inhibiting criterion choice.The functional (8) contour plots are shown in fig. The Newton method converges only in the close neighbourhood of the global maximum. However, modifications of the Newton method using second derivatives allow convergence to the global maximum after 1\u20132 iterations  using the conjugate gradients method, whereas this method required >10 points for identifying the parameters in a more complicated model. The Newton methods required >7 and 12 points respectively, and the coordinate descent method required >10 and 18 points.RL]m using an incorrect binding model. In particular  behaviour was analysed with respect to the evaluation of [see fig. , the funThus, the modification of the functional (8) contour plot from the type in fig. It appears that when an incorrect choice of the receptor binding model has been made, the conjugate gradients method does not lead to convergence, whereas in some cases the Newton method converges to one of the local minima. Therefore, lack of convergence using the conjugate gradients method suggests an incorrect choice of receptor binding model.even when the application of graphical methods is difficult or impossible. As seen here, lack of convergence in the conjugate gradients method indicates that an incorrect choice of model has been made. It is also shown that for the defining the parameters of the correct model, 5\u201310 data points are sufficient.Possible methods have been explored for discriminating among models for receptor binding model and for defining the relevant parameters. The procedure devised allows one to determine the receptor binding model and its parameters,"}
+{"text": "Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP119), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP119 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP119, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP119 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP119 and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP119 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase. However, it is known that a fragment of MSP1 -linked precursor, which is directed to the parasite's surface . The latter comprises two epidermal growth factor (EGF)-like domains and is carried into the interior of the infected-RBC on the merozoite surface 19 has been detected on the surface of the early ring-stage parasite by both IFA 19 that are present in the culture medium at the time of invasion, can be internalized when bound to MSP119 on the parasite surface 19 after invasion has not been studied in any detail.MSP1 is synthesized by intracellular 19 by IFA in the food vacuole of late rings/trophozoites, suggesting that this organelle is able to receive molecules endocytosed from the parasite surface. Although there is no clear morphological evidence for the existence of a classical eukaryotic endosome-lysosome system in Plasmodium, the food vacuole may act as a lysosome-like compartment as it contains proteases , are internalized to form one or more food vacuoles Early after invasion, numerous small food vacuoles form within the ring stage parasite 19 using a combination of metabolic labeling, IFA and IEM techniques applied to highly synchronized parasite cultures, together with antibodies directed against well-defined regions of MSP1. We find that after endocytosis into small food vacuoles in the ring stage, MSP119 persists through the cycle as a coherent component of the food vacuole wall without further processing or addition of newly synthesised MSP1. This behavior indicates that the molecule does not simply follow a classical lysosome-like clearance pathway, but may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.In the present study we have explored in detail the post-invasion fate of MSP1P. falciparum lines 3D7 in vitro as described lbuMAX\u00ae I in RPMI 1640 medium. Parasites were synchronized using combinations of a magnet 19; rabbit polyclonal antibodies raised against either affinity purified MSP1 (Wellcome type) 1919 (i.e. not reacting with MSP119) 19 is position 1526. Rabbit antibodies raised against peptides of CRT P. falciparum antigens, for IEM.A number of antibodies against different regions of MSP1 were used: monoclonal antibody (mAb) 1E1 35S Met/Cys; GE Healthcare, Little Chalfont, UK) essentially as described previously Mature stage parasites (3D7) were metabolically labeled with ProMix  in PBS to identify the nuclei. The films were subsequently mounted in glycerol/PBS solution containing anti-quenching agent  and viewed under oil immersion. The parasites were viewed using a Zeiss Axioplan2 microscope equipped with a Plan-APOCHROMAT 100\u00d7/oil immersion lens and appropriate filters. Images were analysed and processed using Adobe Photoshop  and Microsoft PowerPoint software.Thin blood films were made on microscope slides at intervals from time 0 (when newly invaded erythrocytes had been synchronized and returned to culture), air-dried, frozen without fixation and stored desiccated at \u221220\u00b0C until required. The time-course slides were assayed as follows: samples were thawed and dried quickly, fixed in fresh 1% formaldehyde in PBS for 30 minutes, rinsed in phosphate buffered saline (PBS), permeabilized with 0.1% Triton X-100/PBS for 10 minutes, rinsed in PBS and blocked overnight at 4\u00b0C in 3% BSA/PBS. Cells were probed consecutively with primary antibodies in 3% BSA/PBS, and with Alexa Fluor 488 (green) or Alexa Fluor 594 (red)-conjugated affinity purified goat anti-mouse IgG or anti-rabbit IgG secondary antibody (Molecular Probes) as appropriate at 37\u00b0C for 30 minutes each, followed by three washes in PBS. Dual labeling experiments were performed on thin films and probed successively with different primary antibodies derived from different species (mouse and rabbit). This was followed by staining with appropriate secondary antibody (anti-IgG) conjugated to Alexa Fluor 488 or Alexa Fluor 594. Control incubations were carried out in the absence of primary or secondary antibodies in addition to a range of antibody controls described above. Under no condition was a false fluorescence signal from the hemozoin observed. Finally, the films were stained with 0.5 \u00b5g ml19 with antibody (i.e. prior to invasion) synchronous mature parasites were cultured and allowed to invade in the presence of mAb 1E1: mature schizonts were purified using a combination of gel flotation and Percoll purification. Fresh erythrocytes were added to give a parasitemia of 5% (at 17% hematocrit) and samples were split into two fractions; one-half was placed in normal medium; the other in medium containing 500 \u00b5g ml\u22121 mAb 1E1. Following further culture with gentle shaking for 1 hour, remaining schizonts were removed using a Percoll cushion followed by sorbitol treatment of the pellet. A sample was taken (t\u200a=\u200a0) and smeared for Giemsa staining and for IFA, then the remaining parasites were cultured and smears were prepared every 3 hours over a 48-hour time period. The overall window for invasion was 2 hours.To pre-label MSP1P. falciparum clones 3D7 and C10, and line IT04) were prepared for IEM as described in Schizonts  onwards indicates that the protein is associated with food vacuoles, and that the small food vacuoles had increased in size but decreased in number, suggesting their fusion. The subcellular location of mAb 1E1 bound to MSP119 was identical to that of an MSP119-specific polyclonal antibody that was applied to the parasites post-fixation   , panel B vacuole , panel A19 was detectable in fixed parasites in two independent experiments with rabbit polyclonal anti-MSP119 and mAb 1E1 antibodies at least until 36 and 30 hours post-invasion, respectively, after which time the presence of newly synthesized MSP1 on the parasite surface masked the labeling of internalized MSP119  was used, no labeling was found on any ring structure (data not shown).IEM with the polyclonal anti-MSP1membrane  and occamembrane . Howevermembrane , and, inmembrane , none ofMorphological EM of trophozoites and schizonts, in which a single vacuole is present in each parasite, showed that food vacuole size varied with post-invasion age. In trophozoites and early schizonts the vacuole was rounded or elliptical in section and larger than in rings  19. The persistence of this fragment may therefore also reflect an equal persistence of the associated lipid raft components in the food vacuole wall.The IEM results show that after endocytosis, MSP119 are clustered into one or more patches of high concentration on the vacuole wall, rather than being spread evenly around its perimeter. This suggests that MSP119 molecules and the membrane lipids with which they associate form a coherent unit integrated into the vacuole wall membrane, preserving a group identity separate from other membrane components of the wall. This behavior implies some form of lateral interaction between MSP119 molecules either directly with each other or indirectly through clustering of their linked membrane lipids. Interestingly, inspection of published IEM reports of CRT localization to the food vacuole wall 19 and CRT does not show a total colocalization, suggesting that they cover overlapping or different areas of the food vacuole wall.The IEM results additionally show that in the single vacuole of trophozoites and early schizonts, the molecules of MSP119-attached membrane is only a part. Such an arrangement may not be too surprising, considering what must be the highly dynamic nature of this boundary, which constantly receives new membrane from the cytostomes and probably by fusion of Golgi-derived vesicles carrying proteolytic enzymes These findings indicate that the membrane lining the food vacuole may be a mosaic of different molecular compositions, of which MSP119 increases until at the end it covers most or all of the inner surface of the food vacuole   see . As show19 the results reported here throw new light on the process of endocytosis into the food vacuole at different stages of the cycle. It is plain that food vacuoles formed in rings are in a number of respects different from those of later stages. The avid uptake of MSP119 from the ring surface contrasts strongly with the absence of uptake of full length MSP1 into the schizont food vacuole, as shown by the failure of N-terminal-directed antibodies to detect vacuolar MSP1. It is likely that both GPI-anchored forms of this molecule are associated with cholesterol-rich lipid microdomains 19 is carried into the vacuole by association with such specific lipids. In later stages when it would be wasteful for the parasite to allow vacuolar degradation of newly synthesized GPI-anchored surface proteins destined for the next brood of merozoites, some mechanism must exist at the mature cytostome for ensuring MSP1 is excluded from endocytosis. The molecular basis for this selective process is not at present apparent.Besides the potential functional importance of MSP119 in food vacuoles is merely an aspect of the food vacuole's ability to store undegradable biomolecules, already evident in hemozoin sequestration. However, as MSP119 is located at the inner surface of these organelles, it is likely that any function relates to that boundary. There is experimental evidence that some anti-MSP119 antibodies (though not the anti-MSP119 mAb 1E1 used here) carried into the red cell on the surface of the merozoite can interfere with intracellular development 19 along the food vacuole wall rather than in isolated clumps or vesicles within its lumen (as typical of degraded structures in secondary lysosomes of many eukaryotes: (see 19 is not merely discarded as a functionless end-product.It is possible that the presence of MSP119 suggests that it may serve as a protective protease-resistant protein coat on the inside of the food vacuole, guarding other membrane proteins against the powerful proteases within this organelle. As it covers only part of the wall, it is unlikely to be the only protective agent, and other molecules with similar resistance may also participate, including MSP8, which has a similar EGF domain structure. However, gene disruption experiments have shown that MSP8 is not essential for parasite progress The protease-resistant structure of MSP119 might be involved in nucleating hemozoin crystallization, a role posited for a number of parasite molecules including HRPII, which is either transported directly to the food vacuole or first trafficked to the red cell cytoplasm and then taken back into the food vacuole during feeding 19 has the capacity to nucleate or accelerate hemozoin crystallization.Another possibility is that MSP119 is important in mechanically stabilizing the vacuole membrane especially in late schizonts when the vacuole is packed with sharp-ended hemozoin crystals. Further experiments are needed to test these possibilities.A third possibility is that by virtue of its association with membrane lipids including those forming cholesterol-rich lipid microdomains, MSP1Whatever the true function, the findings reported here point to an important but as yet undefined role for merozoite proteins in the intracellular phase of the parasite's erythrocytic cycle, and they also emphasize our lack of understanding of the degradative pathways within the parasite. Both of these aspects of the parasite's biology deserve attention as potential targets for the development of antimalarial strategies."}
+{"text": "Laminaria digitata induces apoptosis in HT-29 colon cancer cells, as well as the involvement of the ErbB signaling pathway. Cell viability assay revealed that laminarin induced cell death in a dose-dependent manner. Cell cycle analysis revealed that laminarin increased the percentage of cells in the sub-G1 and G2-M phase. Western blot analysis demonstrated that laminarin inhibited the heregulin-stimulated phosphorylation of ErbB2. A decrease in cellular proliferation was also observed; this was found to be dependent on ErbB, which activates c-Jun N-terminal kinase. These findings demonstrate the important role of the epidermal growth factor receptor in colon cancer tumorigenesis, and suggest the potential of laminarin as a bio-functional food with anticancer effects on human colon cancer.Laminarin, found in marine brown algae, is used as a carbohydrate reserve for phytoplankton; however, it is also used in traditional Chinese medicine, and has been shown to have several biological activities, including anticancer activities. In this study, we examined the mechanisms through which laminarin from  Recently, it was discovered that seaweed is composed of certain bioactive compounds, which have antitumor effects. Seaweed is also used in various functional materials and agents. In particular, seaweed polysaccharides, which comprise some of its bioactive compounds, include cellulose and viscous polysaccharides. Seaweed is also comprised of approximately 30\u201360% soluble polysaccharides; these polysaccharides include alginate, laminarin and fucoidan. Of these, laminarin is composed of \u03b21\u20133 and \u03b21\u20136-glucan, and is a storage glucan for brown seaweed .In this study, we investigated whether laminarin has direct anticancer activities in addition to its ability to inhibit cancer cell proliferation. We demonstrate that laminarin induces apoptosis in highly proliferative cancer cells. In a previous study, we showed that activated insulin-like growth factor receptor (IGF-IR) inhibits apoptosis . We showIn this study, we demonstrate that laminarin inhibits the activation of the ErbB pathway. The four members of the ErbB subfamily share a similar structure but have different functions ,8. The o2 and 95% air at 37\u00b0C in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin . The medium was changed every 2\u20133 days.We used HT-29 colon cancer cells  to examine the effects of laminarin. The cells were maintained in a humidified environment comprised of 5% CO3VO4, 1 \u03bcg/ml leupeptin, 1 mM PMSF and 1% Triton X-100]. Subsequently, 50 \u03bcg of boiling sample buffer were added to the total cell lysate, and the samples were boiled for 10 min at 100\u00b0C. Proteins in the extracts were separated by 7.5\u201315% SDS-PAGE and transferred onto polyvinylidene fluoride membranes . The membranes were blocked for 1 h at room temperature in blocking buffer [1% bovine serum albumin (BSA) in TBS-T] then probed with primary antibodies  overnight at 4\u00b0C. The membranes were then washed twice for 15 min each in TBS-T and incubated with peroxidase-conjugated goat anti-mouse or -rabbit antibodies .To prepare a whole-cell extract, the cells were washed in PBS and suspended in extraction buffer . For immunoprecipitation, cell lysates (750 \u03bcg) were incubated at 10\u00b0C with anti-ErbB2 antibodies. After 12 h, protein A-Sepharose beads were added to the cell lysates. The beads were collected by centrifugation for 2 min at 10,000 \u00d7 g and washed 3 times with lysis buffer. The beads were then boiled with the immunocomplex in 1X sample buffer. The eluted proteins were analyzed by SDS-PAGE and western blot analysis.The cells were incubated in SFM for 24 h and stimulated with 100 ng/ml HRG. To prepare a whole-cell extract, cells were washed in PBS and suspended in extraction buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM NaAll variables were compared with an analysis of variance using SPSS software version 10.0 . All values are presented as the means \u00b1 SD. A P-value <0.05 was considered to indicate a statistically significant difference.c into the cytosol.The mitochondrial pathway is a critical apoptotic pathway that involves signaling by Bcl-2 family proteins. The mitochondrial pathway of apoptosis also involves changes in mitochondrial potential and the mitochondrial release of cytochrome c, we examined the cytosolic and mitochondrial levels of cytochrome c. As shown in c in the mitochondrial fraction decreased, whereas the levels in the cytosolic fraction increased and the expression of cytosolic apoptotic protease activating factor-1 (Apaf-1) also increased , ErbB2, ErbB3 and ErbB4. In addition, HRG is co-expressed with ErbB2 in colon cancer, and the autocrine activation of ErbB2 occurs through dimerization with ErbB3 . The extErbB receptor pathway-related proteins play important roles in normal cells; ErbB receptors regulate cell proliferation, metabolism and survival ,14. In tThe HT-29 cells were incubated in SFM with the addition of 5 mg/ml of laminarin for 24 h; the cells were then stimulated with 100 ng/ml of HRG for 0, 1, 5 and 60 min. The effect of laminarin on the HRG-induced association of ErbB2 and p-Akt was examined by immunoprecipitation. As HRG binds with ErbB2, immunoprecipitation was carried out by the addition of ErbB2 antibodies to the cell lysates. The immunoprecipitated cell lysates were analyzed by western blot analysis. As shown in In this study, we demonstrate that laminarin induces apoptosis through an apoptotic pathway involving growth factors and also demonstrate the effects of laminarin on the ErbB signaling pathway in HT-29 colon cancer cells. These findings suggest the important role of EGFR in colon cancer tumorigenesis, as well as the potential value of laminarin as a bio-functional food with anticancer effects on human colon cancer."}
+{"text": "Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in  Chloroplast NADPH-dependent thioredoxin reductase (NTRC) is an NADPH-dependent thioredoxin reductase (NTR) isozyme specifically found in photosynthetic organisms Arabidopsis, and they have found that NTRC and 2-Cys Prx could enhance some environmental stress tolerances Chlorella vulgaris, by interacting with Chlorella 2-Cys Prx. To clarify their involvement and function in the acquisition of freezing tolerance of Chlorella, in the present study, we newly isolated a cDNA clone encoding 2-Cys Prx from Chlorella and examined peroxide reduction activity of Chlorella NTRC/2-Cys Prx antioxidant system by reconstitution experiment using both recombinant mCvNTRC and mCvPrx proteins. Furthermore, the corresponding genes were expressed in yeast to investigate the effect of the mCvNTRC/mCvPrx antioxidant system on several environmental stress tolerances.Several researchers have previously investigated the involvement of the NTRC/2-Cys Prx system in several stress conditions, such as cold temperature, methyl viologen, and heat, by using Escherichia coli BL21(DE3)pLysS/pET-29b(+)/mCvNTRC was used for expression of mature CvNTRC fused with a His-tag in its C-terminal end (His-mCvNTRC) E. coli cultures at 25\u00b0C for 6 h in the presence of 1 mM isopropyl-\u00df-D-thiogalactopyranoside (IPTG) as described previously E. coli JM109 strain was used to express a mature form of the Chlorella 2-Cys Prx gene (mCvPrx). Saccharomyces cerevisiae YPH500  was used for expression of mCvNTRC and mCvPrx genes.+ RNA was isolated from cells of Chlorella vulgaris hardened at 3\u00b0C for 24 h as described previously CvPrx gene was amplified by PCR with primers (5\u2032- TTC TTC TAC CCC CTG GAC TTC AC -3\u2032 and 5\u2032- GGC AGA GAA GTA CTC CTT GG -3\u2032). The primers were designed based on two conserved regions of amino acid sequences of 2-Cys Prx from photosynthetic eukaryotes (2-Cys Prx gene from Chlamydomonas reinhardtii (AJ304856). The amplified fragment was subcloned into a pT7-blue vector  and the plasmid construct was transformed into E. coli followed by sequencing. The full-length sequence of CvPrx was determined by the rapid amplification of cDNA ends (RACE) method using adaptor-specific primers and gene-specific primers based on the obtained cDNA fragment. The RACE fragments were directly sequenced. The resulting sequences of the partial fragment and the RACE fragments were assembled to determine the full-length cDNA sequence. Sequence alignment and phylogenic analysis were performed using the ClustalW program  and the TreeView program Poly(A)karyotes , and nucE. coli JM109. For the efficient expression of His-mCvPrx in E. coli, five rare codons for E. coli were modified as described in the E. coli cultures at 37\u00b0C for 3 h in the presence of 1 mM IPTG.A DNA region (from 161 to 745 bp) corresponding to mCvPrx protein was introduced into a pTrc99A expression vector to form a His-tagged protein (His-mCvPrx), and the construct was transformed into E. coli cells cultured as described above were collected by centrifugation at 8,000\u00d7 g for 5 min, and resuspended in 50 mM sodium-phosphate buffer  containing 1 mM phenylmethylsulfofluoride. The cells were disrupted at 4\u00b0C by sonication using a Tomy Ultrasonic Disrupter UP-201  for 20 min at 48 W with 0.5-s pulses at 0.5-s intervals. Then, the samples were centrifuged at 20,000\u00d7 g for 10 min at 4\u00b0C to recover soluble protein extract. His-mCvNTRC was purified using a HisTrap FF crude column  according to the manufacturer's instructions, and directly subjected to a PD-10 desalting column  to remove imidazole in the eluate. His-mCvPrx was purified using a HisTrap FF crude column and the eluate was treated with 400 \u00b5M H2O2 for 1 h on ice to oxidize thiol (\u2212SH) groups of the protein completely. The treated sample was then subjected to a PD-10 desalting column to remove imidazole and H2O2 in the eluate. Polyclonal anti-mCvPrx antibodies were raised in rabbits using purified His-mCvPrx as an antigen.The 2O2), tert-butyl hydroperoxide (t-BOOH), peroxynitrite (ONOO-), and decomposed ONOO\u2212 (PNdec) were determined by an enzymatic assay 340 was started immediately after the addition of peroxide substrates. The initial rate of NADPH oxidation was calculated from the slope between 0 and 5 s after the addition of the peroxide substrate and corrected for the background oxidation of NADPH observed in the coupled assay without protein.Assays were performed in double beam mode by using an UV-visible spectrophotometer DU800 . ROS-scavenging activities of a protein mixture of His-mCvNTRC and His-mCvPrx against hydrogen peroxide , diluted with 0.3 mM NaOH, and stored at \u221280\u00b0C until use. The precise concentration of peroxynitrite was determined from the absorbance at 302 nm according to the manufacturer's instruction . PNdec was prepared by incubating peroxynitrite diluted in 50 mM NaPi (pH 7.0) for 5 min at room temperature. Decomposition of ONOOmCvNTRC), pPrx (expressing mCvPrx), and pNTRC/Prx (expressing both mCvNTRC and mCvPrx). An empty vector was also transformed into YPH500 to construct a control strain, designated pESC.The DNA region encoding mCvNTRC was introduced downstream of a GAL1 promoter , and the region encoding mCvPrx was introduced downstream of a GAL10 promoter  in a pESC-TRP vector (Stratagene), subsequent to PCR amplification and restriction enzyme digestion of the fragments. The constructed plasmids were transformed into YPH500 by the lithium acetate method g for 10 min at 4\u00b0C, and the supernatant was recovered as a protein extract.Yeast cells were cultured in SR medium  that lacked tryptophan (SR-Trp) and that contained 2% galactose, which induces expression of genes introduced downstream of GAL1 and GAL10 promoters. The cultured cells were collected by centrifugation, and the pelleted cells were resuspended in 50 mM Na-Pi (pH 7.0). The cell suspension was homogenized with an equal volume of 0.5 mm diameter glass beads for 5 min with a vortex mixer. It was then centrifuged at 20,000\u00d7 et al.The protein extracts of yeast transformants were subjected to SDS-PAGE on a 12% polyacrylamide gel 600\u200a=\u200a0.4\u20130.6). The exponential culture was centrifuged, and the pelleted cells were washed twice with 0.9% NaCl and then were resuspended in 0.9% NaCl at the concentration of 2\u20133\u00d7106 cells/mL (OD600\u200a=\u200a0.1). Freezing treatment was performed as follows. An aliquot of the suspension (100 \u00b5L) was transferred to a microcentrifuge tube, placed in a deep-freezer at \u221280\u00b0C for 2 min for ice formation, and then stored in a freezer at \u221220\u00b0C for 24 h. Heat stress treatment was performed as follows. An aliquot of the suspension (1 mL) was transferred to a test tube and incubated at 48\u00b0C in a water bath for 1 h with shaking. Oxidative stress treatment was performed as follows. Menadione  was added at a final concentration of 50 \u00b5M to 1 mL of the suspension in 100 mM Na-Pi . Then, the mixture was transferred to a test tube and incubated at 30\u00b0C for 3 h with shaking. After treatment, cells were appropriately diluted with 0.9% NaCl (for freezing and heat stresses) or 100 mM Na-Pi (for oxidative stress) and plated onto YPD agar plates  followed by incubation at 30\u00b0C for 2 d. Viability (%) was calculated by dividing the counts of stressed cells on YPD agar plates by that of unstressed cells.Yeast cells were cultured in SR-Trp containing 2% galactose to exponential phase , which specifically detects superoxide anion. Dihydroethidium was dissolved in 100% dimethylsulfoxide (DMSO) and added to the cell suspension in 100 mM Na-Pi (pH 7.0) already containing 50 \u00b5M menadione at a final concentration of 2 \u00b5M. The final concentration of DMSO in the suspension was never higher than 0.1%. The suspension containing dihydroethidium was incubated at 30\u00b0C for 3 h with shaking. The treated cells were harvested and mounted onto a slide glass. Fluorescence in yeast cells was visualized using a confocal laser-scanning microscope ECLIPSE E600  with excitation wavelength at 510\u2013550 nm and emission wavelength >590 nm.t test. One-way analysis of variance followed by Tukey's test was used for multiple comparisons. A p<0.05 was considered significantly different.Statistical analysis was performed using GraphPad Prism 5 software for Mac OS X . Single groups were compared by unpaired two-tailed Chlorella 2-Cys Prx was amplified using two primers as shown in 2-Cys Prxs (data not shown). The full-length CvPrx cDNA determined by RACE was 868 bp in length and encoded 239 amino acids, which were deposited at DDBJ/EMBL/GenBank, under accession number AB682671. The deduced amino acid sequence of CvPrx showed homology to the deduced amino acid sequences of the chloroplast 2-Cys Prx genes from Arabidopsis, which are characterized by the two conserved cysteine residues , heat , and manadione-induced oxidative  stress conditions. As shown in Superoxide in yeast cells during menadione treatment was detected by fluorescent microscopy using dihydroethidium as a detection reagent for superoxide anion radicals. As shown in C. vulgaris C-27, a frost-hardy strain Chlorella. Neither 2-Cys Prx nor any type of Chlorella Prx has been isolated and investigated to our knowledge. Moreover, a cell-free extract of Chlorella did not exhibit reduction activity against Synechocystis Prx (sll0755) in the presence of NADPH Chlorella did not have an NADPH-dependent reduction enzyme for Prx. Contrary to the report, information published in the DOE Joint Genome Institute database (http://genome.jgi-psf.org/) suggested that NTRC and 2-Cys Prx are probably encoded in the Chlorella genomic DNA. To characterize the antioxidant system in Chlorella, we first isolated a full-length cDNA sequence of CvPrx, and determined mCvPrx region based on the N-terminal sequence of mCvPrx protein identified previously NTRC is involved in many cellular reactions, such as a cellular protection against stresses , starch synthesis, and photoperiodic development ArabidopsisChlorella cells, mCvPrx was identified as a partner protein of mCvNTRC using in vitro pull-down assay E. coli expression system. Enzyme assay indicated that His-mCvPrx showed cooperating antioxidant activity specifically observed in the presence of His-mCvNTRC , a fluorescent dye for specific detection of H2O2 and alkyl hydroperoxide (data not shown). When organisms are exposed to superoxide anion, many oxidation reactions occur inside the cells and the intracellular superoxide is converted to peroxynitrite through the oxidation reactions In physiological studies of plant NTRC, Serrato mCvNTRC against control , O. locimarinus-2: O. lucimarinus CCE 9901 predicted protein (ABP01316), V. carteri Prx1f: Volvox carteri f. nagariensis female Prx1 (ADI46867), V. carteri Prx1m: V. carteri f. nagariensis male Prx1 (ADI46952), C. reinhardtii: Chlamydomonas reinhardtii 2-Cys Prx (CAC19676), A. thaliana 2-Cys A: Arabidopsis thaliana 2-Cys Prx A (At3g11630), A. thaliana 2-Cys A: Arabidopsis thaliana 2-Cys Prx B (At5g06290). The asterisks indicate two conserved regions used for primer design for amplification of partial CvPrx cDNA fragment.(TIF)Click here for additional data file."}
+{"text": "Objective. This study aimed to examine the potential role of memory T follicular helper (Tfh) cells in patients with neuromyelitis optica/neuromyelitis optica spectrum disorders (NMO/NMOSD). Methods. The percentages of different subsets of circulating memory Tfh cells in 25 NMO/NMOSD patients before and after treatment as well as in 17 healthy controls were examined by flow cytometry. The levels of IL-21 and AQP4 Ab in plasma and CSF were measured by ELISA. Results. The percentages and numbers of circulating memory Tfh cells, ICOS+, CCR7\u2212, CCR7\u2212ICOS+, CCR7+, CCR7+ICOS+ memory Tfh cells, and the levels of IL-21 in plasma and CSF were significantly increased in NMO/NMOSD patients. The percentages of CCR7\u2212 and CCR7\u2212ICOS+ memory Tfh cells were positively correlated with ARR, plasma IL-21, and AQP4 Ab levels. The percentages of CCR7+ and CCR7+ICOS+ memory Tfh cells were positively correlated with CSF white blood cell counts, proteins, and IL-21 levels. Treatment with corticosteroids significantly reduced the numbers of CCR7\u2212ICOS+ and CCR7+ICOS+ memory Tfh cells as well as plasma IL-21 levels in patients with partial remission. Conclusions. Our findings indicate that circulating memory Tfh cells may participate in the relapse and development of NMO/NMOSD and may serve as a new therapeutic target. Memory function .Neuromyelitis optica (NMO) is an autoimmune disease characterized by severe optic neuritis and transverse myelitis . NMO spe+CXCR5+ T cells [ in vitro [T follicular helper (Tfh) cells play a key role in humoral immunity. These cells are defined by their expression of the transcription factor B cell lymphoma-6 (Bcl-6), the cell surface markers CXC-chemokine receptor 5 (CXCR5), inducible costimulator (ICOS), programmed death-1 (PD-1), and CD40 ligand (CD40L) as well as the secretion of interleukin-21 (IL-21) . Dysregu T cells . Most ciin vitro . Therefoin vitro , 20. To Hence, in our study, we investigated the percentages and numbers of different subsets of circulating memory Tfh cells in NMO/NMOSD patients before and after treatment. The levels of IL-21 and AQP4 Ab in plasma and cerebrospinal fluid (CSF) also were examined. Moreover, we explored the potential relationships among values of these measures and clinical outcomes to clarify the potential roles of different subsets of memory Tfh cells in the relapse of NMO/NMOSD.Written informed consent was obtained from all individual participants. The study was approved by the Medical Ethics Committee of the First Hospital of Jilin University, Changchun, China. Twenty-five patients with relapsed NMO/NMOSD were enrolled from the inpatient service of the Department of Neurology, the First Hospital of Jilin University , from July 2014 to June 2015. These patients fulfilled either the Wingerchuk criteria 2006 for NMO  or the dAfter enrollment in this study, all patients were treated with corticosteroids . The patients visited the outpatient office 4\u20138 weeks after treatment for the follow-up. A total of 12 patients returned, and their clinical characteristics are shown in We collected fasting venous blood samples from individual HCs and NMO/NMOSD patients before and 4\u20138 weeks after treatment. One part of each blood sample was centrifuged to prepare plasma samples. The remaining blood was used to prepare peripheral blood mononuclear cells (PBMCs) via density-gradient centrifugation using Lymphoprep . In addition, we collected CSF samples from 15 NMO/NMOSD patients and 8 NND patients when they underwent a lumbar puncture. CSF samples containing blood were excluded. The numbers of white blood cell (WBCs) and lymphocytes in peripheral blood, as well as CSF WBC counts, CSF protein levels, and CSF immunoglobulin G (IgG) levels, were routinely examined in the hospital.6/tube were stained in duplicate with allophycocyanin (APC)-H7-anti-CD3, BV510-anti-CD4, fluorescein isothiocyanate (FITC)-anti-CD45RA, phycoerythrin (PE)-Cy\u21227-anti-CCR7, peridinin-chlorophyll proteins (PerCP)-Cy\u21225.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or proper IgG isotype controls  at room temperature for 30 minutes. After being washed with phosphate-buffered saline (PBS), the cells were analyzed by flow cytometric analysis using a BD FACSAria\u2122 II . The data were analyzed with FlowJo software . We analyzed at least 50,000 events per sample and calculated the numbers of different subsets of circulating memory Tfh cells in individual samples according to the counts of lymphocytes per liter of blood multiplied by the percentage of different subsets of memory Tfh cells in lymphocytes.Human PBMCs at 10The serostatus of AQP4 Ab in all patients was measured through IIFT systems according to the manufacturer's instructions .The levels of plasma and CSF IL-21 were measured by ELISA kits according to the manufacturer's instructions . The detection limit for human IL-21 was 11.99\u2009pg/mL. The levels of plasma and CSF AQP4 Ab were measured by ELISA using a specific kit  in AQP4 Ab-seropositive patients. The sensitivity of this assay was 1.0\u2009ng/mL.U nonparametric tests, and differences between NMO/NMOSD patients before and after treatment were analyzed by Wilcoxon tests. The relationship between variables was evaluated by the Spearman rank correlation test. Statistical analyses were performed using SPSS 19.0 software , and statistical significance was determined according to a two-sided P value <0.05.Data are expressed as medians and ranges. Differences between HCs and NMO/NMOSD patients were analyzed by Mann-Whitney +CD4+CXCR5+CD45RA\u2212 T cells) in NMO/NMOSD patients before and after treatment as well as in HCs  and six patients showed nonremission (NR). To further elucidate the role of corticosteroids in memory Tfh cells and the levels of plasma IL-21, we analyzed the numbers of different subsets of circulating memory Tfh cells and the levels of plasma IL-21 before and after treatment in these 12 patients. We found that the numbers of CCR7+CXCR5+ T cells [+CXCR5+PD-1+ T cells is higher not only in NMOSD patients than in HCs, but also in patients with relapsing NMOSD than in NMOSD patients in remission [+CXCR5+PD-1+ T cell population decreases [+ memory Tfh cells were higher in NMO/NMOSD patients than in HCs. The difference may be because the sample populations that we studied were both small and heterogeneous. Further studies are needed to clarify the accurate role of PD-1+ and ICOS+ memory Tfh cells. Circulating memory Tfh cells can provide support for B cell antibody production [+ memory Tfh cells may be vital memory Tfh cells, associated with the relapse of NMO/NMOSD.Tfh cells are crucial for humoral immunity especially antibody production . Tfh cel T cells . A recenemission . After tecreases . These doduction  and be uoduction . ICOS isoduction . The absoduction . Hence, \u2212 memory (effector memory) T cells and CCR7+ memory  T cells. Upon secondary antigenic stimulation, CCR7\u2212 memory T cells can induce immediate protection in peripheral tissues, and CCR7+ memory T cells can migrate to secondary lymphoid organs where they have an effector function to antigens [\u2212, CCR7\u2212ICOS+ memory Tfh cells were significantly higher in NMO/NMOSD patients than in HCs. The percentages of CCR7\u2212 and CCR7\u2212ICOS+ memory Tfh cells were positively correlated with the ARR. In addition, among AQP4 Ab-seropositive patients, the percentages of CCR7\u2212 and CCR7\u2212ICOS+ memory Tfh cells were positively correlated with the levels of plasma AQP4 Ab. A recent study found that a high percentage of circulating CCR7\u2212PD-1+ Tfh cells exists in systemic lupus erythematosus (SLE) patients, which represented active Tfh differentiation in secondary lymphoid tissues and was related to clinical features of autoimmune disease, suggesting that this subset may participate in the pathogenic antibody response in autoimmune diseases [\u2212 and CCR7\u2212ICOS+ memory Tfh cells may participate in the production of plasma AQP4 Ab in peripheral tissues and be related to disease relapse. Moreover, we also found that the percentages and numbers of CCR7+ and CCR7+ICOS+ memory Tfh cells were significantly higher in NMO/NMOSD patients in our study. The percentages of CCR7+ and CCR7+ICOS+ memory Tfh cells were positively correlated with CSF WBC counts and CSF protein levels. It has been shown that circulating CCR7+ memory Tfh cells migrate to B cell follicles where they provided help to B cells, representing a distinct memory cell subset specialized in supporting the antibody-mediated immune response [+ and CCR7+ICOS+ memory Tfh cells may circulate into the CNS and contribute to the immune response there. Therefore, CCR7\u2212 and CCR7\u2212ICOS+ memory Tfh cells may participate in AQP4 Ab production in the periphery and be related to the relapse of NMO/NMOSD, whereas CCR7+ memory Tfh cells may contribute to immune responses in the CNS of NMO/NMOSD patients.Memory T cells consist of two subsets, CCR7antigens . In the diseases . Accordiresponse . CCR7 isresponse . CCR7 isresponse \u201334. CSF response , 36. Hen\u2212 and CCR7\u2212ICOS+ memory Tfh cells were positively correlated with the levels of plasma IL-21. We also found that the percentages of CCR7+ and CCR7+ICOS+ memory Tfh cells were positively correlated with the levels of CSF IL-21 in NMO/NMOSD patients. Hence, IL-21 may participate in the development and relapse of NMO/NMOSD.IL-21, which is the most important cytokine of Tfh cells, plays a critical role in the survival of Tfh cells and the survival, proliferation, and differentiation of GC B cells . Our res\u2212ICOS+ and CCR7+ICOS+ memory Tfh cells and the levels of plasma IL-21 significantly decreased in NMO/NMOSD patients with PR, but not in those with NR, suggesting that intravenous methylprednisolone therapy has a suppressive effect on memory Tfh cells. A recent study showed that corticosteroids promote Tfh cell apoptosis by regulating IL-21 and IL-6 levels in SLE patients [Corticosteroids are beneficial to NMO/NMOSD patients with PR based on improvement of the EDSS score. The effect of intravenous methylprednisolone on memory Tfh cells and plasma IL-21 is another interesting finding in our study. The numbers of CCR7patients . These d\u2212 and CCR7\u2212ICOS+ memory Tfh cells were positively correlated with the levels of plasma AQP4 Ab, whereas CCR7+ and CCR7+ICOS+ memory Tfh cells were not positively correlated with the levels of CSF AQP4 Ab. It is commonly believed that AQP4 Ab is more abundant in the peripheral blood of NMO patients than in their CSF [+ memory Tfh cells and CSF AQP4 Ab and to determine the precise origin of CSF AQP4 Ab.Surprisingly, we found no differences in the percentages and numbers of different subsets of memory Tfh cells between seropositive and seronegative patients. Other antibodies (such as myelin oligodendrocyte glycoprotein antibodies  and AQP1heir CSF . To date\u2212 and CCR7\u2212ICOS+ memory Tfh cells may serve as new biomarkers for evaluating disease relapse and may participate in the autoantibody production in the periphery. CCR7+ and CCR7+ICOS+ memory Tfh cells play an instrumental role in the autoimmune inflammation lesions in the CNS. Therefore, memory Tfh cells may provide a new insight into the pathogenesis of NMO/NMOSD as well as a new therapeutic target. There are limitations in this study, such as the small sample size and the lack of long-term follow-up and functional studies of different subsets of memory Tfh cells. Nevertheless, further investigation of the precise role played by memory Tfh cells in NMO/NMOSD pathogenesis and development is warranted.In summary, our findings provide clear clinical evidence of the relevance of different subsets of circulating memory Tfh cells in relapse and treatment response to corticosteroids among NMO/NMOSD patients. We speculate that circulating memory Tfh cells may participate in the pathogenic course and relapse of NMO/NMOSD. Furthermore, CCR7"}
+{"text": "Type III interferons (IFNs), also termed lambda IFNs (IFN\u03bbs) or interleukins-28/29, constitute a new addition to the IFN family. They are induced upon infection and are particularly abundant at barrier surfaces, such as the respiratory and gastrointestinal tracts. Although they signal through a unique heterodimeric receptor complex comprising IFNLR1 and IL10RB, they activate a downstream signaling pathway remarkably similar to that of type I IFNs and share many functions with them. Yet, they also have important differences which are only now starting to unfold. Here, we review the current literature implicating type III IFNs in the regulation of immunity and homeostasis in the respiratory tract. We survey the common and unique characteristics of type III IFNs in terms of expression patterns, cellular targets, and biological activities and discuss their emerging role in first line defenses against respiratory viral infections. We further explore their immune modulatory functions and their involvement in the regulation of inflammatory responses during chronic respiratory diseases, such as asthma and chronic obstructive pulmonary disease. Type III IFNs are, therefore, arising as front-line guardians of immune defenses in the respiratory tract, fine tuning inflammation, and as potential novel therapeutics for the treatment of diverse respiratory diseases, including influenza virus infection and asthma. Interferons (IFNs) have a long history. Type I IFNs were first discovered in 1957 as factors that \u201cinterfere\u201d with viral replication . Yet, itType III IFNs comprise four members in humans, IFN\u03bb1/IL-29, IFN\u03bb2/IL-28A, IFN\u03bb3/IL-28B, IFN\u03bb4, and two  in mice . By compAmong the various IFNs, type I IFNs have long been considered to constitute the primary antiviral and antibacterial defense mechanism in the body as they can be produced by almost any cell type upon infection and can signal to almost any cell type to confer protection . In contS. pneumonia, H. influenza, S. aureus, and M. tuberculosis, all of which trigger high levels of IFN\u03bbs. Multiple pattern recognition receptors (PPRs) are involved in this process including endosomal toll-like receptors (TLR), such as TLR3, TLR7/8, and TLR9, and cytosolic sensors, such as RIG-I and MDA-5, recognizing double-stranded or single-stranded RNA, unmenthylated DNA, and other microbial structures , alveolar and interstitial macrophages, and monocytes. Interestingly, although these cells broadly respond to PRR engagement, expression of IFN\u03bbs is selective to specific cell types, most prominently epithelial cells and DCs \u201322, sugg protein  in perox protein , may proin vitro and in vivo studies IFN\u03bb was shown to be as effective as type I IFNs in treating viral or bacterial infections  under control  or because of epigenetic changes that prevent optimal IFN\u03bb gene expression and translation , 69, 70.Deficient IFN production of the respiratory epithelium has also been observed in chronic obstructive pulmonary disease (COPD), another disease characterized by frequent virally induced exacerbations. Bronchial epithelial cells from COPD patients are not capable of mounting a full IFN response upon viral infection . This isOver the last years, major progress in our understanding of the unique functions of IFN\u03bbs, not shared with type I IFNs, has taken place. This has revealed the importance of IFN\u03bbs in front-line antiviral defenses in the body, especially the respiratory and gastrointestinal tracts, acting in synergy with type I IFNs to fine tune immunity for optimal protection and minimal host damage. This has also uncovered the significance of IFN\u03bbs in keeping inflammation under control and preventing exacerbations in asthma, supporting their potential use for the treatment of diverse respiratory diseases. Despite that, key gaps of knowledge exist. Thus, it remains largely unexplored whether IFN\u03bbs are also important in immunity against bacterial or fungal infections of the respiratory tract, or barrier surfaces in general and how these are positioned by comparison to type I IFNs. It also remains unclear whether IFN\u03bbs are important in adaptive immune responses against infections, such as antibody and cytotoxic T cell responses, including immunological memory, which are well known to be affected by type I IFNs. Moreover, it remains to be established whether IFN\u03bbs are important in other chronic respiratory disorders beyond asthma and COPD, and how they can affect the course of the disease process. Further studies toward these directions are, therefore, urgently needed before these highly promising therapeutic candidates can be effectively exploited in the clinic.EA, MS, IG, and OK have contributed to the writing of the manuscript. EA and OK have designed the graphs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Current Hazard Analysis Critical Control Points (HACCP) approaches mainly fit for food industry, while their application in primary food production is still rudimentary. The European food safety framework calls for science-based support to the primary producers\u2019 mandate for legal, scientific, and ethical responsibility in food supply. The multidisciplinary and interdisciplinary project ALERT pivots on the development of the technological invention (BEST platform) and application of its measurable (bio)markers\u2014as well as scientific advances in risk analysis\u2014at strategic points of the milk chain for time and cost-effective early identification of unwanted and/or unexpected events of both microbiological and toxicological nature. Health-oriented innovation is complex and subject to multiple variables. Through field activities in a dairy farm in central Italy, we explored individual components of the dairy farm system to overcome concrete challenges for the application of translational science in real life and (veterinary) public health. Based on an HACCP-like approach in animal production, the farm characterization focused on points of particular attention (POPAs) and critical control points to draw a farm management decision tree under the One Health view . The analysis was based on the integrated use of checklists  and laboratory analyses of well water, feed and silage, individual fecal samples, and bulk milk. The understanding of complex systems is a condition to accomplish true innovation through new technologies. BEST is a detection and monitoring system in support of production security, quality and safety: a grid of its (bio)markers can find direct application in critical points for early identification of potential hazards or anomalies. The HACCP-like self-monitoring in primary production is feasible, as well as the biomonitoring of live food producing animals as sentinel population for One Health. The European White Book for food safety points out the ethic, scientific, and legal responsibility of all food operators, including food primary producers, in guaranteeing the safety of their products. Food safety and traceability have to be ensured at every stage of the food chain, and the primary production is the first critical step . In factDairy farming is among the most complex, and potentially vulnerable, components of farm animal production: the maintenance of good qualitative standards of milk and dairy products still represents a challenge for farmers and manufacturers who, in their turn, ask the scientific community to furnish them with proper tools for hazard identification and risk management. The best strategy to ensure safety calls for implementing preventive approaches, such as good breeding and manufacturing practices or the application of procedures based on the Hazard Analysis and Critical Control Point (HACCP).HACCP was firstly used in food production in the 1970s, providing precise process control measures for each step of the entire food manufacturing process. The Codex Alimentarius Commission has recognized HACCP as an effective tool to improve safety standards; HACCP identifies priority hazards and allows establishing targeted control systems, thus putting focus mainly on preventive measures rather than on end-product testing . HACCP iThe application of HACCP-like systems to animal health and primary production still represent the best approach . The EurCurrently, the European Union recommends primary producers, such as dairy farmers, to apply a HACCP-like program to prevent milk-borne zoonoses; noticeably, the modern concept of zoonoses does include toxicological risks carried over in foods of animal origin . HoweverIn this paper, we define the framework for technology transfer. Indeed, true innovation needs translational activities to make inventions  be sustainably integrated in complex and dynamics real systems. Through field activities in a selected dairy farm in central Italy, we explored individual components of the dairy farm system to define both opportunities and challenges of the BEST technology transfer. The farm-specific scenario is then considered at a broader spatial scale, together with neighboring farms, in order to highlight possible significant aspects associated to managerial or environmental factors.The multidisciplinary and interdisciplinary One Health profile  of the ALERT project is further amplified by the involvements of technological innovation. Farm characterization and risk analysis are basic inputs to establish a targeted grid of probes of the BEST platform in order to monitor a farm-tailored panel of analytical parameters. Indeed, as all health-oriented innovation initiatives the ALERT framework is complex and subject to multiple variables .The dairy farm  object of the present study was selected as representative of a well-conducted, relatively large-sized dairy farm of Central Italy.The characterization of the farm both as an environment, an animal rearing facility and a segment of the food chain was carried out following the seven HACCP principles  during 1(1)Farm position and territorial analysis of the macro-area around the farm. The dataset comprises farm position and area, geo-climatic factors, possible pollution sources , presence of neighboring protected areas, presence of endangered species, land usage, main crops, agricultural techniques, previous mycotoxins alerts, hydro-geographic network, and presence of farms and/or factories within a 20-km buffer around the farm. In order to identify possible health risks from zootechnical activities within the buffer area, among the 40 small size (<300 heads) dairy farms and 1 larger farm (>500 heads) identified, three farms were selected based on structural homogeneity, productive capacity, and lower distance from the chosen farm. Milk quality analysis data of these three farms from 2010 to 2013 were collected  Laboratory Information System) and statistically analyzed .(2)General farming conditions. The dataset comprises animal identification, number of heads for each category, structures, conditions of animal barns , dry period management, biosecurity, and prevention tools.(3)Agricultural, fertilizing, and weeding practices, with particular attention to main crops, pesticides management including the risk of groundwater pollution.(4)Animal nutrition, with particular attention to feed quality, safety, and origin.(5)Animal health and welfare .(6)Milking techniques and milking parlor hygiene.The farm characterization made avail of the checklists elaborated by the Agricultural Agency of the Tuscany region and by National and European Authorities . Herbicides and pesticides treatments are carried out with specific products . Even though treatments are carried out respecting the relevant legal limits, there is the need to monitor the possible pollution of groundwater or crops by the parent molecules or their main by-products, considering also the possible accumulation and mixture effect.Fertilization is performed either with farm\u2019s manure and synthetic fertilizers, such as ammonium nitrate  are purchased outside. There are no different feeding groups for the different production levels; feedstuffs are administered twice a day as unifeed. The unifeed present in the manger is in good condition and particle size is homogeneous. Dry cows are fed only with hay herbage and mineral supplement. The mangers are clean and dry and feed residues are modest. The documents relating to purchased feed and the records of loading and unloading are properly managed and are analyzed once a year. The core and flour are guaranteed as genetically modified organism and aflatoxin-free by the manufacturer.Streptococcus uberis and E. coli  placental retention (8%), (ii) mastitis (5\u20136%) caused by  E. coli , (iii) lThe most used veterinary drugs in the farm are antimicrobials: lincomycin and spectinomycin, marbofloxacin, flunixin meglumine, and amoxicillin. Treated animals are identified on the mantle to ensure the isolation of milk at milking time. The farm is not authorized to hold stocks of drugs; veterinary prescriptions are properly recorded. Nutritional, health, and hygienic status has been assessed for all dry cows, about 10% of lactating cows and 10% of heifers.Cows are milked immediately after calving and from 1 week after calving milk is collected in a buklet (during the first week colostrums is collected separately) up to 305\u2009days. Cows are dried through drastic reduction of the feed  and use of intramammary antibiotics; milking is interrupted abruptly. The whole farm produces an average of 30\u201335\u2009L/head/day, for a total of 8.5\u20139.0\u2009tons/head/year. Cows are milked twice a day by two operators.\u00ae automatic milking machine adopted in the frame of the ALERT activities and integrated in the BEST platform  with electronic recognition of cows through the use of pedometers. The whole milking process lasts about 3\u2009h (mean time of attack-detachment for each cow is 7\u20138\u2009min). The operators do not wear gloves during milking and pre-milking teat dipping is not performed. There is no use of oxytocin, even in primiparous cows.The parlor consists of two herringbone lines, originally 5\u2009+\u20095, then extended to 7\u2009+\u20097, with AfimilkUdder is washed with drinking water  and disinfected with chlorhexidine and finally dried with disposable paper. The first streams of milk are usually discarded.Operators attach the milking clusters ensuring a well-balanced contact with teats. Milk is firstly collected in a small collector tank, filling and emptying every 20\u2009s, which conveys the milk into the main cooling tank.In order to remove/reduce the risk of cross-contamination with contagious mastitis pathogens, a post-milking teat dipping is performed using a filming iodophor disinfectant (IODO PVP FILM) as a barrier preventing bacteria from colonizing teat\u2019s surface and orifice.Disinfection of the milking machine is performed with an acid\u2013alkaline treatment after each milking. Collection time, temperature, and quantity of the milk are properly recorded.The flow diagrams of the production process were drawn. Based on the flow diagrams, critical steps and risk factors for risk management in the farm were identified based on risk assessment.Critical points associated with a potentially occurring hazard impacting on production were identified and classified as control points [critical control points (CCPs)] or points of particular attention (POPAs) Figures  and 2. IAccording to the design of the BEST platform, POPAs are critical points where anomalous trends are measurable and, through anomalous variations in relevant control charting, can drive early risk management procedures in HACCP-like plans .Decision tree is drawn, under the One Health view , with special attention to POPAs and CCPs that can be monitored with the BEST platform.Based on the model presented in Noordhuizen et al. , the farMetals, pesticides, and mycotoxins in feed and well water resulted below the respective legal thresholds or below the limits of quantification/detection, except for pirimiphos-methyl\u2014an organophosphorus pesticide found in one feed sample (0.2\u2009mg/kg). Water (both for drinking or cleaning) showed good microbiological standards , 19 Tab.Zoonotic agents were not detected from any fecal sample.In accordance with EC Regulation 853/04, 37 bulk milk samples were processed for total bacterial count , somatic cell count , fat (%), protein (%), lactose (%), aflatoxin M1 (\u03bcg/kg), and antimicrobial residues. Data show a good milk quality , 21 Tab.The One Health concept applied to toxicant-related zoonoses requires the analysis of risks in the web of interactions at the environment\u2013animal\u2013human interfaces .No environmental pollution sources were identified by the checklists. The farm is located in a not nitrate-prone area that is suited to agricultural activity, and near to protected natural areas . In the Overall, the study farm presented a good standard of farming , agriculBreeding techniques ensure good standards of welfare and animal health. Paratuberculosis is widely diffused in Italy; the farm prevalence can be considered quite low, thus highlighting the possible eradication by mean of the new regional prophylaxis program.Based on the HACCP-like approach and farm management decision tree, the analysis carried out in the sequential POPAs of the farm identified a limited set of farm-specific CCPs. In particular, we consider the following concepts.(1)E. coli). Well water is vulnerable to pollution by pesticides and their degradation products; even though the analyses did not reveal the presence of residues, monitoring is warranted.Well water should be periodically checked for pollution by synthetic fertilizers (ammonium nitrate and urea), as well as for bacterial contamination; indeed, the management of litter could lead to the risk of fecalization of the groundwater, as suggested by previous finding of \u201cenvironmental\u201d bacteria in fore-milk and water ((2)Bulk milk represents the end-stage product of dairy farms. Information gathered on bulk milk is obviously pivotal for food safety . Finally, milk may represent an indicator of the environmental quality, both of surrounding areas out of the farm  and inside the farm as determined by farming management systems . Overall, milk can be considered as a real \u201cOne Health\u201d biomarker as it can provide a cluster of data relevant to food safety, animal health, farming management and environmental quality , thus prDatabases of laboratory analysis provide interesting information for investigation and comparison in other farm systems.The BEST system of early (bio)markers of anomalies can be applied as monitoring system at well water (POPA) and bulk milk (CCPs). The grid of markers  and biomarkers  of the BEST platform (sensors and biosensors) is flexible, so as to host new probes depending on site-specific requirements \u201330. The The use of BEST at watering, milking parlor, and bulk milk is expected to facilitate daily monitoring of farm environment and management, milking efficacy and efficiency, process hygiene, and milk safety. Indeed, the user-friendly and self-instructed (by control charting) BEST system operating on-line and providing timely and continuous information can support the maintenance of production quality  as well Daily maintenance of a good farm management means time and cost-effective preparedness to unwanted and/or unexpected events of both microbiological and toxicological nature. Prevention strategies based on an HACCP-like self-monitoring systems empowering primary food producers  and provThe application of risk assessment using POPAs and CCPs for farm management is a valuable initiative to overcome challenges of translational science in (veterinary) public health. The understanding of complex systems is a condition to accomplish true innovation through new technologies. In the case of One Health technology, biomonitoring of sentinel animals like food producing animals is crucial. The framework discussed in this work demonstrates how the development of an HACCP-like self-monitoring system based on measurable markers in critical points of the primary production chain and in live animals is feasible. Scientific advances in risk analysis can be applied to prevent toxicant-related zoonoses in daily primary production of food, with simultaneous benefit  for the protection of human, animal, and environmental health.Farm owners and farmers have been formally enrolled in the Consortium of the project ALERT and thus they consented to the collection and use of data. According to EU Directive 2010/63 of the European Parliament and of The Council of 22 September 2010 on the protection of animal used for scientific purposes and the Italian law \u201cDecreto Legislativo 26/2016,\u201d the authors can assert that all the animals involved in the study were exclusively submitted to practices respecting animal welfare and undertaken for the purposes of recognized animal husbandry, in accordance with good veterinary practice. Thus, the study does not require any further specification regarding ethics approval by authors.All the authors substantially contributed to: (1) the conception of the work and the acquisition, analysis of interpretation of data; (2) drafting and revising critically the work for important intellectual content, (3) final approval of the version to be published; and (4) agreement on accountability in all aspects of the work, in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest."}
+{"text": "Bipolar disorder is a chronic illness that impairs functioning and affects the quality of life of patients. The onset of this illness usually occurs at an early age, and the risk of relapse remains high for decades. Thus, due to the great clinical relevance of identifying long-term predictors of functioning in bipolar disorder, Strauss and Carpenter developed a scale composed of items known to have prognostic value. To determine the clinical usefulness of the four-item Strauss\u2013Carpenter scale in bipolar disorder, a 1-year prospective follow-up study was carried out. The internal consistency, convergent and discriminant validity, and test\u2013retest reliability of the scale were assessed. We also compared the Strauss\u2013Carpenter scale with the reference scales Global Assessment Functioning (GAF), Clinical Global Impression for Bipolar Disorder, the Modified Version (CGI-BIP-M) and the Sheehan Disability Scale (Sheehan). Additionally, a cut-off point for remission was established.The total sample was composed of 98 patients with a diagnosis of bipolar disorder. The four-item version of the Strauss\u2013Carpenter scale showed to have appropriate psychometric properties, comparable to those of reference scales. The best cut-off point for remission was 14.The four-item version of the Strauss\u2013Carpenter scale has suitable validity and reliability for the assessment of functioning in patients with bipolar disorder. Bipolar disorder is a chronic illness that affects functioning and the quality of life of patients both, during manic and depressive episodes and during remission  of the Strauss\u2013Carpenter scale for patients with bipolar disorder. The reliability, internal consistency, convergent and predictive validity, and prognostic capacity of the scale were assessed. To such purpose, the level of correlation between the score obtained on the four-item scale and two reference variables of functioning was evaluated.A total of 98 patients formed the final sample of the study. All subjects were aged between 18 and 65\u00a0years and met the diagnostic criteria for bipolar disorder type I as assessed by the Structured Clinical Interview for The Diagnostic for the Statistical Manual of Mental Disorders, 4th edition (DSM-IV), Axis I Disorders (SCID-I) , as we considered they were continuous variables. The Spearman correlation was used to assess correlations with the CGI-BIP-M scale, as it has an ordinal measure. The predictive validity of the scale was assessed by calculating Spearman correlations between items of the Strauss\u2013Carpenter scale  and the reference scales at 1\u00a0year. In the case of the total score on the Strauss\u2013Carpenter scale, its relation with the GAF and Sheehan scales was assessed by Pearson correlations. Consistency between values and test\u2013retest reliability of the Strauss\u2013Carpenter was evaluated by comparing baseline and 1-year values by intra-class correlation coefficients (ICC).Finally, ROC curves were used to evaluate the discriminant capacity of the scale. The area under the curve (AUC) and cutoff point for remission were also determined.All statistical analyses were performed using the IMB SPSS statistical software package versions 23 and R 3.1.2 \u00a0years. Most subjects were single (85.4\u00a0%) and had primary education (41.2\u00a0%); seven (7.5\u00a0%) had attempted suicide. Regarding substance use, 27.7\u00a0% used alcohol, 48\u00a0% smoked cannabis and 28.9\u00a0% took other drugs   Table\u00a0.Table\u00a02CTest\u2013retest reliability was calculated using ICC. The total score on Strauss\u2013Carpenter was found to have a good intra-class correlation Table .Table\u00a03Rr\u00a0=\u00a0\u22120.57, respectively). The Hospitalization item (Item 1) and the Symptoms item (Item 4) showed to be significantly correlated with both CGI-BIP-M and Sheehan. Of note, the Hospitalization item was more strongly correlated with the CGI-BIP-M scale (rho\u00a0=\u00a0\u22120.37), whereas the Symptoms item was more significantly correlated with the Sheehan scale (rho\u00a0=\u00a0\u22120.48). Finally, a significant relationship was observed between the Work item (item 2) and GAF and Sheehan scale, but not with the CGI-BIP-M. Again, the strongest correlation was observed with the Sheehan scale (rho\u00a0=\u00a0\u22120.38)  was 0.784 (95\u00a0% CI 0.695\u20130.874), which indicates a good discriminant capacity, as it is close to 1, the maximum value   validated by Rosa et al.  is dividThe results obtained in this study showed that the four-item Strauss\u2013Carpenter scale for patients with bipolar disorder have adequate psychometric characteristics for this population, both in terms of reliability and validity. Therefore, this is an adequate instrument with discriminant and prognostic capacity.\u03b1\u00a0>\u00a00.70 . This confirms the stability and consistency of the first assessment of the test. The result for the Hospitalization item could be explained because one of the exclusion criteria was that patients were not hospitalized at recruitment, but they could have been hospitalized during follow-up. Thus, this item could have more variability in the test\u2013retest.r\u00a0=\u00a0\u22120.573). As expected, this correlation supports the hypothesis on the predictive value of the scale. This agrees with the results published by Ahuir et al. (p\u00a0<\u00a00.01) between Strauss and Carpenter and GAF, CGI and WHO-DAS.Regarding the predictive validity of the four-item scale, the items with the best prognostic value were those that assess social activity and symptoms (Items 3 and 4), which showed a high correlation with the reference scales. The total score on Strauss\u2013Carpenter was also significantly related to the three reference scales, showing a remarkably close relationship with the Sheehan scale  and specificity (the probability that the test correctly detects subjects with a good prognosis of functioning).This study has some limitations such as the small sample size and the short duration of follow-up (a year). Future studies should analyze the psychometric properties of the four-item Strauss\u2013Carpenter scale in larger epidemiological samples and with a longer follow-up. Another limitation is that there was no control group in this study. Future studies should include a control group to confirm the results of this study.The adaptation of the four-item Strauss\u2013Carpenter scale to patients with bipolar disorder has adequate psychometric properties and is an acceptable and very useful instrument, not only for it shortness, but also for its prognostic capacity. This could have great clinical relevance for the clinical and pharmacological management of patients. In addition, the use of this tool facilitates earl intervention to prevent the unfavorable evolution of patients with a worse prognosis."}
+{"text": "We report an open-space microfluidic chip with fluid walls, integrating functions of cell culture and online detection of secreted proteins controlled by the interfacial tension value. via rolling circle amplification (RCA) reaction. In comparison with conventional co-culture detecting platforms, this method features the prominent advantages of saving reagents and time, a simplified chip fabrication process, and avoiding additional assistance for online detection with the help of an interfacial tension valve. On such a multi-functional microfluidic chip, cells  could maintain regular growth and cell viability. VEGF could be detected with excellent specificity and good linearity in the range of 10\u2013250 pg mL\u20131. Meanwhile, VEGF secreted by malignant glioma cells was also detected online and obviously increased when cells were stimulated by deferoxamine (DFO) to mimic a hypoxic microenvironment. The designed biochip with fluid walls provides a new perspective for micro-total analysis and could be promisingly applied in future clinical diagnosis and drug analysis.Despite traditional poly-dimethyl siloxane (PDMS) microfluidic devices having great potential in various biological studies, they are limited by sophisticated fabrication processes and low utilization. An easily controlled microfluidic platform with high efficiency and low cost is desperately required. In this work, we present an open-space microfluidic chip with fluid walls, integrating cell culture and online semi-quantitative detection of vascular endothelial growth factor (VEGF)  Cells as the basic units of life have been widely studied and used to guide clinical medicine.via diverse detection methods.In recent years, microfluidics has developed into an important tool in the field of cell research owing to its unique advantages of portability, miniaturization and integration.Despite PDMS microfluidic chips having been developed extensively in biological analysis, many deficiencies have also emerged. The microfluidic devices mentioned above for cell metabolite analysis tend to require a multi-layer design for the microfluidic chips. Therefore, fabrication of the microfluidic chips typically requires precision equipment and a clean environment, as well as having high reagent costs with low utilization.in situ observation, as well as for semi-quantitative detection of VEGF via rolling circle amplification (RCA) reaction. The two functional parts could be controlled by switching the interfacial tension value between \u201cON\u201d and \u201cOFF\u201d. Based on this strategy, various cells could maintain regular growth. In addition, with the assistance of the RCA reaction for signal enlargement,via oxygen plasma, which would simplify the requirements for equipment and operating processes and reduce the reagent costs of chip fabrication. For the strategy to detect VEGF based on the RCA reaction, it could keep pace with the traditional methods in terms of detection limit and could be valid for clinical samples.In this work, we developed an open-space microfluidic chip with fluid walls, integrating dual functions of cell culture and online detection of the tumor biomarker VEGF. The newly designed microfluidic chip could perform as a platform for cell culture and via covering with an oil phase. One of the chambers was utilized for cell culture and in situ observation. The other one was modified with DNA aptamers in advance for protein capture and fluorescence imaging. The two chambers could be separated or re-connected via the control of the interfacial tension (y-axis direction), the oil occupied the area that the pipette had gone over and the connection was blocked. On the contrary, when the pipette with aqueous solution moved along the connecting channel (x-axis direction), the aqueous solution would re-occupy the hydrophilic area and the two chambers would be re-connected driven by interfacial tension. We applied this device not only for regular cell culture and standard protein sample detection, but also for simulating a hypoxic microenvironment for tumor cells, as well as comparing the dissimilarity of VEGF secretion. Deferoxamine (DFO), an iron chelator, could block the hydroxylation pathway of HIF-1\u03b1, blocking the degradation of HIF-1\u03b1, leading to a hypoxic microenvironment. The remaining HIF-1\u03b1 was then transferred to the cell nucleus, which triggered the transcription of target genes and then promoted the secreting of VEGF  for semi-quantitative analysis -N\u2032-ethyl carbodiimide hydrochloride/N-hydroxy succinimide). Such small molecules keep the height of the bottom surface to a nanometer level with almost no obvious changes  sensor was selected as a surface with high-density modification of carboxyl groups that could efficiently immobilize aminated DNA aptamers. First, AR2G sensors were soaked in the preliminary mixed solution of EDC/NHS to generate ester groups on the surface  test was conducted to study the binding between aptamers and VEGF. An amine-reactive 2nd generation , immunoglobulin G (IgG), insulin, human serum albumin (HSA) and epidermal growth factor receptor (EGFR). From the results in To validate the accuracy and feasibility of the method in our platform, VEGF standard samples of varying concentrations were detected. A large concentration gradient ranging from 10 to 1000 pg mLin situ to verify the stability of VEGF in the aqueous solution. After injection of VEGF solution into the chamber for 6 h, the VEGF concentration in aqueous solution remained stable according to quantitative analysis using a VEGF ELISA kit , an effective chelator for iron ions, could block the degradation pathway of HIF-1\u03b1 and then induce a hypoxic microenvironment in the tumor cells, promoting the secretion of VEGF. Different concentrations of DFO  and another control group with pure cell culture medium were used to represent various hypoxic conditions for cell culture for 12 h. After staining with live/dead reagent, all of the U87 cells retained their cell activity well with nearly 99% cell viability, regardless of the applied drug stimulation . In addiin situ on such a platform. Based on the sandwich immunoassay structure and RCA reaction, this system could detect VEGF over a large concentration range and could achieve a good linear relation in the low concentration range with a detection limit of 10 pg mL\u20131. Meanwhile, we applied our system in a simulated hypoxic cellular microenvironment and confirmed increased VEGF secretion from tumor cells in the hypoxic condition. This proposed biosensor could not only be applied in other biological systems to detect VEGF, but also opens up new horizons for micro-total analysis.We successfully constructed a dual functional microfluidic chip with fluid walls for cell culture and online semi-quantitative analysis of VEGF secretion relying on an interfacial tension valve. Cell morphology could be observed There are no conflicts to declare.Supplementary informationClick here for additional data file."}
+{"text": "MAPs) are a mechanism for managing risk while enabling access to potentially beneficial drugs. Patients and their caregivers have expressed support for these programmes and see patient input as critical to successful implementation. However, they have yet to be systematically involved in their design.Reimbursement decisions on orphan drugs carry significant uncertainty, and as the amount increases, so does the risk of making a wrong decision, where harms outweigh benefits. Consequently, patients often face limited access to orphan drugs. Managed access programmes ; governance ; and evidence collection . They recognized that health\u2010care resources are finite and considered disease or drug eligibility criteria for deciding when to use a MAP .Patients and caregivers co\u2010designed a checklist comprised of six aspects of an ideal A patient and caregiver\u2010designed checklist was created, which emphasized patient involvement and transparency. Further research is needed to examine the feasibility of this checklist and roles for other stakeholders. PAR requires the active involvement of researchers and participants in co\u2010constructing knowledge; promoting self\u2010 and critical awareness ; and building alliances for effective planning, implementation and dissemination of the research.3.1All patients and caregivers at two CORD Regional Forums were invited to participate in the workshops, which were part of the main Forum programme (ie no other sessions were scheduled at the same time). The Forums focused on strategies for sustainable access to therapies and explored personalized approaches to drug access. Presentations were made on assessing therapies for real\u2010world use, strategies for responsible use and different pathways for access, including MAPs. Prior to the Forums, participants had participated in two CORD conferences focused on improving access to therapies for rare diseases and efforts to accomplish this in other countries. They also included presentations on the challenges faced by decision\u2010makers in Canada and discussions around the feasibility of applying international experience to the Canadian context. CORD travel grants were provided to patients and families for the conferences and Forums, minimizing financial barriers to attendance.3.2Workshops built upon findings from research previously undertaken in collaboration with CORD   for further feedback. These teams include clinicians, regulators, provincial drug plan decision\u2010makers and industry representatives. Based on comments received, a final version of the checklist was prepared.4All patients and caregivers who attended the Regional Forum participated in the workshop. They represented a range of disease types  and differing levels of experience within their rare disease communities. Nine patients and three caregivers  participated in the first workshop, and five patients and three caregivers  participated in the second.Through the workshops, four global themes reflecting \u201cnotions\u201d were identified. A notion is an individual's impression of something known, experienced or imagined4.1The four notions are organized below in order of relevant stage in the life cycle of an orphan drug. Refer to Appendix A to see the thematic networks behind each notion Figures \u2010S5 and 4.1.1\u201c[have] a role and\u2026 a responsibility\u201d  within the orphan drug life cycle , and saw themselves as experts in the \u201clived\u201d experience. At the same time, they described challenges in this role, which related to the physician\u2013patient relationship. Patients and caregivers often face pushback from physicians who are \u201cnot open\u2026 [And] don't always listen\u201d  to information patients/caregivers have gathered through their own research and experiences.Patients and caregivers believed that all stakeholders \u201cabsolutely.\u201d They also felt that those who formally or informally contribute to decision making within the life cycle should share their knowledge and insights with the rest of the disease community.In general, patient organizations were viewed as trusted representatives of and important information conduits for the disease community. As such, they are well positioned to identify and inform patients about opportunities to be involved in the life cycle (eg to provide input into coverage decision making). When asked whether there is a role for patient organizations in educating and managing expectations of patients and caregivers around new therapies for which there may be limited clinical evidence, patients and caregivers responded \u201cdidn't read the literature\u201d  and struggled to effectively provide care due to unfamiliarity with the rare disease.With respect to the role of physicians, patients and caregivers felt that physicians should be responsible for ensuring their patients are aware of all treatment options, regardless of the cost. Several expressed frustrations with physicians who 4.1.2Well finally, at least we know I'm not the only one\u2026\u201d \u2013 P5, W2) and its impact on the discovery of effective therapies. They emphasized the importance of on\u2010going collection of natural history and clinical outcomes data. They recognized that while registries may play a role, they require significant resources to implement and maintain. \u201cMany drugs can't support that type of registry\u201d and there are \u201cthe physicians as well\u2026they don't have time to fill out the paper work\u201d  for on\u2010going data collection.Patients and caregivers reiterated the challenges involved in conducting research on rare diseases and orphan drugs, the most significant of which remains the poorly understood natural histories of rare diseases , which is a concern, as they represent an important means of obtaining early access to new therapies. Regardless of location, trials are limited by small patient sample sizes  and a lack of validated outcome measures.Patients and caregivers also identified challenges involved in conducting clinical trials on orphan drugs in Canada. In their view, trials are 4.1.3\u201cnot quite sure how it's going to be used and what the outcomes will be\u2026\u201d . However, they questioned why such processes do not routinely involve specialist physicians \u201cwho know something about the disease and the drug\u201d . They also acknowledged the high cost of orphan drugs as an added challenge for decision\u2010makers, as well as desperate demands from patients and families for access to treatments with \u201cno data to support [them] whatsoever\u2026\u201d . These demands are often exacerbated by \u201cinequality [in access] across Canada\u201d , which they blamed on the lack of a national health\u2010care system. They believed that provincial control of health\u2010care budgets has hindered the implementation of nation\u2010wide programmes, like the pan\u2010Canadian Pharmaceutical Alliance , which could directly affect access to orphan drugs.Patients and caregivers discussed challenges in Canadian coverage decision\u2010making processes that affect their ability to access orphan drugs. They appreciated that coverage decision making is complicated by the significant uncertainties that exist around the clinical benefit of orphan drugs when one is Why don't they have accountability? Why is there no transparency there?\u201d . Patients and caregivers felt that they were purposefully kept in the dark by those involved who \u201cdon't want [patients/caregivers] to know\u201d .There is also a lack of transparency in drug coverage decision making. One participant wondered: \u201cnot a friendly place for [pharmaceutical companies] to come to\u201d . As a result, there is a need for the government to introduce policy mechanisms for bringing new drugs into Canada that provide some security for companies \u201caround how long they have to recoup that money\u201d .Finally, patients and families discussed how Canada represents only a small share of the global drug market and is \u201c4.1.4unique\u201d and do \u201cnot [have] just one experience\u201d  \u201cYou can't do\u2026 one size fits all [with orphan drugs]\u201d . Further, rare diseases are often heterogeneous, with symptoms, severity and response to treatment varying across patients who share a diagnosis ). \u201cYou're dealing with all ages, you're dealing with different responses to treatment, different lifestyle\u2026 at least in our area I would feel very bad as a patient representative to be the only one saying what I think are the right outcomes\u201d .Patients and caregivers explained that no single patient can represent the views of the entire disease group because all patients are \u201c4.2Additionally, patients' and caregivers' described the components that a MAP should contain. Six aspects of an ideal MAP were identified. In considering how to operationalize these components, a checklist was developed, which organized the aspects into three categories relating to accountability, governance and evidence collection Table\u00a0. An anno4.2.1\u201cfor the right purpose\u201d \u2014it must be able to address research questions aimed at determining the right dose of the right drug for the right patient. Individualized treatment protocols, which involve \u201ctrying [the drug] on each individual patient and seeing if it's working for them or not\u201d , may be required.While patients and caregivers viewed MAPs as enabling earlier access to potentially effective therapies, they wanted to ensure that this option is used appropriately or \u201cdon't know when these [opportunities] happen.\u201d It was felt that transparent MAPs would improve patients' acceptance of treatment decisions by helping them \u201cunder[stand] the process a little more and [take] the sting out of why [they] can't get medication\u201d .Transparency in all aspects of the MAP was emphasized. This includes opportunities for patient and caregiver input; as many felt that other stakeholders do not effectively communicate, their requests for input and so the patients 4.2.2\u201ca stipulation that there's patient representation\u201d  from three patient members who: 1) meet a minimum level of experience within the health\u2010care system, 2) have a meaningful role on the committee and 3) are accountable back to the organization they represent to avoid bias and enhance knowledge translation. To this end, they saw a role for patient organizations in selecting patient representatives who \u201cunderstand all [their] needs\u2026 [to] go on [their] behalf\u201d .Patients and caregivers indicated that MAPs should be overseen by a MAP\u2010specific committee with \u201dsomebody in the medical field who understands [the specific disease]\u201d  and the patient community should select that physician. Finally, there was a widely held view that committee meetings should be \u201copen to anybody\u201d  so that all patients/caregivers have the opportunity to provide input into the programme. This is discussed in detail below.They also agreed that committees should include a physician who specializes in the specific rare disease\u2014individual patient and caregiver input in the development of a MAP was stressed ). Further, such input must be collected through a process that is quick, efficient and accessible . Several approaches to collecting feedback were discussed, including online surveys, written documents, videos or face\u2010to\u2010face interviews with the committee. While most agreed that online surveys are an effective and efficient way of gathering input from as many patients as possible, some felt there should still be an opportunity for an individual to present to a committee .The importance of providing opportunities for \u201ca parallel sort of thing where the [Canadian] physician enters [data] into\u2026 [a] US database\u201d . Also, there may be opportunities to learn from other countries that use MAPs and potentially \u201cadopt one of [their] systems\u201d .Given the nature of rare diseases, patients and caregivers proposed collaborating with other countries on certain MAPs, using the experiences of patients across multiple countries. They provided the example of patients who have participated in clinical trials based out of the United States and wondered if for MAPs there could be 4.2.3What do you think? What else can you tell us?\u201d . With respect to continuation criteria, where these could not be determined a priori , participants felt that decisions around continuation on therapy should be made through a conversation between patients and their physicians .Patients and caregivers believed they should have an opportunity to provide input on the outcome measures selected and used as continuation criteria to ensure they are meaningful. They felt that patients should be asked, \u201c\u201cact on the answer\u201d  provided through a MAP was stressed by patients and caregivers. Where the treatment proves ineffective based on previously agreed outcomes, participants indicated it should be discontinued, with decision\u2010makers enforcing follow\u2010through.\u201c..so you do have to set schemes up in such a way (1) in hope of getting an answer and (2) that you're going to act on the results in a reasonable kind of way.\u201d At the same time, the need to a documentation process in place\u201d  to support on\u2010going monitoring with an engaged physician and data collection should begin before treatment is started  to identify \u201cthe stages of that patient journey\u2026 [and] the progressions\u201d  of the disease. Once treatment has begun, registries should collect qualitative and quantitative data related to the impact of the drug.Patients and caregivers felt that MAPs must have \u201c4.2.4\u201clife\u2010threatening or chronically debilitating conditions\u201d  and those for which there are no other legitimate alternatives . Drugs that are innovative (ie offer a new mechanism of action) or high cost were also seen as priorities. When asked whether disease prevalence alone is a sufficient criterion to make a drug a priority for a MAP, patients and caregivers both responded \u201cno.\u201d While there was broad agreement from patients and caregivers on these criteria, some wondered \u201cwhy we\u2026are even thinking about excluding [drugs]\u201d , arguing that the use of MAPs for all drugs may make the health\u2010care system more efficient.Patients and caregivers recognized that health\u2010care resources are finite and that it is infeasible to have a MAP for every drug. As such, they also considered possible disease or drug eligibility criteria for deciding when to use a MAP. They included drugs that treat 4.3In addition to the 4 notions described above, 3 overarching \u201csentiments\u201d emerged. These captured why patients and caregivers felt MAPs were a reasonable solution for addressing the uncertainties that coverage decision\u2010makers face. While the workshops were not intended to gather information on why patients and caregivers supported the use of MAPs, these sentiments were embedded throughout their discussions around what MAPs should look like.\u201cMaybe they don't want us to know\u201d \u2013 P4, W1). Some did not trust their physicians to know which therapies were most appropriate. One patient described receiving a prescription that she later \u201cfound out\u2026 [they] should have never\u2026 had\u201d . Also, at times, they felt that their physician chose not to inform them of all their treatment options because of the costs. \u201cPhysicians [make] treatment decisions based on the cost of the drug\u201d , not on its potential effectiveness, assuming \u201c[the patient] can't afford it.\u201dFirst, trust in other stakeholders is often lacking. Patients and caregivers questioned the meaningfulness of many outcome measures used in decision\u2010making processes that they feel are opaque  so if they are offered access to a drug in a trial or a MAP \u201c[they'll] sign anything\u201d  to participate.The second \u201csentiment\u201d was desperation. Patients and families described feeling desperate to find a treatment for their disease, particularly when no alternatives exist. They become willing to try almost anything and accept risk thresholds that are much higher than those accepted by their health\u2010care providers. They I'm not giving up for anything. And if my son doesn't make it, I'll also be fighting for the other ones\u201d . This theme was apparent in both patients' and caregivers' enthusiasm around the workshop dedicated to the design of an ideal MAP and in their discussions about uncertainty and the difficulty it creates for decision\u2010makers. They recognized that these uncertainties are an issue, but felt there are ways they could be involved to help reduce them , such as identifying meaningful outcome measures  and contributing to decision\u2010making committees.The third \u201csentiment\u201d that emerged was hope. Patients and caregivers were steadfast in their belief that access to orphan drugs can be improved. One caregiver said: \u201c5The work described in this study contributes to the growing body of literature supporting the inclusion of patients in the assessment of new health technologies to inform reimbursement decisions. To date, much of the attention has been focused on the development of generic guidance documents for patient involvement in HTA, such as those developed by the European Patients Academy (EUPATI).The MAPs checklist is an example of a tool co\u2010designed by patients and caregivers to not only improve access to high\u2010cost drugs for rare diseases, but also generate the kind of evidence needed to inform appropriate reimbursement (ie right drug for the right patient at the right time). To our knowledge, no other such tool exists. However, some jurisdictions, such as England and Wales, have already implemented MAP\u2010like schemes [ie patient access schemes (PAS)]. While PAS proposals from pharmaceutical companies are not co\u2010designed by patients or caregivers, their input is sought during the review process managed by the National Institute for Health and Clinical Excellence (NICE).Additionally, a checklist for evaluating access with evidence development (AED) schemes, which serve a similar purpose to MAPs, has been published.While much of this work focussed on what an ideal MAP should look like, general discussions around the current context of orphan drug access, the challenges that patients and caregivers face and, ultimately, why MAPs was viewed as an appropriate solution also took place. The three sentiments identified from these discussions  have been documented in published literature. One study found that patients with lower levels of trust in their physician were more likely to want an autonomous role in treatment decision making.It was also recognized that MAPs will not address all of the issues that patients and families face with respect to managing rare diseases . Studies on the reasonableness of patients and their willingness to accept limits have been documented in other studies.5.1Both workshops were held at national events hosted by CORD, and it is possible that the individuals who chose to participate in these events were not representative of the rare disease population in Canada. However, CORD is comprised of over 80 different rare disease patient organizations and covers travel expenses for patients and caregivers to attend their events, reducing the likelihood of bias.6The MAP checklist co\u2010designed by patients and caregivers offers a tool for informing the development and evaluation of such policy options, which aim to improve access to drugs where there is a high degree of uncertainty in the available evidence. Future research is needed to examine the feasibility of this checklist and roles for other stakeholders.A. Young, D. Menon, J. Street, W. Al\u2010Hertani and T. Stafinski report no conflict of interests.\u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "Electronic health records (EHRs) can provide researchers with extraordinary opportunities for population-based research. The National Health Insurance system of Taiwan was established in 1995 and covers more than 99.6% of the Taiwanese population; this system\u2019s claims data are released as the National Health Insurance Research Database (NHIRD). All data from primary outpatient departments and inpatient hospital care settings after 2000 are included in this database. After a change and update in 2016, the NHIRD is maintained and regulated by the Data Science Centre of the Ministry of Health and Welfare of Taiwan. Datasets for approved research are released in three forms: sampling datasets comprising 2 million subjects, disease-specific databases, and full population datasets. These datasets are de-identified and contain basic demographic information, disease diagnoses, prescriptions, operations, and investigations. Data can be linked to government surveys or other research datasets. While only a small number of validation studies with small sample sizes have been undertaken, they have generally reported positive predictive values of over 70% for various diagnoses. Currently, patients cannot opt out of inclusion in the database, although this requirement is under review. In conclusion, the NHIRD is a large, powerful data source for biomedical research. The increasing availability, size, and detail of electronic health records (EHRs) offer unprecedented opportunities for research. The advantages of EHRs include increased statistical power, speed, wide breadth, relatively low cost, representative population coverage, completeness of follow-up, and the ability to assess interventions in routine clinical care . LinkingAmong national EHR databases all over the world, the National Health Insurance Research Database (NHIRD) of Taiwan is unique. This large database, which contains data from 23 million residents of Taiwan, was previously described by Chen et al. . HoweverTo increase the affordability and accessibility of health care, in 1995, the Taiwanese government initiated a single-payer health insurance system, known as National Health Insurance (NHI). NHI has a contract with most healthcare facilities in Taiwan, and it is mandatory for physicians to upload the claims data from each visit to the National Health Insurance Ministry. Notably, the primary care system in Taiwan is different from that of many other countries. Referrals from general practitioners are not required to receive specialist care; therefore, patients with non-emergency health concerns can either visit local private or public clinics or go directly to specialists at hospital outpatient departments . As a prIn 2000, the anonymous and encrypted sampling dataset from this national insurance system was first released for use in research, under the regulation and maintenance of the National Health Research Institutes of Taiwan. From 2000 to 2013, the National Health Research Institutes made available to researchers general sampling datasets with 1 million subjects, as well as disease-specific sampling datasets. In 2016, these insurance data were moved to the Data Science Centre of the Ministry of Health and Welfare of Taiwan, where data are regulated and managed by the government . The regThe NHIRD is a powerful for observing chronic diseases and assessing the effects of treatments. For instance, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are relatively prevalent in Taiwan. Previous studies using the NHIRD demonstrated that the use of statins, medicines for decreasing low-density lipoprotein in the blood, was associated with a decreased incidence of hepatocellular carcinoma (HCC) in HBV and HCV patients ,9. AnothThe basic structure of the NHIRD data is shown in The NHIRD data are released in three forms. The first form is a general dataset containing 2 million patients. Two million subjects are collected using stratified random sampling by age, sex, and the registry of regions from the full database population. They were sampled at three different time points: 2000, 2005, and 2010 . Each daThe second form of NHIRD data are disease-specific databases. These databases contain complete claims data of all patients with a certain health condition. For instance, all patients with a diabetes diagnosis from 2002 to 2015 are included in the diabetes database. As of 2018, there are 13 disease-specific databases available for research . These dThe third form of NHIRD data is the full population dataset, which has been available for research since 2016. The full population dataset covers the entire Taiwanese population from 2000 to 2016, which comprises approximately 23 million people. Researchers can apply for complete claims data, including inpatient and outpatient records, investigations, and treatment, which can be linked with hospital information, birth certificate applications, death records, the cancer registry dataset, and the major illness datasets. Furthermore, the full population data can also be linked with individual datasets, a feature that will be introduced later. These released datasets are a valuable source for epidemiological research.Since 2016, under the authorisation and regulation of the Ministry of Health and Welfare, NHIRD data can be more widely linked with other public surveys at the Data Science Centre. These datasets include governmental surveys, disease registries, health surveys, social reporting system data, and welfare registry data. Detailed descriptions of these databases are given in The NHIRD is a nationally representative cohort that contains detailed registry and claims data from all 23 million residents of Taiwan. This huge database provides researchers with powerful and generalisable real-world evidence for biomedical studies. For instance, a molecular epidemiological study has suggested that aristolochic acid (AA), an ingredient in Chinese herbal remedies, was correlated to HCC in Taiwan and other Asian countries . SimilarThere are some issues with the NHIRD. First, the NHIRD lacks comprehensive validation, although some validation studies of the clinical diagnoses in the NHIRD have been done. Some of these validation studies used national disease registries as the reference standard, which is more convincing. Other studies used hospital-based records to validate the diagnoses found in the NHIRD and have reported relatively high positive predictive values (over 70%) . HoweverResearchers can access NHIRD data after ethical and scientific review processes. Prior to applying, researchers must obtain approval from the institutional review board. Notably, the applicant must be Taiwanese or be affiliated with a Taiwanese research institute. Applicants should submit their research proposal to the Data Science Centre. Proposals should include specific methods and variables required for their analyses. The cost of accessing data depends on the number of variables requested and the time period that they require the data for analyses; for example, accessing one variable for 1 year would cost 200 new Taiwanese dollars. After receiving an application, the Ministry of Health and Welfare reviews the legitimacy of the proposal, which is later reviewed by a scientific committee consisting of three experts. If one of the committee members disagrees with the proposed use of the data, then the researchers must submit a revised proposal to a higher advisory committee for a second review.After receiving approval, researchers must go to the branches of the Data Science Centre to perform their data analyses. The analyses of NHIRD data are complicated, and there is no structural training course for using the NHIRD. Therefore, a mock dataset containing 100,000 subjects is provided by the Data Science Centre to help researchers in writing statistical analysis syntax. When researchers enter the Data Science Centre, they are allowed to use provided computers and software including SAS, Stata, R, and SPSS to conduct their data analyses .Ethical review board approval is mandatory when applying to use NHIRD data. There are 27 institutional review boards capable of issuing approvals, and all are supervised and regulated by the Ministry of Health and Welfare . To protThe NHIRD of Taiwan contains a large quantity of claims data and has the potential for multiple data linkages. Although more validation research is needed, and regulatory work to protect privacy is ongoing, this nationwide cohort is a valuable resource for medical research."}
+{"text": "Tsc1 and Depdc5. Unlike former models that induced limited mTORC1 upregulation, hepatic deletion of both Tsc1 and Depdc5 (DKO) produced strong, synergistic activation of the mTORC1 pathway and provoked pronounced and widespread hepatocyte damage, leading to externally visible liver failure phenotypes, such as jaundice and systemic growth defects. The transcriptome profile of DKO was different from single knockout mutants but similar to those of diseased human livers with severe hepatitis and mouse livers challenged with oxidative stress-inducing chemicals. In addition, DKO liver cells exhibited prominent molecular pathologies associated with excessive endoplasmic reticulum (ER) stress, oxidative stress, DNA damage and inflammation. Although DKO liver pathologies were ameliorated by mTORC1 inhibition, ER stress suppression unexpectedly aggravated them, suggesting that ER stress signaling is not the major conduit of how hyperactive mTORC1 produces liver damage. Interestingly, superoxide scavengers N-acetylcysteine (NAC) and Tempol, chemicals that reduce oxidative stress, were able to recover liver phenotypes, indicating that mTORC1 hyperactivation induced liver damage mainly through oxidative stress pathways. Our study provides a new model of unregulated mTORC1 activation through concomitant upregulation of growth factor and nutrient signaling axes and shows that mTORC1 hyperactivation alone can provoke oxidative tissue injury.mTORC1 is a protein kinase important for metabolism and is regulated by growth factor and nutrient signaling pathways, mediated by the Rheb and Rag GTPases, respectively. Here we provide the first animal model in which both pathways were upregulated through concurrent mutations in their GTPase-activating proteins,  Regulation of mTORC1 is believed to be mediated by two small G proteins, Rheb and Rag5. The tuberous sclerosis complex (TSC) and the GAP activities Towards Rags 1 complex (GATOR1) are GTPase-activating proteins (GAPs) that regulate Rheb and Rag, respectively5. TSC, consisting of the TSC1 and TSC2 proteins, mediates growth factor and energy signals to mTORC17, while GATOR1, consisting of DEPDC5, NPRL2 and NPRL3 proteins are essential for amino acid sensing9 and stress response10 of the mTORC1 pathway.Mammalian target of rapamycin complex 1 (mTORC1) is a protein kinase complex that promotes cellular anabolism in response to insulin/growth factor stimuli and nutrient abundance9. DEPDC5 is also implicated in various human pathologies including brain and liver diseases15. Genetic variations in the DEPDC5 locus were associated with hepatitis C virus (HCV)-induced hepatocellular carcinoma in a Japanese population13, HCV-induced fibrosis progression in a European population14, and hepatitis B virus (HBV)-related hepatocarcinogenesis in a Chinese population15. However, whether DEPDC5 regulates liver homeostasis and how it affects liver disease progression has not been investigated in an intact animal model.DEPDC5 is a component of GATOR1 that is critical for binding and inhibiting Rag3. mTORC1 activation is important for upregulating protein translation by phosphorylating two substrates: p70 ribosomal protein S6 kinase (S6K) and translation initiation factor 4E-binding protein 1 (4E-BP1)1. mTORC1 also upregulates lipid and nucleic acid synthesis while downregulating autophagic catabolism through inhibition of unc-51-like autophagy activating kinase (ULK1)4. Therefore, mTORC1 regulation is thought to be critical for maintaining metabolic homeostasis in the liver3. Indeed, disrupting mTORC1 through liver-specific deletion of Raptor, an essential subunit, induced spontaneous liver damage associated with inflammation and fibrosis16. This accelerated liver carcinogenesis upon administration of diethylnitrosamine (DEN), a chemical hepatocarcinogen16. Activating mTORC1 through hepatocyte-specific deletion of Tsc1 (Tsc1\u0394hep) also produced liver inflammation and carcinogenesis in aged mice, but these pathologies were not obvious in young mice18.mTORC1, the DEPDC5 and TSC1 target, is an important metabolic regulator in the liver10, DEPDC5 could be an important regulator of mTORC1 in hepatocytes. To understand the genetic role of DEPDC5 in the liver, we generated Depdc5\u0394hep mice, which have hepatocyte-specific deletion of the Depdc5 gene. Similar to Tsc1\u0394hep mice, Depdc5\u0394hep mice showed slight elevation in mTORC1 activity and exhibited mild inflammation and fibrosis in advanced age. However, when Depdc5\u0394hep mice were crossed to Tsc1\u0394hep mice, a much more striking phenotype was observed. Although individual deletions of Depdc5 or Tsc1 in the liver only slightly upregulated mTORC1 with no gross phenotypes, hepatocyte-specific Depdc5 and Tsc1 double knockout (DKO) mice had robust mTORC1 activation that induced prominent hepatocyte damage. Consequently, serious liver failure associated with jaundice, hepatomegaly, fur discoloration and growth suppression were observed by 8 weeks of age. Transcriptomic analyses with RNA-seq and subsequent protein analyses indicated that DKO livers suffer excessive ER stress and oxidative stress leading to metabolic dysregulation, DNA damage and inflammation. Among these outputs, oxidative damage was the most critical in producing DKO pathologies, while ER stress signaling protected hepatocytes by suppressing mTORC1 in a negative feedback mechanism.Given the importance of DEPDC5 in nutrient and stress-dependent mTORC1 regulationAlb-Cre/Depdc5F/F (Depdc5\u0394hep) mice lost hepatic Depdc5 expression and slightly upregulated the level of phosphorylated S6 (p-S6), a downstream marker of mTORC1 , which provokes hepatocellular death most prominently in zone 3, compared to littermate controls  mice were born at the expected Mendelian ratios, their growth was severely suppressed, and their fur was gray and patchy by two months old , which facilitates nascent protein folding in vivoion Fig. . This waios Fig.  and moreios Fig. .In light of these observations, we questioned if TUDCA actually relieved ER stress in DKO mice. Immunoblotting showed that despite aggravated phenotypes, TUDCA generally reduced hepatic ER stress Fig. , top. MaTsc1\u0394hep, Depdc5\u0394hep, and DKO mice through RNA sequencing 28. Genes upregulated in late-stage fibrosis during HCV infection  and Redds (Ddit4 and Ddit4l), which are stress-inducible negative feedback regulators of the mTORC1 pathway29, were also upregulated in DKO livers  staining which visualizes superoxide radicals37. DKO livers had pronounced elevation of DHE staining intensity  is important for reducing superoxides and suppressing their toxic effects38. Genes whose hepatic expression is induced by DQ treatment38 were highly enriched in clusters 1 and 2 . Endogenous superoxide dismutase , another antioxidant that scavenges superoxide radicals  did not cause gross pathologies in young mice, although it promoted age-associated liver inflammation and carcinogenesis in one-year-old mice18. A physiological role for Depdc5 in the liver was not formerly investigated until the current study. Here, we showed that Depdc5 deletion in mouse liver upregulated hepatic mTORC1 most prominently in zone 3, where oxygen and nutrients are relatively scarce. Since GATOR1 is important for suppressing mTORC1 in nutrient-depleted conditions8, it is plausible that GATOR1 is critical for regulating mTORC1 in zone 3 hepatocytes. After maturation and aging of Depdc5\u0394hep mice, mTORC1 upregulation became more pronounced and resulted in phenotypes similar to Tsc1\u0394hep mice, such as mild inflammation and decreased fat levels. Consistent with their similar mild phenotypes, Depdc5\u0394hep and Tsc1\u0394hep mice had similar transcriptomic profiles that were only moderately different from the wild-type profile.Rheb and Rag are regulated by their respective GAPs, TSC and GATOR1Depdc5\u0394hep mice with Tsc1\u0394hep mice, we showed that mutations in both Depdc5 and Tsc1 generate a synergistic genetic interaction and produce a very strong hyperactivation of mTORC1. This provides genetic evidence in animal models confirming that the Rheb and Rag pathways indeed interact for mTORC1 regulation in a physiological context. mTORC1 hyperactivation in DKO mice resulted in liver dysfunction associated with prominent hepatocyte injury and fibrosis by two-months of age. This led to dramatic elevation of liver damage markers in the serum. Excessive bilirubin accumulation in serum led to an externally observable jaundice phenotype. In addition, since the liver is the primary source of insulin-like growth factors that are essential for systemic growth, liver failure in DKO mice also suppressed growth. At the liver transcriptome level, specific stress response pathways, such as oxidative stress, inflammation, DNA damage and cell death pathways were strongly upregulated. All of these striking phenotypes were not manifested in either Tsc1\u0394hep or Depdc5\u0394hep single knockout strains or in any formerly described models of mTORC1 activation, such as Deptor knockout mice45. Therefore, our current work provides a unique model of unregulated mTORC1 activation and shows that mTORC1 hyperactivation by itself can disrupt hepatocellular homeostasis, provoking liver injury and failure.By crossing 47. Since Akt is an mTORC1 upregulator, Akt inhibition can limit mTORC1 activation. However, at the same time, inhibition of insulin-AKT signaling can also precipitate metabolic insulin resistance. Correspondingly, DKO mouse livers exhibited strong insulin resistance, and hepatocytes from DKO mice did not activate AKT in response to insulin. Although the DKO liver suffers strong insulin resistance, the blood glucose level was rather strongly decreased due to the deterioration of hepatocyte homeostasis and subsequent reduction in hepatic glucose output.mTORC1 is regulated through multiple negative feedback loops. mTORC1 hyperactivation is known to inhibit Akt through S6K- or Grb10-mediated feedback inhibition of insulin signaling48 and Depdc549 pathways. In the current work, we found that Sestrins expression levels were substantially elevated after deletion of Tsc1, Depdc5 or both. AMPK, one of the downstream effectors of Sestrins inhibiting mTORC110, was subsequently activated in these tissues. Redd1 (Ddit4) and Redd2 (Ddit4l), which inhibit mTORC1 through Tsc1/Tsc2 upregulation29, were also upregulated in mTORC1-activated liver tissues. Therefore, it is possible that, in our mTORC1 activation models, Sestrins and Redds may have resulted in negative feedback inhibition to limit mTORC1 activities.In addition to the feedback loop involving insulin signaling, Sestrins can also provide a negative feedback mechanism for the mTORC1 pathway. In Drosophila, Sestrin is an important feedback inhibitor of the mTORC1 pathway through Tsc1/2It was quite striking that all of the liver pathologies in DKO mice were almost completely rescued by only 10 days of rapamycin treatment. Liver/body weight ratios were restored to normal levels, and liver damage markers in the serum also recovered close to clinically normal ranges. Although rapamycin was historically considered a growth attenuator, rapamycin-mediated normalization of liver homeostasis actually promoted systemic growth in this specific DKO model. Upon rapamycin treatment, necrotic and fibrotic lesions in DKO mice disappeared, and hepatocellular ER stress, oxidative stress and apoptosis were all relieved. Therefore, mTORC1 is indeed the major conduit of how the Rheb and Rag pathways pathogenetically interact to produce liver injury and failure.23. DKO mouse livers exhibited upregulation of ER stress signaling at the protein level, confirming that mTORC1 hyperactivation in DKO mice indeed precipitated unfolded protein accumulation and ER stress. However, the ER stress response pathway was not overrepresented in the DKO transcriptome, raising questions of whether the ER stress pathway is important for DKO pathologies. Indeed, TUDCA, a chemical chaperone that effectively suppressed hepatocellular ER stress in DKO mouse liver, was completely ineffective in rescuing DKO liver pathologies. Instead, TUDCA-treated DKO mice increased mTORC1 activation, further potentiating liver pathologies in DKO mice to the point of fatality. Even in the surviving mice, TUDCA administration increased expression of fibrogenic markers and more extensively damaged hepatocytes. It is possible that ER stress signaling somehow limits mTORC1 activation, reducing its negative consequences on liver health. These data also indicate that ER stress signaling is not the major mechanism of how hyperactive mTORC1 disrupts hepatocellular homeostasis.mTORC1 upregulation increases protein synthesis, which can put a burden on protein folding machinery and therefore induce accumulation of unfolded proteins in the ER, also known as ER stress51, inhibiting autophagic elimination of dysfunctional mitochondria52, and suppressing the superoxide-scavenging action of Sod153. Indeed, DKO livers experienced severe oxidative stress associated with excessive accumulation of superoxide radicals and exhibited a transcriptomic profile that is similar to DQ-induced oxidative stress and Sod1 loss. This high level of oxidative stress can damage cellular macromolecules including DNA. Consistent with this, the DKO transcriptome also exhibited upregulation of some DNA damage response genes. Administration of chemical antioxidants that scavenge superoxide radicals, such as Tempol and NAC, effectively reduced hepatic oxidative stress. Surprisingly, 10 days of antioxidant administration was sufficient to normalize almost every liver pathology parameter observed in DKO mouse liver and even restored normal growth. Since mTORC1 signaling itself was not suppressed by chemical antioxidants, these results indicate that hyperactive mTORC1 signaling provokes liver failure primarily through the induction of superoxide radicals that injure hepatocytes.In addition to inducing ER stress, mTORC1 hyperactivation can elevate oxidative stress by altering mitochondrial metabolismIl654 and Cd4455, TGF-beta signaling targets genes Acta2, Mmp2 and Timp256 and Hippo-Yap target genes Ctgf57 and Notch258, were all upregulated in DKO mice. These signaling pathways were implicated in inflammation-dependent acceleration of carcinogenesis in previous studies59. Consistent with the finding and former studies, we found that the DKO mice spontaneously developed HCC at 5 months, a relatively early age.At the tissue level, mTORC1 hyperactivation produced crosstalk with a number of additional pathways. For instance, NF-kB target genes such as DEPDC5 gene can accelerate HBV/HCV-associated liver pathologies such as hepatic fibrosis14 and carcinogenesis15. HBV and HCV infections upregulate mTORC1 by activating PI3K-AKT signaling and/or inhibiting TSC, both of which subsequently activate Rheb61. Genetic variations suppressing DEPDC5 function would upregulate Rag signaling, and this would synergistically interact with the HBV/HCV infection that elevates Rheb signaling. Concomitant upregulation of both Rag and Rheb axes would lead to mTORC1 hyperactivation that can precipitate oxidative liver pathologies, as observed in the DKO mice described here. Furthermore, we found that our DKO liver transcriptome is closely related with human HCV and NASH transcriptomes. Therefore, our DKO mice provide a novel mouse model for investigating the role of human DEPDC5 variations in accelerating liver pathologies associated with HCV and NASH. However, the DKO model currently described here does not involve an actual viral infection or virus-associated activation of adaptive immunity. Therefore, additional studies should be conducted in the context of actual HBV and HCV infection to gain a more direct translation of our findings into the corresponding human liver pathologies.Our observations also provide an explanation of how human genetic variations in the Tsc1 and Depdc5 genes provokes prominent upregulation of mTORC1, disrupts hepatocellular homeostasis, and subsequently precipitates oxidative injury and subsequent liver failure. Our work provides a valuable model for examining the consequences of mTORC1 hyperactivation, understanding human liver pathologies associated with HCV, NASH and DEPDC5 variation, and developing therapeutic strategies for treating such pathologies with mTORC1 inhibitors or antioxidant compounds.In conclusion, we show that the Rag and Rheb pathways are both required for maximum mTORC1 activation in tissues. Correspondingly, double knockout of the Depdc5F/F (EM: 10459) mice, originated from the HEPD0734_3_G10 embryonic stem cell clone, were obtained from the European Mouse Mutant Archive. Depdc5F/F mice were bred to Albumin (Alb)-Cre to produce hepatocyte-specific knockout mice. DKO mice were generated by interbreeding Depdc5F/F and Tsc1F/F mice62, then breeding progeny with Alb-Cre mice. Depdc5 single knockout experiments were done in C57BL/6 background. Tsc1 mice were originally produced in a 129S4/SvJae background62 but were backcrossed to C57BL/6 for more than three generations for DKO experiments. To minimize genetic and environmental variations, littermate controls were used throughout the study, and mice were cohoused. For instance, Depdc5\u0394hep (Alb-Cre/Depdc5F/F) and Depdc5F/F littermates were used for Depdc5 single knockout experiments. Alb-Cre/Tsc1F/+/Depdc5F/+ and Tsc1F/F/Depdc5F/F breeders produced control (Alb-Cre negative mice and Alb-Cre/Tsc1F/+/Depdc5F/+ mice), Tsc1\u0394hep (Alb-Cre/Tsc1F/F/Depdc5F/+), Depdc5\u0394hep (Alb-Cre/Tsc1F/+/Depdc5F/F), and Tsc1\u0394hep/Depdc5\u0394hep  littermates that were analyzed for genetic interaction assays. Alb-Cre/Tsc1F/F/Depdc5F/F males and Tsc1F/F/Depdc5F/F females produced DKO littermate cohorts for drug intervention experiments. Mice were maintained in filter-topped cages with cob bedding and given free access to autoclaved regular chow/low fat diet , high fat diet , and water, as previously described63. When indicated, freshly made rapamycin (10\u2009mg/Kg body weight), tauroursodeoxycholic acid , N-acetylcysteine  or vehicle  solutions were administered once daily through intraperitoneal (i.p.) injections for the last 10 days. A superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl  was administered to mice through drinking water. Acetaminophen  was administered through a single i.p. injection after 12\u2009h of fasting. Glucose (1\u2009g/Kg glucose) and insulin (0.65\u2009U/Kg insulin) tolerance tests (GTT/ITT) were done according to previously described procedures25. For acute insulin response studies, mice were put under a surgical plane of isoflurane anesthesia. First, one part of the liver was collected as an untreated control. Then 0.8\u2009U/Kg insulin, diluted in PBS, was injected intravenously through the vena cava. After 5\u2009min, the other parts of the liver were collected as an insulin-treated sample. Information regarding mouse number, age, gender, diet duration, drug dose, route and frequency are indicated in the corresponding Figure and Figure legends. All animal procedures were ethically approved by the Institutional Animal Care & Use Committee and overseen by the Unit for Laboratory Animal Medicine at the University of Michigan.Antibodies for DEPDC5 were generated from Pocono Rabbit Farm & Laboratory using bacterially expressed recombinant proteins. We obtained COL1A1 (sc-293182), Pro-COL3A1 (sc-166316), PECAM-1 (sc-376764), MMP-2 (sc-53630), MMP-3 (sc-21732), MMP-9 (sc-393859), LOX (sc-373995), MMP-13 (sc-515284), CTGF (sc-365970), S6K (sc-230), eIF2\u03b1 (sc-11386), ATF4 (sc-200 and sc-22800), and TIMP-3 (sc-373839) antibodies from Santa Cruz Biotechnology, Actin (9E10) antibody from Developmental Studies Hybridoma Bank, phospho-Thr389-S6K (9234), pThr172-AMPK (2535), pThr37/46-4E-BP (2855), 4E-BP (9452), pSer51-eIF2\u03b1 (3398), pThr980-PERK (3179), PERK (5683), PDI (3501), BIP (3177), CHOP (2895), pSer473-AKT (4060), AKT (4691), pSer236/239-S6 (2211) and S6 (2317) from Cell Signaling Technology, \u03b1-smooth muscle actin  antibody from Abcam, and F4/80 (MF48000) antibody from Invitrogen. Acetaminophen, NAC and Tempol are from Sigma, TUDCA is from Cayman Chemical, and rapamycin is from LC labs.63. In brief, paraffin-embedded liver sections were incubated with primary antibody (1:100), followed by incubation with biotin-conjugated secondary antibodies  and horseradish peroxidase (HRP)-conjugated streptavidin . The HRP activity was visualized with diaminobenzidine staining. Hematoxylin counterstaining was applied to visualize nuclei. For \u03b1-SMA and Ki-67 staining, Alexa Flour 488 or 594-conjugated secondary antibodies (Invitrogen) were used to visualize primary antibody staining. Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assays were performed using In Situ Cell Death Detection Kit-TMR-Red (Roche). Dihydroethidium (DHE) staining was performed using freshly frozen liver sections and DHE  as formerly described25. To visualize collagen fibers, liver sections were stained with saturated picric acid containing 0.1% Sirius Red . For Oil Red O staining, OCT-embedded frozen liver sections were dried and stained with fresh 0.5% Oil Red O solution for 15\u2009min then rinsed with 60% isopropanol. Reticulin staining was performed using a kit from Polyscience (25094), following the manufacturer\u2019s recommendation. Histology samples were analyzed under an epifluorescence-equipped light microscope from Meiji.Liver tissues were fixed in 10% buffered formalin, embedded in paraffin and subjected to hematoxylin and eosin (H&E) staining and immunohistochemical staining, as previously describedCells and tissues were lysed in radioimmunoprecipitation assay (RIPA) buffer ). Lysates were clarified by centrifugation, and protein concentration was normalized using Bio-rad protein assay dye reagent. Protein lysates were boiled in SDS sample buffer for 5\u2009min, separated by SDS-PAGE, transferred to PVDF membranes and subjected to immunoblotting procedures. 5% blocking grade non-fat milk (170\u20136404 from Bio-Rad) in TBST was used for membrane blocking and antibody incubation. 1X western blocking reagent (11 921 673 001 from Roche) in TBST was used for phospho-specific primary antibody incubation. Primary antibodies from Santa Cruz Biotechnology and Developmental Studies Hybridoma Bank were used at 1:100, and all the other primary antibodies were used at 1:1000. HRP-conjugated secondary antibodies were purchased from Bio-Rad and used at 1:2000. Chemiluminescence was detected using LAS4000 (GE) systems.Blood was obtained by cardiac puncture and separated by centrifugation to obtain serum. Serum chemistry markers associated with liver cytotoxicity  or liver function  were obtained through standard operating procedures using the Liasys clinical chemistry system (AMS Alliance) within the In Vivo Animal Core of the Unit for Laboratory Animal Medicine.Tsc1F/F/Depdc5F/F; n\u2009=\u20093), Tsc1\u0394hep , Depdc5\u0394hep  and DKO  mice, were submitted to BGI for mRNA enrichment, library construction and sequencing (BGISeq 50SE), and processed through standard experimental and analytical pipelines. Each sample produced more than 20\u2009M clean reads, that were mapped to the mm9 reference genome using STAR64. Then, Cufflinks was used to generate Fragments Per Kilobase of transcript per Million mapped reads (FPKM) table65, supplied as Supplementary Table k-means clustering analyses. The accession number for the RNA-seq data reported in this paper is GSE136684.In total 10\u2009\u00b5g of DNAase I-treated total RNA, purified from liver tissues of control 28. From the HCV dataset, disease progression-associated fold changes of differentially expressed genes  were compared with the DKO-induced gene expression fold changes. The oxidative stress-upregulated gene list was obtained from livers of mice acutely treated with Diquat (DQ) for 12\u2009h38. From the same dataset, DQ-treated and Sod1-knockout induced fold changes of differentially expressed genes  were compared with the DKO-induced gene expression fold changes. The ER stress-upregulated gene list was obtained from tunicamycin-treated mouse embryonic fibroblasts26. Cytokine and chemokine pathway gene lists were generated by selecting relevant genes from the list of genes whose names begin with Ccl/Ccr, Cxcl/Cxcr, Il/Ilr, Ifn/Ifnr and Tnf/Tnfr. The fibrosis-associated gene list was generated by selecting relevant genes from the list of genes whose names begin with Col, Mmp, Timp and Tgfb. Cytochrome P450 and major urinary protein gene lists were generated by selecting relevant genes from the list of genes whose names begin with Cyp and Mup, respectively.As formerly describedt-test . When multiple parameters were assessed, the Holm\u2013\u0160\u00edd\u00e1k method was used to compare groups . A two-way ANOVA was used to evaluate the effect of Tsc1 and Depdc5 mutations and assess interactions and synergy between them , and statistical significance between two individual groups were assessed through Tukey\u2019s multiple comparison test . The effect of drugs on body weights was assessed through repeated measures (RM) 2-way ANOVA to evaluate the interaction between treatment and time (####P\u2009<\u20090.0001). Differences in individual data points were assessed through Sidak\u2019s multiple comparison test . Survival curves were compared with a log-rank test. Statistical significance of gene enrichment in a specific cluster was calculated using Fisher\u2019s Exact test. Correlations between RNA-seq datasets were assessed by computing nonparametric Spearman correlation (r); P\u2009<\u20090.0001 for all correlation observations. GraphPad Prism 8 was used for all statistical analyses except k-means clustering analyses and gene enrichment analyses, which were performed using R.Immunoblot images were quantified by densitometry, and protein expressions were expressed as relative band intensities. Histological images were analyzed by densitometric or fluorometric methods as appropriate. When indicated, data are shown as mean\u2009\u00b1\u2009SEM. Statistical significance between two groups was calculated using a Student\u2019s Supplementary Table S1Supplementary Table S2Supplementary Information"}
+{"text": "Presurgical planning for brain tumor resection aims at delineating eloquent tissue in the vicinity of the lesion to spare during surgery. To this end, noninvasive neuroimaging techniques such as functional MRI and diffusion-weighted imaging fiber tracking are currently employed. However, taking into account this information is often still insufficient, as the complex nonlinear dynamics of the brain impede straightforward prediction of functional outcome after surgical intervention. Large-scale brain network modeling carries the potential to bridge this gap by integrating neuroimaging data with biophysically based models to predict collective brain dynamics. As a first step in this direction, an appropriate computational model has to be selected, after which suitable model parameter values have to be determined. To this end, we simulated large-scale brain dynamics in 25 human brain tumor patients and 11 human control participants using The Virtual Brain, an open-source neuroinformatics platform. Local and global model parameters of the Reduced Wong\u2013Wang model were individually optimized and compared between brain tumor patients and control subjects. In addition, the relationship between model parameters and structural network topology and cognitive performance was assessed. Results showed (1) significantly improved prediction accuracy of individual functional connectivity when using individually optimized model parameters; (2) local model parameters that can differentiate between regions directly affected by a tumor, regions distant from a tumor, and regions in a healthy brain; and (3) interesting associations between individually optimized model parameters and structural network topology and cognitive performance. Individualized medicine is increasingly put forward as an important means to advance medical care. In this regard, the neuroinformatics platform The Virtual Brain holds great promise, given its direct focus on simulation of subject-specific brain activity. Reliable prediction of patient-specific large-scale brain dynamics would open up the possibility to virtually lesion structural connectomes, making computational models unique predictive tools to investigate the impact of diverse structural connectivity alterations on brain functioning, including those purposefully induced by surgery. Results of this study establish the basis for this purpose, by demonstrating individual specificity of biophysical model parameters, differences in local model parameters dependent on distance from a tumor, and associations between model parameters and structural network topology and cognitive performance.Presurgical planning for brain tumor resection aims at delineating eloquent cortical areas and white matter pathways in the vicinity of the lesion to spare during surgery, to preserve essential brain functions. In brain tumor patients, localization of brain function often cannot be inferred from anatomic landmarks alone, since mass effects can distort normal topography, and disease processes such as brain shift or plasticity can induce relocation of functions . TherefoHowever, taking into account this preoperative neuroimaging information is often still insufficient, as the complex nonlinear dynamics of the brain impede straightforward prediction of functional outcome after surgical intervention. For example, fMRI cannot differentiate cortical areas that are essential for a particular function and should be surgically preserved from expendable areas which merely correlate with but are not essential for functionality .Large-scale brain network modeling carries the potential to bridge this gap by integrating neuroimaging data with biophysically based models to predict collective brain dynamics, from which functionality could be inferred. A future application of this framework could then entail the presurgical virtual exploration of the effects of different neurosurgical approaches based on individual patient data, to identify an optimal surgical strategy .As a first step in this direction, an appropriate computational model has to be selected, after which suitable model parameter values should be determined that lead to plausible brain dynamics. Different approaches can be used to simulate activity of nodes  in large-scale brain network models varying from detailed spiking neuron models , over soIn this study, we simulated large-scale brain dynamics in brain tumor patients and control subjects using The Virtual Brain TVB; , an openThe objectives of this study were (1) to evaluate the importance of constructing personalized virtual brain models using individually optimized model parameters and subject-specific structural connectomes; (2) to determine if the individually optimized model parameter values differ between brain tumor patients and healthy controls; and (3) to elucidate the relationship between these model parameters on the one hand, and cognitive performance and structural network properties on the other hand.In this study, we included patients who were diagnosed with either a glioma, developing from glial cells, or a meningioma, developing in the meninges . Both tyPatients were recruited from Patients were recruited from Ghent University Hospital (Belgium) between May 2015 and October 2017. Patients were eligible if they (1) were at least 18 years old, (2) had a supratentorial meningioma (WHO grade I or II) or glioma (WHO grade II or III) brain tumor, (3) were able to complete neuropsychological testing, and (4) were medically approved to undergo MRI investigation. Partners were also asked to participate in the study to constitute a group of control subjects that suffer from emotional distress comparable to that of the patients.BTC_preop.We collected data from 11 glioma patients , 14 meningioma patients , and 11 healthy partners . Patient characteristics are described in 10.1523/ENEURO.0083-18.2018.t1-1Table 1-1A) and glioma patients (B). Download Table 1-1, TIF file.Percentage of voxels per node that was affected by a tumor (only for tumor nodes), per subject, for meningioma patients . Next, resting-state functional echo-planar imaging (EPI) data were obtained in an interleaved order . After the first 4 control subjects, 5 meningioma patients, and 2 glioma patients were scanned, the fMRI protocol was accidentally changed to a TR of 2400 ms, resulting in a TA of 7:19 min. This has been taken care of in subsequent analyses by inclusion of an additional covariate. During the fMRI scan, participants were instructed to keep their eyes closed and not fall asleep. Finally, a multishell high-angular resolution diffusion-weighted MRI (DWI) scan was acquired . In addition, two DWI b = 0 s/mm2 images were collected with reversed phase-encoding blips for the purpose of correcting susceptibility-induced distortions  to obtain a subject-specific parcellation of each subject\u2019s brain into 68 cortical regions (34 per hemisphere). T1-weighted data of all control subjects were subjected to the default recon-all processing pipeline, which includes the following steps: intensity normalization, skull stripping, removal of non-brain tissue, brain mask generation, cortical reconstruction, segmentation of subcortical white matter and deep gray matter volumetric structures, cortical tessellation of the gray matter/white matter and gray matter/pial boundary, and construction of a probabilistic atlas based cortical parcellation into 68 regions according to gyral and sulcal structure . Second, the Normalisation tool of the BCBtoolkit . Specifically, the following operations were applied: motion correction using MCFLIRT , part of FSL . First, raw diffusion-weighted MRI images were corrected for several artifacts. In particular, DWI images were denoised  and MRtrix3 (2) and per tissue type  using the MRtrix3 script dwi2response dhollander  was performed using dynamic seeding generating 30 million streamlines per subject  yielded a decaying degree distribution while ensuring all subjects\u2019 network remained fully connected. This approach is similar to the one adopted by TVB  was usedG) was optimized. This parameter rescales each subject\u2019s structural connectivity, which is given by relative values, to yield absolute interaction strengths. In particular, values of G were varied from 0.01 to 3 in steps of 0.015. For each value of G, individual resting-state BOLD time series were generated with the same duration and sampling rate as the subject\u2019s empirical resting-state fMRI acquisition, using the Balloon\u2013Windkessel hemodynamic model \u2014controlling the strengths of connections from inhibitory to excitatory mass models within each large-scale region i\u2014were tuned in each iteration of the parameter space exploration, to clamp the average firing rate at \u223c3 Hz for each excitatory mass model. 3 Hz was chosen as attractor value, as it is the \u201cintrinsic frequency\u201d of an isolated neural mass model according to derivations from a spiking network . Second, median iJ was calculated in patients across a subset of tumor regions (tumorJ) to investigate possible local alterations in biophysical model parameters in the direct vicinity of the lesion. In glioma patients, tumor regions were defined as those cortical areas of the individual FreeSurfer parcellation that showed at least partial  overlap with the tumor mask. In meningioma patients, tumor regions consisted of regions that were  displaced because of the tumor\u2019s mass effect. To estimate which regions were displaced by the meningioma, patients\u2019 anatomic images were transformed to MNI space (using FSL FLIRT with 12 DOF), and this transformation was applied to their tumor mask. Then, the overlap between subjects\u2019 tumor mask in MNI space and the fsaverage Desikan\u2013Killiany atlas (Jnon-brain) for further analyses: global efficiency, modularity, and participation coefficient.Structural network topology was assessed with various graph theory metrics of integration , segregation , and centrality , as well as graph density, using the Brain Connectivity Toolbox . After iGlobal efficiency relates to the capacity of the network to rapidly integrate specialized information from distributed brain regions and is defined as the average inverse shortest path length . ModularCognitive performance of patients and control subjects was assessed using the Cambridge Neuropsychological Test Automated Battery . In particular, four cognitive domains were examined that have been identified by previous studies to be affected by brain tumors: sustained attention, working memory, information processing speed, and executive functioning . One gliBefore the actual test administration, the Motor Screening Task (MOT) was used to screen for sensorimotor or comprehension difficulties that could limit valid data collection. Subsequently, the main tasks were administered in random order to avoid sequence bias. Specifically, the Rapid Visual Information Processing (RVP) task was used to assess sustained attention, the Spatial Span (SSP) task measured working memory capacity, the Reaction Time task (RTI) evaluated participants\u2019 mental response speed, and the Stockings of Cambridge (SOC) task assessed planning accuracy.As cognitive performance can be affected by several factors, results were corrected for participants\u2019 motivation, level of emotional distress, lesion volume, age, and sex. Likewise, previous studies have shown that graph theory metrics can be affected by various factors such as age and gender , handednz-scores for subsequent analyses using the mean and SD of the respective metric in the group of control subjects, for the ease of interpretation.Linear regression models were then constructed for every outcome variable  as a function of these confounders. Of note, only main effects of the confounders were considered, since inclusion of interaction effects between the covariates would considerably reduce the degrees of freedom. Residuals of these models were further transformed to TR on the construction of FC matrices and the parameter space exploration. First, we simulated BOLD time series for every subject, using the subject\u2019s optimized global coupling value and (a) a TR corresponding to the subject\u2019s empirical rs-fMRI TR, and (b) the other TR . Results showed that the similarity between the upper triangular part of both simulated FC matrices per subject was rather high . Further, we investigated in two subjects (one control subject and one glioma patient) whether the parameter space exploration was comparable when using a different TR . Results again showed a very limited impact of TR on the parameter space exploration.Additionally, we investigated the effect of First, we compared the optimized model parameters between glioma patients, meningioma patients, and control participants using one-way analysis of variance tests and Kruskal\u2013Wallis rank sum tests, depending on whether the normality assumption was violated. Afterward, optimal model parameters were related to structural network topology and cognitive performance using linear regression. All analyses were performed on a Cooler Master Ubuntu 14.04 desktop PC.https://github.com/BrainModes/The-Hybrid-Virtual-Brain, and all scripts for postprocessing can be found at https://github.com/haerts/The-Virtual-Brain-Tumor-Patient. All code is also available as All code used for this study is freely available. The optimized TVB C code can be found at 10.1523/ENEURO.0083-18.2018.ed1Extended Data 1Extended Data 1, ZIP file.Code used for simulation of large-scale brain dynamics and scripts for subsequent postprocessing analyses. Download r = 0.68, 95% CI 0.45\u20130.82, p < 0.0001). No significant differences in prediction accuracy of empirical functional connectivity were found between healthy controls, meningioma patients, and glioma patients  = 0.32, p = 0.73) or according to lesion size  = 1.20, p = 0.28).In this study, we simulated large-scale brain dynamics in brain tumor patients and control subjects using the Reduced Wong\u2013Wang model  as impleF = 6.34, p = 0.0005; post hoc Tukey tests showed that the highest similarity between individuals\u2019 simulated and empirical FC was obtained when model parameters were individually tuned on the individual or control average SC matrix . When ndiv SC\u201d A or usinndiv SC\u201d B, predicndiv SC\u201d D vs. \u201cInndiv SC\u201d A; diff =ndiv SC\u201d D vs. \u201cCOndiv SC\u201d B).F = 3.44, p = 0.044). Furthermore, a trend toward increased group means in tumor regions was observed, although this difference did not reach statistical significance  = 2.72, p = 0.081). In healthy brain regions, median local inhibitory connection weights did not differ between patients and controls  = 1.27, p = 0.293). However, local inhibitory connection strengths strongly depend on the number of connections a brain region has, which in turn tightly correlates with region size. That is, larger cortical areas encompass on average more white matter fiber bundles , and hence need more local inhibition to balance global excitation. Additional analyses showed that tumor regions were significantly larger compared to non-tumor regions  = 15.11, p = 0.0001), but that the number of connections per cortical area were not significantly different in tumor regions compared to non-tumor regions (X2(1) = 2.29, p = 0.130). Given the high dependence between region in-strength and size (r = 0.80), we regressed out only the effect of region size. Results now revealed opposite effects (X2(2) = 14.4, p = 0.0007) and more variable  = 5.27, p = 0.010) in tumor regions compared to healthy regions/brains. Moreover, local inhibitory connection strengths in non-tumor regions were now also increased  = 6.51, p = 0.039) compared to healthy controls  = 1.42, p = 0.26).We then sought to determine if the individually optimized biophysical model parameter values differ between glioma patients, meningioma patients, and control participants.  effects C, with mcontrols D. With rcontrols E, no sigF = 0.13, p = 0.88 for reaction time; F = 0.35, p = 0.71 for sustained attention; F = 0.17, p = 0.84 for planning accuracy; F = 0.38, p = 0.69 for spatial span length). With regard to structural network topology, a significant group difference was found in the participation coefficient  = 3.94, p = 0.029; Post hoc (Tukey) testing revealed that participation coefficients were higher in glioma patients compared to healthy controls (p = 0.026). Group differences in global efficiency and modularity were not significant  = 0.57, p = 0.57 for global efficiency; F = 1.26, p = 0.30 for modularity; Before relating the biophysical model parameters to cognitive performance and structural network topology, we examined whether group differences were present in these variables. As can be seen in t = \u20134.42, p < 0.001, \u03b72 = 0.34) and the median feedback inhibition control parameter across non-tumor regions in brain tumor patients . Specifically, higher values of global efficiency were associated with lower values of both modeling parameters, as illustrated in r = \u20130.67 and \u20130.45 for the global coupling parameter and the feedback inhibition control parameter, respectively). No significant associations were found between the modeling parameters and modularity or participation coefficient.Next, we aimed to elucidate the relationship between the individually optimized modeling parameters on the one hand, and structural network topology and cognitive performance on the other hand. Linear regression analysis showed that global efficiency of the structural network was significantly associated with the global scaling factor  and sustained attention  and patients\u2019 reaction time  that using personalized virtual brain models significantly improves prediction accuracy of simulated functional connectivity; (2) that local inhibitory connection weights can differentiate between regions directly affected by a brain tumor, regions more distant from a brain tumor, and regions in a healthy brain; and (3) that individually optimized model parameters correlate with structural network topology and cognitive performance.Individualized medicine is increasingly put forward as an important means to advance medical care. In this regard, TVB holds great promise, given its direct focus on the simulation of subject-specific brain dynamics. Reliable prediction of patient-specific large-scale brain dynamics would open up the possibility to virtually lesion structural connectomes, making computational models unique predictive tools to investigate the impact of diverse structural connectivity alterations on brain functioning. That is, computational modeling would enable us to investigate what types or extent of damage the brain can withstand, and conversely, which kind of distortions can be expected after brain lesions, including those purposefully induced by surgery.Therefore, the first aim of this study was to verify the added value of personalized virtual brain models. Results showed that individual optimization of the modeling parameters significantly increased the prediction accuracy of individual functional connectivity. However, whether model parameters were tuned on the individual SC matrices or on the average SC matrix across all control subjects did not yield a significant difference, corroborating findings in previous research e.g., . Future In the next step, we investigated which factors are associated with the individual model parameters, examining the relative contribution of the presence and type of brain tumor, structural network topology, and cognitive performance.With regard to the effect of the presence and type of brain tumor, results revealed no significant differences in the feedback inhibition control parameter between tumor regions and healthy regions/brains. However, this local parameter is highly dependent on the size of the cortical area that is modeled, and additional analyses showed that regions affected by a tumor were on average larger than non-tumor regions. After taking into account this confounding effect of region size, results showed median local inhibitory connection strengths that are much lower and more variable in tumor regions compared to healthy regions/brains. Moreover, local inhibitory connection strengths (controlled for region size) in non-tumor regions were increased compared to healthy controls. Hence, these results suggest that brain tumors have a distinct focal and distant effect on the local inhibitory connection weights, beyond what could be expected from their region size.A previous computational modeling study also reported alterations in inhibitory over excitatory coupling in chronic stroke patients . It is dIn contrast to previous computational modeling studies that found increased global coupling values in chronic stroke patients , our resWe then turned to structural network properties and measures of cognitive performance as possible predictors of the individual biophysical model parameters. Initial descriptive analyses of these metrics revealed no significant differences in cognitive performance between brain tumor patients and healthy controls. Although most patients with slow-growing brain tumors exhibit normal clinical examinations , previouDespite a lack of large group differences in cognitive performance and structural network topology, several interesting associations across groups were found between these variables and the individually optimized biophysical model parameters. First, results showed a strong negative relation between global efficiency of the structural network and global coupling, corroborating the finding of Furthermore, negative associations were found between local inhibitory connection weights in regions directly affected by a brain tumor and reaction time and sustained attention. This implies that brain tumor patients who performed worse on sustained attention and reaction time tasks on average had higher local inhibitory connection weights in regions directly affected by the tumor. This finding is a bit counterintuitive, since the feedback inhibition control parameter was significantly lower in brain tumor patients compared to healthy controls, which would imply a higher cognitive performance in brain tumor patients relative to healthy controls. However, as noted in the Results, the correlation between local inhibitory connection strength and especially sustained attention is mainly driven by a few outlying observations; hence, caution is advised in interpreting this finding. Future research using a larger sample size would be required to clarify this association.In interpreting the results of this study, some important limitations should be taken into consideration. First, the sample size is rather small, limiting the statistical power of the analyses. In addition, substantial intersubject variability is present in both patient groups, caused by (among other factors) heterogeneity in lesion etiology and size. It is likely that these two factors interfere with separating clinical groups based on computational model parameters, cognitive performance, and structural network topology. As more efforts are undertaken to make clinical datasets publicly available, future studies would benefit from using larger sample sizes.2 and a state-of-the-art processing pipeline, future studies could investigate whether prediction accuracies of individual SC matrices improve when using data of even better quality . Another contributing factor to the low variability in individual SC matrices might be the relatively coarse parcellation schemes currently applied in computational modeling studies. With increasing computational power, future studies can test finer and more elaborate parcellations, for example based on multimodal data such as in Second, simulated and empirical functional connectivity were only moderately related after optimization of the model parameters. Moreover, using individual structural connectomes did not yield better predictions of individual functional connectivity patterns compared to using a control average structural connectivity matrix. This is, however, a limitation of all current computational modeling studies and a matter of much debate. A recent study by Related to this issue, the robustness of the obtained results to different parameter space exploration criteria has to be investigated. In this study, the link-wise Pearson correlation between empirical and simulated FC was maximized. Atlhough this method is routinely employed in large-scale modeling studies, other methods are worth exploring. For example, similarity could be sought at the modular level, maximizing the cross-modularity between simulated and empirical functional connectivity . These aAs pointed out in the Introduction, the next step entails prediction of postsurgical outcome. To this end, longitudinal research is required to investigate whether individually optimized model parameters can reliably predict postsurgical functional connectivity. This would be a major step toward presurgical virtual exploration of different neurosurgical approaches and identification of an optimal surgical strategy."}
+{"text": "Interestingly, the biochar-captured N was as plant available as the mineral nitrate, except for the highest dosage. Refuting our hypothesis, no significant amounts of N were extractable at the end of the study from the biochar\u2013soil mixtures with repeated-extraction protocols. Thus, N captured by biochar may improve the N use efficiency in agriculture. Further research should evaluate the role of biochar particle size, root morphology, mycorrhization, and soil moisture (variations) for nitrate retrieval from biochar particles by plants because the captured biochar N was less available in the field as under present controlled conditions.Biochar may serve as a tool to sustainably mitigate climate change via carbon sequestration and by improving soil fertility. Biochar has shown to retain nitrate in its pores, which increases with an organic coating of the inner surfaces and residence time in soil (\u201caging\u201d). Here we investigated the plant accessibility of the captured nitrate in field-aged biochar, as a pre-requisite for developing carbon-based N fertilization techniques with environmental benefits. Based on previous results, we hypothesized that part of the biochar-captured nitrate would remain unavailable for plants. A two-factorial greenhouse experiment was designed, where the N was applied either as Ca(NO In December 2015, the COP21 stated in the Paris Agreement that humanity intends \u2018to keep atmospheric temperature rise to well below the 1.5\u00a0\u00b0C by 2050\u2019. To achieve this goal, it is not only necessary to curb anthropogenic CO2 emissions drastically within the next 30\u00a0years but also to additionally remove about 300\u00a0Gt of carbon from the atmosphere until 2,100 . Among various NETs, natural climate solutions (NCS) such as afforestation/reforestation, combustion/pyrolysis5 and soil C sequestration  offer multiple co-benefits, supporting the UN\u2019s sustainable development goals (SDGs3). NCS involving soil management strategies play an important role among the available spectrum of NETs. It is estimated that if the full potential of carbon sequestration in the biosphere (vegetation and soil 155 and 178\u00a0Pg respectively) is accomplished6, it will reduce the atmospheric CO2 concentration by more than 150\u00a0ppm7. For a soil SOC increase, recommended soil carbon sequestration strategies have to be adopted between 2020 and 21006.Since preindustrial times , anthropogenic CO12 and is considered to be one of the potential NETs in the IPCC 1.5\u00a0\u00b0C special report published in 201813.Biochar is a solid by-product of pyrolysis, the thermal conversion of biomass at temperatures of 350\u2013900\u00a0\u00b0C at low to absent oxygen conditions. It has been proposed as a carbon sequestration strategy with environmental co-benefits when used in soils\u22121 to soils, resulted in no yield increases in temperate latitudes and an average 25% yield increase in the subtropics and tropics14. Another meta-analysis of 153 peer-reviewed studies reported that crop plant productivity is dependent on the consortium of biochar and soil properties15. However, using biochar at \u2265\u200910\u00a0t\u00a0ha\u22121 is economically challenging since the yield increase does not necessarily repay the investment, as long as services to the commons such as N2O emission reductions, reduced nitrate leaching17 or C sequestration3 are not paid for by society. Hence, strategies for achieving agronomic benefits need to be developed that allow using lower cost-effective dosages, e.g. by blending biochar with organic fertilizers and applying it at very low rates (0.5\u20132\u00a0t\u00a0ha\u22121)20; or by reducing biochar production costs by using local residue biomass and simple, clean techniques21. As an example, rice husk biochar treated with urea-H2O2 was applied as a slow N release fertilizer (17.63% slower release than ordinary fertilizer) in a pot experiment with cabbage, with the additional benefit of cadmium immobilization, resulting in significant yield improvements22. A successful commercial strategy has been to create a compound fertilizer that comprises 20% biochar, 5% clay, and 75% NPK with application rates of the biochar at approximately 100\u00a0kg\u00a0ha\u2212124.Recently, a global meta-analysis of 105 studies revealed that the use of pure biochar, applied in large doses of \u2265\u200910\u00a0t\u00a0ha.25 observed that co-composted biochar particles improved plant growth to a much greater degree compared to pure biochar, since they were loaded with nutrients, in particular nitrate, which was surprising because biochar does not have a large anion exchange capacity26. Subsequently, Haider et al.27 demonstrated that the nitrate entrapped in field-aged or co-composted biochar particles was partly un-exchangeable by standard extraction methods ; in the field, this caused significant nitrate retention in the top soil28, a finding underlined by results of a recent meta-study16. Joseph et al.29 found that capture of nitrates after composting followed a series of complex reactions that involved, movement into the pores, then surface adsorption and incorporation into an organomineral layer. Moreover, Hagemann et al.30 showed that co-composting enriched biochars with nitrate (the magnitude depending on the biochar type) and that the observed nitrate capture was related to the formation of an organic coating on biochar surfaces31 as well as the formation of organo-mineral complexes29. This is often termed \u201caging\u201d and does not necessarily mean that the biochar-C itself was oxidized, but rather that the biochar acquired/sorbed non-biochar (aromatic) dissolved carbon and nitrogen species32.Kammann et al28. The study also showed that there is a slow release of nitrate from picked biochar particles from the field site  where no N fertilizer was applied to one crop (summer barley (Hordeum vulgare L.) over the vegetation period in 2014. The hypothesis was that the biochar-amended, nitrate-enriched plots should show higher biomass yields as have higher mineral N in the 0\u201390\u00a0cm soil profile. The results obtained in the unusually hot summer 2014, however, refuted this hypothesis; no yield increase occurred compared to the control without biochar28.Our field experimental results showed significant nitrate retention in biochar-amended sandy topsoil over time14. Hence, we picked naturally nitrate-loaded biochar particles from the field experimental site and used them as nitrate (carbon) fertilizer, comparing biochar-captured nitrate to the same amount of calcium nitrate in a fully randomized pot study with two different plant species under controlled conditions in the greenhouse.These findings instigated the current study. We hypothesized that part of the nitrate captured in the biochar particles would not be plant-accessible and assumed that the biochar would retain a certain amount of N, which would subsequently be unavailable for plant N uptake; and that this feature may be the cause for the sometimes slightly negative effects that pure, production-fresh biochar use can have on crop yields in temperate soils\u22121), the synthetic N source produced greater (p\u2009\u2264\u20090.001) dry matter yields (+\u200999%), compared to N delivered from BC-aged. There was a greater (p\u2009\u2264\u20090.001) fresh to dry mass ratio between N sources at 352\u00a0kg\u00a0ha\u22121, indicating a higher moisture content in the plant material in the BC-aged treatment (see supplementary information). Interestingly, up to the second-highest rate of application in quinoa (N at 176\u00a0kg\u00a0ha\u22121), there was no significant difference in the aboveground biomass production between equal N supplies from the synthetic versus the BC-aged N sources , where the synthetic N fertilizer again produced greater dry biomasses (+\u200920%), compared to N applied as BC-aged.The dry matter yield of quinoa Fig.\u00a0A and rye3\u2212 and NH4+) increased with increasing rates of application  captured by biochar particles during its field aging was not easily extractable by standard methods, nor readily plant-available under field conditions28. Therefore, the remaining N from both sources of N application was assessed by repeated extractions. No significant difference in the form of remaining N (NO3\u2212 or NH4+) was found between the two sources of N in the soil mixtures used in the study  is presented in Table The fresh biochar had a slightly higher concentration of dissolved organic carbon (DOC) than the BC-aged and the highest DOC concentration was measured in fresh biochar in the 2nd extraction Table . It suggThe BC-aged had a much higher concentration of dissolved nitrogen [sum of dissolved organic nitrogen (DON) and dissolved inorganic nitrogen (DIN)] and most of this nitrogen was released in the first extraction Table . The higThe surface of the fresh biochar has a relatively high concentration of micron and submicron mineral particles embedded in the carbon matrix Figure . The minThere is a range of micron and submicron minerals that have been deposited on the surface during biochar ageing Fig.\u00a0. The are28 we hypothesized that only part of the captured N would be accessible by growing plants. Moreover, we expected that a higher percentage of biochar-N would be taken up by plants when N was a limiting resource (lower application rates), triggering root growth and foraging for N , and a lower fraction when N was applied at larger doses.This study aimed to test for the first time if N (largely nitrate) captured in field-aged biochar particles would be bioavailable to plants under controlled conditions. Based on earlier field results35. The use of comparably large amounts of biochar in this study (used as \u201cbiochar-nitrate/N\u201d) does not contradict this aim, because it is in line with recent studies of concentrated use of biochar in the root zone (conservation farming approach36) with locally high dosages, but overall as low as 2\u20134\u00a0t\u00a0ha\u22121. Recently, Schmidt et al.37 reported that the use of biochar\u2013urine mixtures, placed with compost concentrated in the root zone at dosages of 1\u20132\u00a0t\u00a0ha\u22121, produced on average double yields in 21 field trials in fertile soils in Nepal . One of the open questions was if the temporary uptake and re-delivery of N from biochar particles could mechanistically explain part of the success of this novel approach, i.e. if low-molecular mineral or organic N captured by biochar would be plant-available, and to what degree.The background of the study is the shifting paradigm of using biochar as a carbon compound/carrier material for developing carbon fertilizers instead of applying large amounts, production-fresh and untreated, to soilsOur results reveal that the captured N in field-aged biochar was nearly completely plant available to both, perennial ryegrass and quinoa under controlled conditions, which essentially contradicts our initial hypotheses that part of the N would remain biochar-bound, and that the amount that was retrieved by plants would depend on the N dosage.28 was less plant available in the field study than in this pot study. We assume that in contrast to this study, N-carrying biochar particles were spread widely in the field soil when plowed in homogenously in 2012, with the plant roots having to grow long distances towards the nearest N containing biochar microsites. In contrast, in this 200\u00a0g soil\u2009+\u2009biochar mixture in a pot (which was more similar to a concentrated root zone application), roots were in close proximity to biochar particles .25, also using quinoa, that plants can utilize N (nitrate) from naturally loaded (aged/coated) biochar particles. The combined observations of the field study and this pot study, however, suggest that a more concentrated root zone placement together with the nutrients  may be favorable in terms of N capture and release to crop plants, as described by Schmidt et al.37.Our study is in line with results by Kammann et al\u22121 (i.e. near the optimum N requirements of quinoa and ryegrass), while the highest (352\u00a0kg\u00a0N\u00a0ha\u22121) N application with field-aged-BC did not further improve yields, posing the question if upper limits exist of beneficial biochar concentrations in the plant root zone.The plants\u2019 fresh and dry biomass yield was statistically similar up to 176\u00a0kg\u00a0N\u00a0ha.39. In general, quinoa has a high N uptake efficiency40. However, in a study with high and low N supply to different quinoa genotypes, Bascu\u00f1\u00e1n-Godoy et al.41 observed 25% less carbon assimilation, smaller and thinner leaves by some of the tested genotypes with high N application, which may explain the growth anomaly observed in the present study at the highest N application dose via BC-aged  when the N was supplied from BC-aged, compared to the synthetic N application; the latter, however, showed untypical morphology Figure . Soil mid Figure . This ma\u22121) would equal an application dose of 33.92 t ha\u22121 of biochar (when applied homogeneously and plowed in) and it might be argued that such a large biochar dosage may change the soil\u2019s pH value, impacting micronutrient availability. However, since the pH of the aged biochar was even lower than that of the field soil , the biochar addition is unlikely to have impacted (micro) nutrient availability via pH changes.The biochar amount of 6.03\u00a0g , largely depend on the soil water availability; and with the ion uptake gradient that a root-biochar contact is able create. Classical chemical nitrate sorption/desorption processes of nitrate from aged, organically coated biochars may play a small role30, but chemically aged (oxidized) biochars can lose some of their  anion exchange capacity42. Hence, the main mechanism of nitrate capture is unlikely to be chemical sorption to biochar surfaces per se. Hence, the release pathway (and delivery to plant roots) also needs to be different. Previous studies showed that repeated extraction cycles and/or longer extraction times increased the amount of nitrate that could be retrieved from N-enriched, aged or co-composted particles31. Thus, the constant water supply of 60\u201365% WHC in the pots, together with the root proximity to the particles, may have provided ideal conditions for nitrate extraction by plant roots. We hypothesize that plant roots will create a concentration gradient between a higher nitrate concentration in the biochar pores and a lower concentration at the root surface where nitrate is taken up, thus draining pores over time, given that enough water is present to allow nitrate diffusion in (water filled) pores43. The main factors determining the movement of nitrate during pore drainage (by plant N uptake) may hence be (1) diffusion along this gradient, (2) the biochar properties that determine surface (water) interactions, and (3) the clogging of the pores by minerals or organic molecules. Biochar properties include (1) pore size distribution , (2) surface properties such as induced dipole forces of the biochar-coating combination  or (3) general water movement characteristics on inner biochar (pore) surfaces43. While Joseph et al.29 assumed a blocking of biochar pores with organo-mineral layers during composting, essentially trapping nutrients inside biochar pores, Hagemann et al.30 showed that the organo-mineral coating itself (which formed on biochar particles during composting) was highly enriched in N.The question is why the nitrate has nearly completely been drained from the biochar particles in this study, while this did not occur in the field study of Haider et alRegardless of the mechanism of capture or exact location of nitrate capture, our study demonstrates for the first time that the biochar-captured nitrate was largely extractable by plant roots. This may aid the development of biochar-based fertilization techniques or for buffering N fluctuations for crops in soils, as long as the soil moisture conditions allow nutrient flow from biochar particles to plant roots. However, the soil water potential (pore size distribution of the soil\u2013biochar continuum) may limit the extractability. We hypothesize that moisture/soil suction thresholds will exist where nitrate-enriched biochar particles can no longer be mined by plant roots, which should be tested in future studies.2) 6.3 contains 0.59% total organic carbon, 0.06% total nitrogen, 7.42\u00a0mg\u00a0kg\u22121 nitrate, and 0.531\u00a0mg\u00a0kg\u22121 ammonium. The soil available phosphorous contents is 92.2\u00a0mg\u00a0kg\u22121  as the extractants), available potassium content is 124.5\u00a0mg\u00a0kg\u22121 (CAL extractants), and Magnesium is 35.5\u00a0mg\u00a0kg\u22121.The soil used in this experiment was collected from the plow layer of an experimental site at the Research Station of the Institute for Plant Breeding and Agronomy I, Justus Liebig University Giessen, Germany. The Research Station is located at 49\u00b0\u00a045\u2032\u00a0N and 8\u00b0\u00a029\u2032\u00a0E, 90\u2013145\u00a0m (above sea level) at Gross-Gerau in the federal state of Hesse. The sandy soil at the experimental station was formed from the sand deposits of the river Rhine flowing in the west of the experimental site. The river Main is flowing in the North and, the eastern side of the experimental area is surrounded by the Odenwald Mountains. The site is rain-fed but frequently irrigated with sprinkler irrigation in case of longer drought periods during the summers. The average (56\u00a0years) precipitation and temperature of the region are 600\u00a0mm and 9.8\u00a0\u00b0C, respectively. The soil was characterized as silty sand, containing 85.2, 9.6, and 5.2% sand, silt, and clay respectively. The soil with a pH of  was used for biochar production. The overall feedstock was comprised of small wood chips, bark, twig pieces (70%), and needles (30%). Biochar was commercially produced by Pyreg GmbH, D\u00f6rth, Germany, at pyrolysis temperatures of 550\u2013600\u00a0\u00b0C. The detailed chemical properties, particle size distribution, and elemental composition of BC are provided in Haider et al.28. Fresh BC was applied to the soil surface and incorporated to 15\u00a0cm depth in April 2012. The pre-existing crop rotation and conventional fertilizer regimes of maize (Zea maize L.), wheat (Triticum aestivum L.), barley with the intercrop pea (Pisum sativum L.) are described in Haider et al.28.A feedstock mixture of two different tree species 27 and calculated with the maximum amount of 5,305.53\u00a0mg\u00a0kg\u22121  mineral N contained in BC-aged, a conservative approach to the experimental set-up detailed below.Biochar particles were retrieved at the end of the fourth major crop Maize in 2015 by sieving field soil samples after air-drying and picking particles from the sieved larger non-biochar detritus. At the time of application  biochar particle size fraction ranged >\u20096.3% to\u2009<\u20090.1% see details in Haider et al. 2015. Particles of 2\u20135\u00a0mm from field aged and laboratory-stored biochar (saved from the field-applied production lot (Fresh-BC), kept in an air-tight container) were ground to powder and analyzed for total carbon and nitrogen with a CN analyzer . Fresh and field aged BC contained 0.55% and 0.98% N, respectively, where the fresh biochar naturally contains heterocyclic but no mineral N, whereas the aged biochar had additionally loaded itself with mineral N \u2009+\u200950\u00a0g quartz sand to cover the seeds. The N application doses equalled 0, 44, 88, 176 and 352\u00a0kg\u00a0N\u00a0ha\u22121 (on soil weight basis with a bulk density of 1.5\u00a0kg\u00a0L\u22121) obtained from two different sources (A) analytical grade calcium nitrate (Ca(NO3)2) by Sigma-Aldrich, and (B) from captured mineral N in BC-aged. The BC-aged required to meet mineral N doses  was equivalent to 0, 4.22, 8.49, 16.99, and 33.92\u00a0Mg\u00a0ha\u22121. Increasing rates of N application were selected to test, (a) what level of N, supplied by N-carrying aged biochar, would equal a certain amount of analytical grade mineral N and thereby, (b) to determine how much of the biochar-bound N may not be plant available. Each treatment pot was supplied and thoroughly mixed with a 7.0\u00a0mL of micronutrient solution containing the following nutrients per Litter: 0.86\u00a0g boron (H3BO3), 6.4\u00a0g copper (CuSO4\u2009\u00d7\u20095 H2O), 0.06\u00a0g molybdenum (ammonium molybdate), 8.2\u00a0g manganese (MnSO4\u2009\u00d7\u2009H2O), and 14.3\u00a0g zinc (ZnSO4\u2009\u00d7\u20097 H2O). Ten seeds of quinoa, respective 0.35\u00a0g of ryegrass seeds were sown into each pot on Dec 18, 2015. After the emergence, only three well-growing quinoa plants of equal size per pot were maintained; perennial ryegrass seedlings were all maintained. The water holding capacity of the soil or soil\u2013biochar mixture was determined beforehand by the procedure described by Kammann et al.45, and experimental pots were adjusted to 65% of the maximum water holding capacity every second day on weight loss basis. The pots , were installed in CRD with a factorial arrangement.The experiment followed a completely randomized design (CRD) with two different plant species  in a controlled climate chamber at the Experimental Station of the Institute of Plant Nutrition, Justus Liebig University Giessen, Germany. The treatments for each crop were designed as follows: five levels of nitrogen supply (Control: no nitrogen addition), and 4, 8, 16, 32\u00a0mg\u00a0N\u00a0potQuinoa was harvested 38\u00a0days after sowing on January 26, 2016. Ryegrass was harvested twice, at 30th and 60th of sowing on January 18 and second on February 16, 2016, respectively. The total fresh mass of all plant parts (aboveground) per pot was recorded and plant biomass was oven-dried at 70\u00a0\u00b0C for 48\u00a0h. Total dry mass was noted, and plant samples were ball milled for nutrient analysis. Total carbon and nitrogen analyses of the plant material were performed with a CN analyzer . Pot N uptake was calculated by multiplying the harvested biomass per pot with the N concentration in the plant material.min (NO3\u2212 and NH4+). During the first extraction, we followed the standard 2\u00a0M KCl extraction method46. A 20\u00a0g soil was weighed in 80\u00a0mL 2\u00a0M KCl (1:4 w/v soil/KCl), shaken for 1\u00a0h at 100\u00a0rpm, and filtered (round filter \u00f8 70\u00a0mm S&S type 595). After the first extraction, the filter papers containing the soil and the biochar particles were placed in new extraction bottles and another 80\u00a0mL of fresh 2\u00a0M KCl solution was added. This time, samples were shaken in a hot-water bath (80\u00a0\u00b0C) for 24\u00a0h to extract potentially trapped nitrate from the biochar particles27. Again, the extractant was collected by filtering the extraction solution. The concentration of mineral N (NO3\u2212 and NH4+) was quantified colorimetrically using an auto-analyzer . Unfortunately, we were unable to extract the soil after the ryegrass harvest due to difficulties to remove the very fine roots properly.After the harvest of quinoa, the quartz sand at the top surface of each pot was removed and plant roots were gently recovered. Fresh soil samples were sieved (2\u00a0mm) and extracted twice for N.29 and Archanjo et al.47.To determine where and what type of nitrogen is existing in the fresh and field-aged biochar samples were analyzed using X-ray Proton spectroscopy (XPS), FEI corporation NanoSEM 230 scanning electron microscope (SEM) and JOEL ARM transmission electron microscope. Details of the techniques and equipment are given in Joseph et alTo determination the dissolved organic carbon from biochar samples, 1\u00a0g of fresh and field-aged biochar samples were finely ground and added to 10\u00a0mL distilled water. The solutions were regularly stirred at 50\u00a0\u00b0C for 24\u00a0h and subsequently, filtered to separate the solid and liquid phases [first extraction (1Et)]. The pH value was adjusted before measuring the DOC content from the samples. Liquid Chromatography\u2014Organic Carbon Detection (LC\u2013OCD) was performed on the solutions and results were analyzed using customized software .To measure the concentration of DOC in the second extraction (2Et), 10\u00a0mL distilled water was added to the solid phase obtained from the first extraction . Likewise, 10\u00a0mL of distilled water was added to the solid phase from the second extraction  to obtain third extraction (3Et). The concentration of TC/TN was also measured in each step using Multi N/C Analyser.P\u2009\u2264\u20090.05. Data were tested for normality and homogeneous variances with the Shapiro\u2013Wilk test and Brown-Forsythe test, respectively. Where needed, data were transformed  before statistical analysis. The Tukey\u2019s Honest Significant Difference (HSD) test was carried out to identify treatment level effects such as N application dose or N source  at the individual treatment levels (P\u2009\u2264\u20090.05).Results were analyzed separately for both plant species  because of their different growth habits. We employed two-way ANOVA for both data sets by using SigmaPlot 13.0  at a significance level of Supplementary Information."}
+{"text": "There is an urgent need to search for new sorbents of pollutants presently delivered to the environment. Recently biochar has received much attention as a low-cost, highly effective heavy metal adsorbent. Biochar has been identified as an efficient material for cobalt (Co) immobilization from waters; however, little is known about the role of Co immobilization in soil. Hence, in this study, a batch experiment and a long-term incubation experiment with biochar application to multi-contaminated soil with distinct properties  were conducted to provide a brief explanation of the potential mechanisms of Co (II) sorption on wheat straw biochar and to describe additional processes that modify material efficiency for metal sorption in soil. The soil treatments with 5% (v/w) wheat straw biochar proved to be efficient in reducing Co mobility and bioavailability. The mechanism of these processes could be related to direct and indirect effects of biochar incorporation into soil. The FT-IR analysis confirmed that hydroxyl and carboxyl groups present on the biochar surface played a dominant role in Co (II) surface complexation. The combined effect of pH, metal complexation capacity, and the presence of Fe and Mn oxides added to wheat straw biochar resulted in an effective reduction of soluble Co (II), showing high efficiency of this material for cobalt sorption in contaminated soils.  In recent decades, industry\u2019s reliance on cobalt as a material essential for enabling technological development has caused considerable growth in the use of cobalt and accidental release of this metal into the environment. Metal ore mining and the smelting process , alloys and chemicals containing cobalt (Co), sewage effluents, and urban and agricultural runoff (phosphate fertilizers and pesticides)  have bee2+, Mg2+, K+, Na+, P, NH4+, NO3\u2212), and the total contents of trace elements  were determined to describe the properties of the material. The total surface area was determined using a BET  specific surface area analyzer Gemini VII 2390 Series . The cation exchange capacity and exchangeable cations  were determined according to the modified method described by Munera-Echeverri et al. [3) solution at a pH of 8.5 had a similar pH to WSBC. This extractant decreased calcium in solution , and this decrease enhanced the dissolution of Ca-phosphates. The nitrate content was analyzed according to the ISO 14256-1:2003 procedure on a UV-Vis Cary 60 . The pH values were measured at a ratio of 1:5 (w/v) in deionized water after the sample was shaken for 1 h at 130 rpm with a calibration check pH meter . The ash content was determined by weight loss after combustion at 750 \u00b0C for 6 h in a muffle furnace according to ASTM D7348-13 [3 was determined following the Scheibler method with a calcimeter [Biochar was produced from wheat straw (WSBC) at the pyrolysis temperature of 550 \u00b0C and time remaining in the reactor 60 s. The BET surface area, cation exchange capacity (CEC), pH in deionized water, CNHSO elemental composition, ash and carbonates content (% volume/dry weight), exchangeable cations and anions content  at afforested sites at different distances and locations from the copper smelter in SW Poland , expecting cobalt enrichment from smelter emissions. In the samples, pre-scanning studies for a wide range of heavy metals, including cobalt, were done on a microwave plasma-atomic emission spectrometer MP-AES 4200  after microwave sample digestion in 70% nitric acid (1:10 w/v ratio). The total concentration of cobalt remained in a wide range from 4.5 to 74 mg/kg, depending on the distance from the smelter and the soil type. Two soils with the highest Co concentrations, differing in texture, were chosen for the incubation experiment. Large soil samples, 15 kg each, were collected of Cutanic Luvisol and Fulvic Brunic Arenosol according to the FAO (Food and Agriculture Organization of the United Nations) guidelines  at a disAfter soils were incubation with 5% (w/v) wheat straw biochar, the (Community Bureau of Reference (BCR) sequential extraction procedure was applied to measure the following four fractions of cobalt in the tested soil: exchangeable and bound to carbonates (Fraction 1), reducible or bound to Fe and Mn-oxides (Fraction 2), oxidizable or bound to organic substances (Fraction 3), and residual (Fraction 4). Acetic acid, hydroxyl ammonium chloride, hydrogen peroxide plus ammonium acetate, and aqua regia stages of the sequential extraction procedure were applied to the soil samples, respectively ,44,45. Tt-tests were used to test for significant differences in cobalt fractions between biochar treated and untreated soils (p < 0.05). The obtained data were compiled using Microsoft Excel 2016 and Statistica Statsoft 13.3.The metal sorption batch experiments were performed in triplicate and the soil sorption experiments were performed in six replicates. The data are presented as the mean values with the relative standard deviation (RSD). Student\u2019s The elemental composition of the tested wheat straw biochar is presented in 2, with the cation exchange capacity (CEC) of 63 cmolc/kg. The total ash content was 32.4% and the contribution of calcium carbonates was 3.07% (w/dw), however almost 90% of exchangeable cations in CEC was a K+, followed by Ca2+, Mg2+, and a very low content of Na+ and NH4+  groups. This change confirms that the O-H groups take part in Co (II) and Cu (II) complexation on wheat straw biochar surface. The carboxyl peak observed for a pure wheat straw biochar at 1624 cm\u22121 was shifted to much smaller values, i.e., 1583 cm\u22121 or 1570 cm\u22121 in the spectra of BC treated with salts , Cu (II), and mix Co (II) + Cu (II) biochars . From thth salts .\u22121, characteristic for C=O carboxylic group, can be explained by the interaction with Co (II) and Cu (II) ions with free carboxyl groups on the biochar surface and change to carboxylates, which indicates the important role of carboxyl groups on the biochar surface in metal binding. To provide more details about the type of the metal binding to carboxylic group on the biochar surface at 1624 and 1420 cm\u22121, a calculation of \u0394 according to Nakamoto [The decrease in the wavenumber of the peak 1624 cmNakamoto  was perf2O or OH groups. The results of the FT-IR analysis also showed that metal ions complexation could be related to an abundance of carbonates and polysaccharides such as moieties in biochar, since changes of peak at 1080 cm\u22121 related to Si\u2013O, C\u2013O, and S=O groups were observed in metal spiked biochar. Very low concentrations of sulfur in biochar , and bridging . The cal biochar  can limirmations .Wheat straw biochar was applied to soils to verify the hypothesis that application of this material can immobilized cobalt ions in soil. Copper fractionation for tested soils was described in the previous paper  and is nThe application of 5% (w/v) wheat straw biochar affected cobalt speciation in both tested soils. In sand + 5% WSBC treatment, a significant decrease in exchangeable cobalt Fraction F1 (F1) from 17.4% to 7.3% was determined. The observed reduction in Co content in F1 was mainly balanced by their increased content in Fraction 2 (F2), reducible or bound to Fe and Mn-oxides  by 9.4%.The application of SEM-EDX proved that the metal ions can be bound on the biochar surface. Examination of the SEM-EDX element map, 2/g, depending on pyrolysis (slow or fast), although, in most research, lower values can be found with good efficiencies for heavy metal removal [Biochar application can have a direct or indirect impact on cobalt immobilization/mobilization process in soil. Indirectly, biochar can affect soil sorption properties and pH, reducing the presence of metal in exchangeable and soluble forms in soil solution. The direct effect can be related to biochar properties such as sorption capacity, oxygen functional groups, and mineral components content  increasing or supporting soil sorption capacity for cobalt. All mentioned processes were considered in our study. The tested wheat straw biochar had high SSA as compared with other straw-derived biochars produced under similar conditions, as porosity of biochar significantly increases between 400\u2013600 \u00b0C . Gul et  removal ,53. Cati removal . Wheat s removal . The whe removal ,54. Bioc removal , modifyi removal ,19,55. O removal . Wheat s removal . In othe removal ,58, simi removal ,60. As o removal . Similar removal . Sun et  removal  showed t removal , suggest removal . Zhang e removal  observed removal . These f2+ removal as compared with materials produced at high temperatures with a more aromatic structure. However, biochar applied to soil vs. solution undergoes many abiotic and biotic processes causing sorbent oxidation, called biochar aging [As oxygen-containing functional groups are predominant mechanisms of divalent cations the result of our study suggests that biochars produced at lower temperatures or oxidized during pretreatments  have betar aging , which car aging  and genear aging . Uchimiyar aging  describear aging  observedp > 0.05) in biochar treated soils. The Mn and Fe oxides and organometallic moieties such as Fe\u2013O\u2013C can be formed on the biochar surface during pyrolysis. Most of the Fe in biochar is present in crystalline phases ranging from zerovalent iron to ferric oxides [2 which could explain the high stability of Co forms after WSBC application to loam soil. Pan et al. [Findings of our study showed that wheat straw biochar can support soil sorption complex acting as a source of several mineral components, for example, Fe and Mn oxides ,70, silic oxides . The rolc oxides ,75,76,77c oxides . Kabata-c oxides  describen et al.  describeThe results of our study showed that wheat straw biochar has good removal efficiencies in single-metal systems determined in a batch experiment with cobalt salts in the solution. However, the capacities of biochar for cobalt sorption can be modified in multiple-metal systems due to the competition between the heavy metals present in soil and lower stability of the material due to cation exchange and the surface oxidation process that biochar undergoes under soil conditions. As biochar can affect soil properties, changing conditions of metal immobilization, and as soil conditions can have an impact on surface properties of biochar, predictions about biochar efficiency for metal sorption in soil are difficult and need further recognition. 2+, decreasing mobility and availability of this element in soil.The results of the experiments showed that wheat straw biochar is an efficient sorbent of CoThe dominant mechanisms of cobalt sorption on biochar surface are related to oxygen functional groups present on the biochar surface, mainly carboxylic and hydroxyl groups.Biochar can support soil sorption complex with mineral components such as Fe and Mn oxides which increase efficiency of cobalt immobilization.The efficiency of cobalt removal from soil can be modified by biochar oxidation and interactions with soil constituents which makes the process more complex as compared with cobalt removal from aqueous solutions.The results from this study suggest that application of biochar is a feasible strategy for remediation of cobalt contaminated soils, reducing health risks related to human exposure to Co from anthropogenic sources.Further and more complex studies are necessary to recognize the problems of cobalt soil pollution, potential risks, and remediation solutions, including biochar application."}
+{"text": "Saccharum officinarum L.) cultivation leaves behind around 20\u00a0t\u00a0ha\u22121 of biomass residue after harvest and processing. We investigated the potential for sequestering carbon (C) in soil with these residues by partially converting them into biochar . First, we modified the RothC model to allow changes in soil C arising from additions of sugarcane-derived biochar. Second, we evaluated the modified model against published field data, and found satisfactory agreement between observed and predicted soil C accumulation. Third, we used the model to explore the potential for soil C sequestration with sugarcane biochar in S\u00e3o Paulo State, Brazil. The results show a potential increase in soil C stocks by 2.35\u2009\u00b1\u20090.4 t C ha\u22121\u00a0year\u22121 in sugarcane fields across the State at application rates of 4.2 t biochar ha\u22121\u00a0year\u22121. Scaling to the total sugarcane area of the State, this would be 50 Mt of CO2 equivalent year\u22121, which is 31% of the CO2 equivalent emissions attributed to the State in 2016. Future research should (a) further validate the model with field experiments; (b) make a full life cycle assessment of the potential for greenhouse gas mitigation, including additional effects of biochar applications on greenhouse gas balances.Sugarcane ( Saccharum officinarum L.) is the world's largest crop by production quantity, with a total of 1.8 billion tonnes of cane produced globally per annum in more than 90 countries1. Sugarcane fields were traditionally burned to facilitate manual harvest2. However, to avoid air pollution, in many countries the fields are now mostly left unburned and harvested mechanically. This \u2018green harvesting\u2019 leaves large quantities of biomass (hereafter referred as \u2018trash\u2019) in the field3. Although trash provides a mulch that can benefit soil fertility and the growth of subsequent crops, it can also increase the risk of fire, pest proliferation, and reduced soil warming and drying in the spring4. Currently it is typical for all the trash to be left on the field, although studies into sustainable rates of removal have been made5. A potential alternative use of the trash is for energy generation, substituting for fossil fuels7. Another option is to make biochar, which potentially provides greenhouse gas (GHG) removal as well as returning carbon (C) and nutrients to the soil9. It is also argued that biochar has additional GHG abatement potential through effects on crop production, including reduced requirement for manufactured fertilizer7.Sugarcane (11. The effects on existing soil organic carbon (SOC) may lead to increased SOC mineralization (\u2018positive priming\u2019) or decreased mineralization (\u2018negative priming\u2019)13. Studies report negative, positive and no priming effect14, sometimes with a change in the direction of priming over time, typically from increased SOC mineralisation in the first year or so, to decreased mineralization thereafter12. In a recent meta-analysis, Wang et al. (2016) found a wide range of priming effects depending on biochar and soil characteristics; but they found consistently large priming effects in low-fertility sandy soils which are typical of many sugarcane areas.Predicting the potential of biochar for these purposes requires allowance for the wide range of biochar types that can be created, and the variable effects of soil conditions on biochar decomposition, and vice versa. The properties of biochar vary according to pyrolysis conditions and other manufacturing parameters as well as the nature of the biomass \u2018feedstock\u201915. Dil and Oelbermann adapted the CENTURY model to evaluate the long-term effect of biochar by representing it as 95% lignin added to the \u2018slow C pool\u2019 in CENTURY16. However, this pool has a turnover time of 10 to 50\u00a0year17 which is at least an order of magnitude faster than typical biochar turnover18. Archontoulis et al. developed a biochar sub-model for the Agricultural Production System Simulator (APSIM), and its simulations compared favourably with some experimental observations, but it lacked wider calibration and validation19. Lychuk et al. developed a sub-model to integrate biochar into the Environmental Policy Integrated Climate (EPIC) model by allowing for the effect of biochar on the initial soil properties represented in the model, but not explicitly for biochar turnover20. Mondini et al. modified the RothC SOC model to better describe the decomposition of exogenous organic matter, such as biochar, but without specific modifications for biochar21. Overall, none of the existing models of biochar turnover in soils is suitable for our purposes, as these models were not optimized for biochar-amended soils.Attempts to model long-term increases in SOC following biochar application have relied on data from short-term studies2; and the combined model predicts long-term changes in SOC as a function of biochar properties and soil, cropping and environmental conditions. We evaluate the model against published data and use it to make predictions for\u00a0S\u00e3o Paulo State in Brazil using available data on sugarcane production and relevant soil and climate conditions. S\u00e3o Paulo is a suitable case study because Brazil accounts for 40% of global sugarcane production and S\u00e3o Paulo accounts for 55% of the national production22 with 96% of its sugarcane fields now mechanically harvested23. We compare three scenarios in which either 100%, 50% or 25% of the available sugarcane residues are used to produce biochar, with the remaining residues added to the fields as fresh material. Thereby we provide the first assessment of the carbon capture potential of sugarcane biochar at a regional scale, accounting for different climatic and soil characteristics.In this paper, we develop and test a biochar sub-model for the RothC model with which to assess the potential for excess sugarcane trash and the bagasse residues to produce biochar for in-field soil C sequestration. The sub-model divides the biochar C into fresh plant material, which is fed into RothC, and recalcitrant material, which slowly decomposes to CO24 is presented in Fig.\u00a024. Our model reaches a loss of 4% of BC-C after 5\u00a0years without leaching and 12% with leaching, with no modelled interaction with application rate.Comparison of simulated values against the experimental observations from Liu et al.19 to validate their APSIM biochar sub-model (Supplementary Information). The evaluation results using that prior dataset19  and the dataset used by Archontoulis et al.25. The good level of fit provides some confidence in the model predictions.The extent of this evaluation exercise is limited but we consider it the best available at the current time, owing to the general paucity of long-term data. The intensive investigation of biochar is rather recent compared to the heritage of long-established field experiments26. This region is characterized by a clayey soil  and mild climate 30.Figure\u00a0Under Scenario 1 Fig.\u00a0B\u2013D, therFigure\u00a02 removal.Including biochar-induced priming reduces the predicted increases in SOC Fig.\u00a0. Since p31 of sugarcane fields in S\u00e3o Paulo State , or 31% of the 159 Mt CO2e emissions attributed to the State in 201632. On the other hand, the least promising scenario  could sequester 2.5 Mt of C per year over the State (9.1 Mt of CO2e), or 6% of the 159 Mt CO2e attributed to the State in 201632. These numbers indicate the sequestration potential of biochar application on the sugarcane fields of S\u00e3o Paulo. As far as we are aware, there are no long-term field experiments on the C sequestration potential of sugarcane biochar in Brazil or elsewhere, with which to test our model predictions. This needs to be done in future.These three scenarios result in a wide range of potential C sequestration over all 5.77\u00a0Mha39. In general, yield responses are smaller for perennial crops, such as sugarcane, compared to annual crops38. In any case, the management of sugarcane plantations in S\u00e3o Paulo is such that yields are already apparently optimized and there is little room left for improvement. According to the IBGE (Instituto Brasileiro de Geografia e Estat\u00edstica), yields stabilized around 200740.We have not allowed for possible increases in sugarcane yield over time with biochar incorporation. The literature reports wide ranges in yield effects of biochar depending on crop type, soil conditions, climate, and biochar characteristics2O emissions during nitrification and denitrification in soils, at least in the first year following application42. Meta-analyses of field studies indicate 28%\u2009\u00b1\u200916% lower N2O emissions with various types of biochar application43, but the wide range of results indicate this is one of the most uncertain components of the GHG balance6.Biochar addition may affect N4 emissions from soils, particularly if flooded or acidic or both44. However, biochar applied to unflooded neutral or alkaline soils can increase CH4 emissions. Taking landscape diversity and CH4 uptake (oxidation) into account, it has also been suggested that biochar does not affect net CH4 release45.Similar analysis suggest biochar may decrease CH41, reduced fertilizer requirement due to the phosphorus and potassium available in the applied biochar46 and reduced nitrogen leaching losses47.Other biochar aspects that potentially influence the GHG balance include the potential reduced need for irrigation due to improved soil water holding capacity with biocharWhile the model provides an efficient way to predict potential increases in SOC stocks in sugarcane fields following biochar addition, this constitutes only part of the GHG balance of the overall practice. Emissions during biochar production, transport and application and those discussed above should be considered in a full Life Cycle Assessment of the integration of biochar into sugarcane systems, so as to provide a more accurate figure for the carbon sequestration potential.\u22121\u00a0year\u22121 is left on the field to assist active cycling of organic matter50. We assumed that trash amounted to 140\u00a0kg DM per tonne of harvested cane51, in agreement with previous work5. Likewise, we assumed that the potentially available bagasse (pith and rind from cane progressing) amounted to 140\u00a0kg DM per tonne of harvest cane52. We used these data to determine the input of fresh C to the field for three different scenarios and a baseline  is divided between two pools of differing decomposability (DPM and RPM) which decompose to two SOC pools of differing decomposability (BIO and HUM), which then inter-convert  is treated as fresh plant material and added to FPM pool in RothC (flux IB\u2009=\u2009\u03b1IB). The remaining, recalcitrant material (RBC) decomposes very slowly releasing CO2. The products of such slow decomposition will have a minor impact on soil C pools (BIO ad HUM) and can be neglected for simplicity. HenceCRBC is the concentration of RBC and kRBC is its decomposition rate constant.In the biochar sub-model, a proportion, 13 and which was consistent with previous assessments55. Based on a sugarcane biochar specific incubation study, we assumed that the remaining fraction would decompose at a rate of 11.9% over 100\u00a0year18, i.e. mean residence time\u2009=\u2009840\u00a0year, consistent with 560\u2009\u00b1\u2009480\u00a0year calculated by Wang et al.13. Hence kRBC\u2009=\u20090.00119\u00a0year\u22121. This k value is consistent with recent IPCC guidelines for estimating the change in SOC stock in mineral soils from biochar amendment56.We nominally parameterised the coupled models for biochar produced by slow pyrolysis of bagasse and trash at 550\u00a0\u00b0C. Lacking sufficient specific data we considered 3% of the applied biochar C as the portion reflecting the degradability of fresh plant material , based on a recent meta-analysis of all biochar types13 meta-analysis, which reported a mean 21% increase in SOC turnover with biochar applications to sandy soils; and (b) by 91% based on increased sucrose turnover with sugarcane bagasse biochar in an incubation experiment with a simulated soil57. We consider the latter to indicate the maximum possible positive priming effect of biochar, since most SOC is highly stabilised and unlikely to respond as the labile material. Since published meta-analyses encompass short-term study of large doses and single additions, our permanent increase of C turnover across all pools is highly conservative with respect to SOC.We explored the sensitivity of the model to positive priming of SOC turnover by biochar by increasing the RothC rate constants of all pools except RBC by (a) 21% based on Wang et al.\u2019s58, coded in R59. We amended the SoilR code to obtain output that better corresponded to the original RothC model for its own calibration data (see Supplementary Information). At steady state, before any biochar application, the \u2018inert\u2019 C stock for S\u00e3o Paulo soils was calculated according to Falloon et al. (1998)60. Model results are presented using ArcGIS 10.5.1.61. To smoothen the values between climate stations, we interpolated the meteorological data using inverse distance weighting, which uses the distance of known points to unknown points to estimates their values62. The graphical representation of the results were created using R package \u2018ggplot2\u201963 from R software version (3.5.1)59.We used the RothC model from the package \u2018SoilR\u2019\u22121\u00a0year\u22121 (7 t DM ha\u22121\u00a0year\u22121) is left on the field. The remaining trash is supplied to a combined heat and power plant, along with 100% of the bagasse. The total fresh organic C input to the field is 6.57 t C ha\u22121\u00a0year\u22121, since the C from trash is supplemented by below ground inputs  as well as the application of 100% vinasse and filter cake  are supplied to the pyrolysis plant, which yields 2.46 t biochar C ha\u22121\u00a0year\u22121 to be returned to the sugarcane fields . These are repeated annual applications. The other inputs are unchanged. In Scenario 2, only half of the potential biochar production from trash and bagasse is realised (i.e. 1.23 t biochar C ha\u22121\u00a0year\u22121\u2014from 60% of the available bagasse). The remainder is left in (or returned to) the field, increasing fresh organic C input from 6.57 in Baseline and Scenario 1 to 9.73 t C ha\u22121\u00a0year\u22121. In Scenario 3, the maximum potential biochar production is further diminished to one-quarter of the potential, amounting to 0.62 t biochar C ha\u22121\u00a0year\u22121 (from 30% of the available bagasse). The remainder is left in (or returned to) the field, increasing fresh organic C input to 11.1 t C ha\u22121\u00a0year\u22121.In Scenario 1, 1.29 and 4.62 t C ha65. Scenarios 1, 2 and 3 are run for 5, 10, and 20\u00a0year, starting at the steady-state (baseline) C stock. A maximum of 20\u00a0year was chosen to match the higher experimental biochar additions tested in any cropping system66.All the scenarios include a loss of 5% of the trash dry matter during the stalk collection and transport68. The relevance of the coupled biochar sub-model is not diminished relative to RothC, as it contains parameters derived from meta-analysis and reflecting all published biochar research, so although it would have been ideal to calibrate against sugarcane crop data, it was not essential. Since there are to our knowledge no published data on long-term field experiments on biochar in sugarcane, we instead used data from a wheat\u2013maize study in China24. In this study 0, 1.16, 3.48 or 5.79 t of rice straw biochar C ha\u22121 were applied to both the wheat and following rice crop in each year. This study is useful as it is both long-term and involves repeated additions of biochar to all land, representative of our modelled scenarios. Data required for the model evaluation are reported in the Supplementary Information  after one year73, depending on the biochar particle size69, rainfall amount69 and soil texture and porosity70. Biochar movement to subsoil layers or beyond will not reduce net C sequestration and may even increase it compared with more-transient storage in the topsoil75, but could introduce errors in field experiments with limited depth of sampling. For the purpose of model evaluation by deriving a relation between percentage downward loss and soil texture using data reported in the literature73, a probable loss could be derived for sandy loam soil of the wheat\u2013maize experiment used in calibration data24. The value was 5.18% of the applied biochar per year.The biochar literature suggests that some biochar is transported after topsoil application to sub-soil layers by rainfall (infiltration) and/or bioturbationSupplementary Information 1."}
+{"text": "This study aimed on the increasing nitrogen use efficiency (NUE) of maize via the use of high temperature produced biochar (700\u00a0\u00b0C). Maize was grown to maturity on two contrasting soils  in pots with a treatment of biochar co-applied with ammonium sulphate stabilised by a nitrification inhibitor  or un-stabilised. The combination of biochar with ammonium sulphate containing DMPP increased maize biomass yield up to 14%, N uptake up to 34% and NUE up to 13.7% compared to the sole application of ammonium sulphate containing DMPP. However, the combination of biochar with un-stabilised ammonium sulphate (without DMPP) had a soil-specific influence and increased maize biomass only by 3.8%, N uptake by 27% and NUE by 11% only in acidic Cambisol. Further, the biochar was able to increase the uptake of phosphorus (P) and potassium (K) in both stabilised and un-stabilised treatments of ammonium sulphate. Generally, this study demonstrated a superior effect from the combined application of biochar with ammonium sulphate containing DMPP, which improved NUE, uptake of P, K and increased maize biomass yield. Such a combination may lead to higher efficiency of fertilisation practices and reduce the amount of N fertiliser to be applied. From N sources, plants can take up N mainly in the form of ammonium (N\u2013NH4+) and nitrate (N\u2013NO3\u2212). However, their availability in soils is limited and accounts for only 2% of the total soil N content. Due to the growing demand in crop production, the use of inorganic N fertilisers has dramatically increased over the past 50\u00a0years2, resulting in crop yields enhanced by 30\u201350%3. However, overall nitrogen use efficiency (NUE) of applied fertilisers by cereals is typically ranging from 30 to 50%4.Nitrogen (N) is usually the most growth-limiting nutrient of crops, so crop production is highly dependent on the N soil supply capacity5 and fertilisers are applied in an excessive amount to cope with the low NUE. This excessive use of inorganic fertilisers causes numerous problems related to soil chemistry, root growth, losses of nitrogen and may result in soil degradation. Low NUE is mainly caused by the decline in the availability of nitrate and ammonium at the later stages of crop growth due to the losses of these species through volatilisation, denitrification and leaching6. Recently, N fertilisers containing nitrification inhibitors are in use to reduce the fast oxidation of NH4+ and its subsequent leaching to reduce the N losses from soil9. Biochar (BC) is among the materials often cited as the effective soil additive being able to induce N sorption and reduce losses12. Moreover, BC application has been recommended for the restoration of degraded and acidified soils due to the excessive use of N fertilisers13. BC is also known to increase soil pH14, increase the soil content of base cations  and increase the cation exchange capacity (CEC) of soils18. The reduction of both NH3 volatilisation and NO3\u2212 leaching due to the adsorption effect of BC has also been reported21. This effect of BC arises from the alkaline components of BC, including ash and carbonates of Ca2+, Mg2+ and K+23, unique physical properties (high porosity and surface area) and chemical properties (negatively and positively charged surface)24. Ammonium sulphate can be more efficiently combined with biochar than other N forms of fertilizers to improve NUE. Urea-N is available to plants after the hydrolysis, increasing soil pH25, so its co-application with biochar could further increase soil pH28 making nutrients like phosphorous less available. Fertilizers containing both ammonia and nitrate forms are less effective to regulate N soil transformation25.Such low NUE leads to environmental issuesHence, we hypothesised that soil application of biochar in the combination with ammonium sulphate (AS) stabilised with 3,4-dimethylpyrazole-phosphate (DMPP) could increase the nitrogen use efficiency (NUE) and yield of maize biomass. Further, this study aimed to elucidate the mechanisms and chemical changes caused by the co-application of these materials.29, where an identical biochar (wood chips pyrolysed at 700\u00a0\u00b0C) was used. The relatively higher biochar production temperature (700\u00a0\u00b0C) was preferred due to the need for producing relatively stable biochar with relatively lower ammonium and a higher nitrate sorption capacity. Out of the ten soils used in the previous studies, two contrasting soils were chosen: (1) Chernozem  a soil characterised by a neutral pH and a decline in the concentration of exchangeable Ca and cation exchange capacity (CEC) after the application of BC and (2) Cambisol  soil was selected for its acidic pH and an increase in the concentration of exchangeable Ca and CEC after BC application. Detailed characteristics of the soils and BC are presented in Table Two soils with desired properties were selected based on our previously published work chosen:  ChernozeThe pot experiment was set up using 5\u00a0kg (dry weight) soil in 6-L pots in a precipitation-controlled vegetation hall. Nine treatments were set up Table  to achie\u22121 of soil and corresponded roughly with the N application rate of 600\u00a0kg\u00a0N\u00a0ha\u22121 in field conditions. In this study, stabilised ammonium sulphate (SAS) was bought from COMPO EXPERT GmbH (Germany) with the product trade name NovaTec Solub 21 having  and 21% N). The corresponding un-stabilised ammonium sulphate (USAS) treatment was fertilised using ammonium sulphate ((NH4)2SO4; 21% N) from the AGRO CS Group . Fertilisers were applied in the form of powder and were thoroughly mixed with soil. After preparing all the treatments, five maize seeds were sown per pot and thinned to three plants per pot two weeks after sowing. Each pot was regularly irrigated to 60% of the soil maximum water holding capacity. Soil solution was collected over the vegetation period using Rhizon MOM suction cups as described by Refs.31. Maize aboveground biomass was harvested 115\u00a0days after sowing, oven-dried (65\u00a0\u00b0C) and ground to a fine powder before analyses. After the harvest, soil samples were collected and analysed for the available content of mineral N.The nitrogen fertilisation rate represented 207\u00a0mg\u00a0N\u00a0kg2 (w/v\u2009=\u20091/5). The available content of nutrients in both soil and biochar were determined by the use of inductive coupled plasma-optical emission spectrometry  after the extraction of samples with 0.01\u00a0M CaCl2 in 1:10 (w/v) for 2\u00a0h32. The available content of inorganic N (nitrate and ammonium nitrogen) were measured by the Skalar San Plus System continuous flow segmented analyser  after extraction of samples with 0.01\u00a0M CaCl2 (w/v\u2009=\u20091/10) for 2\u00a0h33. The total content of C and N were determined by the use of a CHNS elemental analyser . The total organic carbon (TOC) was determined according to Sims et al.34, i.e. spectrophotometrically following the oxidation of organic matter (OM) with K2Cr2O7. Determination of cation exchange capacity was done according to Gillman et al.35 by a three-step saturation of samples (1\u00a0h for each agitation) with 0.1\u00a0M BaCl2 solution and collecting the extracts for determination of exchangeable cations  by ICP-OES. The pellet remaining after extraction was used for subsequent release of Ba2+, where it was agitated with 0.02\u00a0M MgSO4 for two hours, and then CEC was calculated based on the amount of Mg2+ retained by the soil or biochar. The pseudo-total contents of elements  in both soils and biochar were determined by ICP\u2013OES after microwave assisted aqua regia extraction36.The measurements of soil and biochar pH were done using an Argus pH meter  with a transistor CupFET probe after the extraction of samples with 0.01\u00a0M CaCl3\u00a0 and H2O2  in an Ethos 1 microwave-assisted wet-digestion system , and P, S, Mg and Ca concentrations in the digests were determined by ICP-OES. The concentrations of K were determined using flame atomic absorption spectrometry . The total concentrations of N in maize tissue were determined by the Kjeldahl method .The concentration of nutrients in maize biomass was determined after the digestion of plant samples with concentrated HNOp\u2009<\u20090.05 followed by the Tukey test to assess the effect of the individual treatments. The interactions of the variables  on maize biomass and yield component were analysed by a multivariate analysis of variance, MANOVA. The repeated measure analysis of variance rANOVA was implemented to describe the within-subject effect of sampling time and between-subject effect of soil, biochar, fertiliser and their interaction on the pH and nutrient content of soil solution. The uptake of nutrients (mg per pot) by maize was calculated as Eq.\u00a0 at NFT is the N or S uptake in fertilised treatment, NCT is the N or S uptake in corresponding control, non-fertilised treatment, and Nap is the amount of N or S applied in a pot in the form of ammonium sulphate.Nitrogen use efficiency (NUE) and sulphur use efficiency (SUE) was calculated according to Eq.\u00a0.2\\documep\u2009>\u20090.001), followed by type of soil  and the application of biochar , see supporting information (SI p\u2009=\u20090.001) effect.The biomass yield in the SAS and USAS treatments was up to 5 times higher than in the NoAS treatments. The effect of biochar on maize biomass was not significant in the case of NoAS and USAS treatments Fig.\u00a0. Howeveration SI . The intp\u2009=\u20090.05) increment of N uptake of 26% , then soil  and biochar  on the uptake of nitrogen. More interestingly, there was also a significant interaction effect for fertiliser and biochar , soil, biochar and fertiliser  and an interaction between soil and biochar   SI . The appp\u2009<\u20090.05) increments of P uptake by 58 and 54%, respectively on the neutral Chernozem and significant (p\u2009<\u20090.05) increments of 14 and 18% in the USAS and SAS treatments of the acidic Cambisol, respectively. The uptake of P for the 2% BC treatments of USAS and SAS was significantly higher for the Chernozem soil than for the corresponding treatments of Cambisol. The application of fertiliser had the highest effect  on the uptake of P (SI p\u2009<\u20090.001) and the interaction effect of soil with biochar , then the fertiliser with biochar . The interaction between soil, fertiliser and biochar also had a significant  effect.In both soils, the application of biochar without fertiliser was not able to induce any significant changes in P uptake Fig.\u00a0. However of P SI . It was p\u2009<\u20090.001) on the uptake of K, then fertiliser , biochar , the interaction of soil with fertiliser , fertiliser with biochar  and the interaction effect of soil, fertiliser and biochar  (SI p\u2009<\u20090.05) increment of K uptake in all treatments  of both soils  SI . The appp\u2009<\u20090.001) then soil type , biochar , the interaction of soil with fertiliser  and fertiliser with biochar  (SI p\u2009<\u20090.05) reduced the uptake of Ca in all treatments   at both the 1 and 2% application of biochar for the SAS and USAS, while it was significant only at the 2% biochar application rate at NoAS treatments. The declines for the 2% BC rate were 17, 26 and 27% for the NoAS, USAS and SAS, respectively. The uptakes of Ca in the Chernozem soil CON as well as the 1 and 2% BC of both the USAS and SAS were significantly higher than corresponding treatments of the Cambisol soil  SI . The app588, .001p\u2009<\u20090.001), then soil  and biochar , the interaction of soil with fertiliser  and fertiliser with biochar  (SI p\u2009=\u20090.05) decline in all treatments of both soils, while only 1% caused a decline in the SAS and USAS of Chernozem soil. The declines in Chernozem soil at the 2% biochar rate were 17, 27 and 17% for NoAS, USAS and SAS, respectively; while in the Cambisol, the declines were 19, 17 and 13 for the NoAS, USAS and SAS, respectively. Inversely, the uptakes of Mg in the Cambisol soil CON as well as 1 and 2% BC for both USAS and SAS were significantly higher than the corresponding treatments for Chernozem soil  SI . As that4, .001 Sp\u2009<\u20090.001) then soil  and the interaction of soil with biochar , biochar  and the interaction of fertiliser with biochar   SI . The appp\u2009<\u20090.001), then time of sampling   SI . The eff.001) SI . At the 3\u2013 N concentration was detected in the control treatment of USAS in the Chernozem and all USAS treatments of the Cambisol between 7 and 14 DAS, while in the remaining treatments, the concentration of NO3\u2013 N was rather decreasing over time . The concentration of NH4+\u2013N in soil solution was significantly affected by soil, biochar, fertiliser, the period of sampling and the interaction of soil with biochar .The concentration of P was significantly higher in the Chernozem soil as compared to the Cambisol Fig.\u00a0. Only thh DAS SI . All theh DAS SI . The grep\u2009<\u20090.001 (SI p\u2009<\u20090.001. The concentration of Ca showed a decline over time except for a significant increment in control treatment of USAS Ca from 959\u00a0mg\u00a0L\u22121 (7 DAS) to 1555\u00a0mg\u00a0L\u22121 (14 DAS) in the Chernozem  to 65\u00a0mg\u00a0L\u22121 (14 DAS)  SI .37. This output is in complete disagreement with the findings of Sheikhi et al.38, Fuertes-Mendiz\u00e1bal et al.39, and Keiblinger et al.40, where authors presented the negative interaction of DMPP with biochar.The highest maize aboveground biomass was achieved by the combination of 2% BC and stabilised ammonium sulphate (SAS) identically on both soils Fig.\u00a0. Moreove4+ oxidation by DMPP in soil with a higher proportion of clay is expected due to the sorption of DMPP by clay minerals and their reduced effect41, while the opposite is true in soils with a high proportion of sand and further prolongation by biochar is expected. Secondly, the nitrification inhibitory effect of DMPP is much higher in acidic soil as compared to alkaline soils42, which is again further prolonged by the application of biochar. This was noticeable for the USAS and SAS treatments of Cambisol at 7 and 14 DAS , whereas there was a significant decline in the content of NH4+\u2013N. This was not true in the case of the neutral Chernozem soil, which indicates that NH4+ was being slowly nitrified in the biochar treatments of acidic Cambisol, especially in the SAS and USAS treatments accompanied by NO3\u2013\u2013N availability even after the harvest of the maize, which is beneficial for the next cropping season.Other important finding is that the biochar-induced increment of NUE was higher in the acidic Cambisol fertilized by DMPP treated ammonium sulphate. This could indicate the better interaction of biochar with DMPP treated ammonium sulphate in the acidic soils as compared to neutral or alkaline soils. The first reason for the better joint effect of biochar with DMPP treated ammonium sulphate in the acidic Cambisol could be due to the acidic pH (4.8) and higher sand content of the Cambisol (26.1%) than the Chernozem (13.2%). The short delay of NHDAS Fig.\u00a0. This almaize SI . There wp\u2009=\u20090.05) correlated (r\u2009=\u20090.95) in the Chernozem soil and (r\u2009=\u20090.89) in Cambisol. This phenomenon could indicate that the co-precipitation of Ca and S resulted in the decline of both Ca and S in the biochar treatments of USAS and SAS. The increment in S adsorption and the formation of S-Ca precipitate in the Ca-rich condition is evident43. Biochar could also decrease the availability of S due to the sorption of SO42\u2212 by electrostatic interaction with the charged surface of biochar44. The decline in the content of sulphate by biochar application has been reported due to the formation of weakly soluble CaSO445.The uptake of S was higher in the SAS and USAS treatments of the Cambisol than that of the Chernozem. This is in agreement with the high content of available S in the Cambisol soil solution of the USAS and SAS treatments. The application of biochar induced a decline in the uptake of S in the SAS and USAS treatments. The application of biochar in the fertilised treatments reduced sulphur use efficiency up to 1.22%, meaning that there were always lower uptakes of S in the biochar treatments of SAS and USAS than in the controls in both soils. This is mainly due to the low availability of S in the soil solution of biochar treatments SI . The dec46. Again, the multivariate analysis (SI p\u2009<\u20090.001) compared to soil type. There was also a significant interaction effect of biochar and fertiliser  revealing the highest effect of biochar to reduce the uptake of S is in fertilised treatments, while it had insignificant effects in NoAS treatments.The content of S in the treatment without fertiliser was lower compared to ammonium sulphate treatments (SAS and USAS). This is mainly due to the release of sulphate from applied ammonium sulphate and the improved uptake of S in the soils having higher contents of N. The decrease of sulphur uptake in low N available conditions has been described by Clarkson et al.lysis SI  confirme\u22121 was able to increase maize P uptake47. The main reason for the increment of P uptake could be the biochar-induced weakening/inhibition of phosphate anions  adsorption by the Al/Fe (hydr)oxides of soils48. The adsorption of soil HPO42\u2212 or PO43\u2212 by Fe (hydr)oxides is expected to be lower at the relatively higher pH induced by biochar. This is due to the repulsion of negatively charged HPO42\u2212 and/or PO43\u2212 by the negatively charged surface sites of the ferrihydrite and as a result of OH\u2212 ion competition on the negatively charged sorption sites at the higher soil pH induced by biochar49. The increment of soil pH due to the release of Ca2+, Mg2+ and K+ from biochar could effectively reduce the solubility of reactive Al3+ oxides, and this could reduce the sorption of P in acidic soil50. The biochar-induced increment of soil pH is evident and significant especially in the SAS and USAS treatments of acidic Cambisol at least at the seventh DAS  and their subsequent replacement by the exchangeable Al3+ and H+ on the exchangeable sites of biochar as well as the binding of surplus H+ ion to the negatively charged  surface functional groups of biochar51. Thus, the better expression of pH increment in the SAS and USAS treatments of acidic Cambisol than the neutral Chernozem is simply due to the greater effectiveness of biochar to increase soil pH in soils having low pH, CEC and exchangeable Ca2+.The uptake of P was generally higher in the Chernozem soil than in the Cambisol due to the higher availability and total content of P in Chernozem soil Table  and a su+ to the soil solution by NH4+ from the applied ammonium sulphate  and extractable contents of K (2278\u00a0mg\u00a0kg\u22121) from the biochar used in this study compared to the Chernozem (65\u00a0mg\u00a0kg\u22121) and Cambisol (32\u00a0mg\u00a0kg\u22121) soils. Biochar could serve as a potential source of K, and this results in the subsequent increment of K uptake53. The release and improvement of K uptake by crops after biochar application have been previously reported55. Similarly, the improvement of K availability and the subsequent increment of K uptake by maize was reported after the application of 2% vineyard pruning biochar56.Based on the multivariate analysis of variance SI , the higphate SI . Based o57. The study of Horie et al.58 confirmed that the class II high-affinity potassium transporter (HKT) was involved in the transport of K, Ca and Mg, and hence preferentially transporting K over the divalent cations (Ca2+ and Mg2+), leading to the suppression of Mg and Ca uptake in K-rich environment. The highest effect of fertiliser 2SO4. Further biochar induced a decline of Ca content in the soil solution of the neutral Chernozem and an increment in the acidic Cambisol. Biochar is principally capable of increasing available Ca content in soils having lower original Ca content than the biochar used, while biochar could induce a decline of Ca content when added to soils having higher Ca contents than the biochar applied15. The neutral Chernozem had a much higher content of exchangeable Ca2+ (253\u00a0mmol\u00a0kg\u22121) than the acidic Cambisol (72\u00a0mmol\u00a0kg\u22121) and biochar (176\u00a0mmol\u00a0kg\u22121)  used in this study. Therefore, the weakly adsorbed NH4+ could slowly nitrify and become available for the plant uptake at the later crop growing stages. This directly goes with the intended use of DMPP, which slows the nitrification of NH4+. Some previously published works of other studies seem quite opposing to our finding in some ways, which is attributed only to the higher production temperature of biochar (700\u00a0\u00b0C) used in this study. For example, the adsorption of DMPP by lower temperature produced biochar was reported for the biochars pyrolyzed at 450\u00a0\u00b0C38, 500\u00a0\u00b0C39, 400\u00a0\u00b0C and 525\u00a0\u00b0C40. In those studies, the presence of low NH4+ concentration in the treatments containing DMPP with biochar seems holding back the intended use of DMPP to limit the process of nitrification. However, the low availability of NH4+ in soils where low temperature produced biochar was applied is expected due to the strong sorption of NH4+ by easily available negatively charged oxygen containing functional groups of low temperature produced biochar61. Further, the lower temperature produced biochar can adsorb DMPP. Fuertes-Mendiz\u00e1bal et al.39 reported the adsorption of DMPP driven by the oxygen containing functional groups, more specifically carboxyl groups, of the lower temperature produced biochar (500\u00a0\u00b0C) used in their study. This is in the agreement with the finding of Keiblinger et al.40, reported a greater adsorption of DMPP by the biochar produced at 400\u00a0\u00b0C than the biochar produced from the same feedstock at the higher temperature (525\u00a0\u00b0C). The occurrence of the phenomena (adsorption of NH4+ and DMPP by biochar) is expected to be very low in our study due to the loss of oxygen containing functional groups proportional with the rise in production temperature. The clear decline of oxygen containing functional groups with the rise in temperature is evident62. Therefore, the use of high temperature produced biochar is a choice for the overall better performance of biochar-DMPP combination.As discussed above, the positive impact of high temperature produced biochar co-application with DMPP treated ammonium sulphate fertilizer on the NUE and biomass of maize is mainly attributed due to the weak adsorption of NHThe interaction effect of biochar with ammonium sulphate containing DMPP (NovaTec Solub 21) on the biomass and yield component of maize was studied on two soils with contrasting properties. The outcome revealed the effectiveness of biochar co-application with ammonium sulphate containing DMPP to induce a significant increment of maize biomass as well as the uptake of N, P and K.Co-application of biochar with ammonium sulphate containing DMPP was able to increase maize biomass by 10%, nitrogen use efficiency by 13.7%, the uptake of P by 54%, and the uptake of K by 57% compared to a single application of ammonium sulphate containing DMPP. The interaction of biochar with ammonium sulphate containing DMPP was more effective to increase maize biomass, N uptake and K uptake in the acidic Cambisol, while P uptake increased in the neutral Chernozem. The application of biochar also induced a decline in the uptake of Ca and Mg because of the antagonistic effect of K. Additionally, biochar induced a decline of S uptake when co-applied with ammonium sulphate. In the case of un-stabilized ammonium sulphate, biochar was not able to induce a significant change in maize biomass, while there was an increment in N uptake only in the acidic Cambisol, an increment in the uptake of K in both soils and a decline in the uptake of Ca, Mg and S. Furthermore, the effect of biochar was also pronounced in the soil solution by increasing the concentrations of K, Mg in the soil solution of both soils, while there was an increment of Ca in the acidic Cambisol and a decline in the neutral Chernozem.Generally, the interaction effect of biochar on the maize biomass, NUE and uptake of N was much higher when combined with ammonium sulphate containing DMPP than its co-application with un-stabilized ammonium sulphate and a single application of both stabilised and un-stabilized ammonium sulphate. Hereafter, we conclude that the application of high temperature produced biochar with ammonium sulphate containing DMPP could increase crop yield and improve nitrogen use efficiency due to a greater extent by the reduction of nitrogen losses.Supplementary Information"}
+{"text": "Fenton-like system formed in a natural soil environment deemed to be significant in the aging process of biochar. Aged biochars have distinct physico-chemical and surface properties compared to non-aged biochar. The aged biochar proved to be useful soil amendment due to its improved elements contents and surface properties. The biochar aging process resulted in increased surface area and pore volume, as well as carbon and oxygen-containing functional groups  on its surface, which were also associated with the adsorption behavior of 2,4,6-trichlorophenol . The biochar aging increased the adsorption capacity of 2,4,6-TCP, which was maximum at pH 3.0. The 2,4,6-TCP adsorption capacity of aged-bush biochar (ABB) and aged-peanut shell biochar (APB) was increased by 1.0\u201311.0% and 7.4\u201338.8%, respectively compared with bush biochar (BB) and peanut shell biochar (PB) at the same initial concentration of 2,4,6-TCP. All biochars had similar 2,4,6-TCP desorption rates ranging from 33.2 to 73.3% at different sorption temperatures and times. The desorbed components were mainly 2,4,6-TCP and other degraded components, which were low in concentration with small molecule substance. The results indicated that the aged-biochar could be effective for the long-term remediation of naturally organic polluted soils. Organic pollution is usually affected by the physical and chemical properties of the sorbent on which sorption\u2013desorption reactions occur. Biochar properties  affect the infiltration of wastewater into soils which are crucial for protecting water resources and minimizing health hazards2.Trace organic compounds act as pollutants in waste water and pose a major threat to the human health and ecosystem security/safety3. Because of the specific surface characteristics , abundant microchannels and stable aromatic chemical structure 4, biochar has been widely used as an environmentally friendly amendment for the treatment of wastewater and soil pollution5. For example, wood chip biochar  had a high ability to remove 2,4\u2010dinitrotoluene (DNT) and 2,4\u2010dichlorophenol (DCP) from an aqueous solution6.Biochar is carbonaceous and porous material which is produced from waste biomass through pyrolysis technique7. The biochar has ability to decrease organic pollutant\u2019s bio-availability and transport through increased sorption and/or degradation8. The sorption strength of organic pollutants depends on physical and chemical properties of the biochar, which vary between different feedstock materials and aging time durations in amended soils10. Increasing adsorption capacity with aged biochar would decrease the available organic pollutants concentration, thereby limiting their activity and promoting biodegradation over time2. Biochar also decreases degradation of organic pollutants such as pesticides in soils, often attributed to increased pesticide sorption and decreased pesticide bioavailability. For example, Loganathan et al.11 found that biochar amended soils contained higher non-desorbable fractions of atrazine compared with control which contained more rapidly mineralizing atrazine.In the environment, biochar interacts with environmental constituents or factors and undergoes a series of biogeochemical reactions (i.e. aging process) that result in alteration of the biochar\u2019s surface properties over time12. Biochar in soil is oxidized with aging as it acts primarily as a reducing agent over a long period of time. In order to accelerate aging process, the simulating experiments had been done in the laboratory to assess the aging effects on the biochar properties13. The nano-iron/nickel modified biochar effectively removed up to 71.6% of 2,4,6-trichlorophenol  from the natural waste water treatment14. Zhuang et al.15 also reported that Fe3O4 activated-hyacinth biochar removed up to 98.9% of 2,4,6-trichlorophenol from waste water by enhancing fermentation performance. Biochar aging is also a natural process that alters biochar properties to immobilize the heavy metals pollutants in different environment13. In the natural aging process, wheat straw biochar significantly reduced bioavailability of heavy metals concentration in paddy soils16. The 12-month aging of the biochar increased the Cd adsorption capacity in the soil17.The biochar undergoes physical and chemical changes over time in soils, as a result of which its pore structure and particle size are changed, influencing its recalcitrant and pollutants sorption ability18. The modified poplar catkins biochar exhibited maximum adsorption capacities of 384\u00a0mg\u00a0g\u22121 for chloramphenicol in wastewater19. Moreover, Fenton-like oxidizing agents (H2O2 and NaClO) modified biochar showed a high ability to sorb phenanthrene in waste water20.Abiotic processes were more important than biotic processes for the initial oxidation of fresh biochar, which was used to simulate the natural aging process in the laboratory experiment21. To protect soil and surface waters from contamination, knowledge of organic pollutants degradation and sorption\u2013desorption processes is necessary. During plant nutrient uptake, root sorption governs the bioavailability of organic pollutant such as 2,4,6-TCP to plant in soil solution. It is well documented that the biochar has a greater ability to sorb the organic pollutant, but mechanisms of the aged biochar, its properties and sorption of the organic pollutant are less explored. Moreover, there is a limited research on the simulated aging of biochar and role of biochar\u2019s surface characteristics when it is applied to polluted soil. So, we hypothesize that surface properties and acidic functional groups of the biochar improve with the aging process, which in turn increases sorption ability of the organic pollutant onto biochar. Therefore, in this study, aging of biochar, derived from peanut shell and bush, was simulated by chemical oxidation with citric acid/FeCl3 and citric acid/\u03b1-Fe2O3 systems. In an effort to investigate the impact of aging treatment on the surface chemistry of biochar and the associated adsorption capacity and sorption\u2013desorption processes, we try to understand the aging mechanisms in the Fenton-like system that occur in the natural soil environment.The governing mechanisms for biochar in sorbing of organic pollutant are its high external and internal surface area and oxygen-containing (e.g. carboxyl and hydroxyl) surface functional groups, which enhance the biochar\u2019s sorption ability to organic pollutants by promoting \u03c0\u2013\u03c0 and electrostatic interactions, especially for the aging biochar\u22121) was prepared by dissolving 2,4,6-TCP in methanol, followed by storage at 4\u00a0\u00b0C in the dark to avoid photo-chemical degradation.The 2,4,6-TCP (>\u200998%) was provided by Jiangsu Academy of Agricultural Sciences. A 2,4,6-TCP stock solution  and bush (roadside green belt plant of school) were air dried and crushed into small pieces, and then further dried in the oven at 105\u00a0\u00b0C. The dried peanut shell and bush materials were pyrolyzed at 450\u00a0\u00b0C under anaerobic conditions in the vacuum tube furnace  and the prepared biochars were dried at 105\u00a0\u00b0C for further experimental use in the laboratoryes Table .3 and citric acid/\u03b1-Fe2O3 mixtures were chosen to oxidize peanut biochar and bush biochar as mineralization models of biochars in acidic soil.Chemical aging was conducted to simulate different natural field conditions. For this purpose, the citric acid/FeCl\u22121 Fe3+  solution, and then adding 400\u00a0mL of 0.01\u00a0M buffer solution , after that UV irradiation was applied by a photochemical reaction apparatus  with 500\u00a0W high pressure Hg lamp for 6\u00a0h13. The biochar was then separated with 0.45\u00a0\u03bcm glass filter paper (Whatman #934-AH). To remove residual solution, aged biochars were washed repeatedly with deionized water for 1\u00a0h until pH of the filtrate was stabilized. The aged biochar was dried in the air-circulating oven at 105\u00a0\u00b0C for 4\u00a0h. All treatments were run in triplicate.The Fenton-like aging process was initiated by immersing 05\u00a0g biochar in 100\u00a0mL 0.30\u00a0mM L13.The C, H, N and S contents were analyzed by Vario EL Cube instrument . Oxygen content was calculated as the difference between the sum of C, H, N, S and ash mass fractions\u22121 by using a Waltham Nexus-670 FTIR Spectrometer  with a resolution of 1.0\u00a0cm. The biochar pellets were prepared by using KBr powder.The FTIR spectra were recorded in the region of 400\u20134000\u00a0cm13. The spectra were separated using Systat Peakfit, Version 4.12 5.XPS was used to analyze surface properties in approximately 10\u00a0nm depth of biochars. XPS measurements were conducted using an ESCALAB 250Xi , equipped with a focused monochromatized Al K\u03b1 radiation (h\u03bd\u2009=\u20091486.6\u00a0eV). The X-ray spot size was 500\u00a0\u03bcm. Survey scan spectra in the 1351\u20130\u00a0eV binding energy range were recorded with a pass energy of 100.0\u00a0eV, others in 20.0\u00a0eV16.Scanning electron microscope with energy dispersive spectroscopy (SEM\u2013EDS) was carried out on Nova NanoSEM 450  and AZtec X-MaxN 80 . SEM was operated at 15\u00a0kV with a probe current of 0.6 nA to which a Bruker silicon drift detector EDS system had been interfaced\u22121 were prepared from the stock solution. The 2,4,6-TCP solution and biochars were mixed in a 100\u00a0mL glass flask, and sealed with tape and shaken at 180\u00a0rpm for 2\u00a0h in a water bath at 25\u00a0\u00b0C, 35\u00a0\u00b0C, and 45\u00a0\u00b0C. The background electrolyte of the mixed solution was 0.01\u00a0mol\u00a0L\u22121 NaNO3, and the pH was adjusted with 0.1\u00a0M NaOH or HCl22. After reaching the equilibrium during the sorption experiment, the solution was filtered through 0.22\u00a0\u03bcm nylon membrane. The 2,4,6-TCP concentrations were analyzed by high performance liquid chromatography  with a UV detector equipped with a reverse phase column, 4.6\u00a0mm\u2009\u00d7\u2009150\u00a0mm XDB-C18 column 9.A series of batch sorption/desorption experiments were performed in triplicates to evaluate the 2,4,6-TCP adsorption/desorption capacity of aged biochars. For the adsorption experiment, different 2,4,6-TCP concentrations, ranging from 5 to 160\u00a0mg\u00a0L\u22121) is the equilibrium liquid-phase concentrations of 2,4,6-TCP, Qe (mg\u00a0g\u22121) is the amount of 2,4,6-TCP adsorbed per gram of biochar at the equilibrium state, and Kf (g\u00a0mg\u22121) are rate constants for Freundlich sorption isotherm model, 1/n is the Freundlich constant related to the surface heterogeneity.The adsorption capacity of 2,4,6-TCP was calculated according to the following Freundlich sorption isotherm equations:\u22121 NaNO3 to desorb the 2,4,6-TCP from the biochars and shaken for 2\u00a0h at 25\u00a0\u00b0C. After shaking the solution was filtered, and the 2,4,6-TCP concentration in the filtrate was measured as described above. At the end of the desorption experiment, parts of the solution were adjusted for the pH (<\u20092 with HCl) and extracted with 50\u00a0mL dichloromethane  and was dried with anhydrous sodium sulfate. The extract was analyzed for 2,4,6-TCP and the degradation products using Thermo Trace DSQ II Gas Chromatography-Mass Spectrometry (GC\u2013MS) .The biochars adsorbed with 2,4,6-TCP were washed with distilled water three times to remove the surface 2,4,6-TCP, followed by addition of 50\u00a0mL 0.01\u00a0mol\u00a0LP\u2009<\u20090.05. All statistical analyses were carried out using SPSS, version 20.0 .All data were expressed as means\u2009\u00b1\u2009one standard deviation. Differences between the treatments were examined using a two-way analysis of variance (ANOVA), with statistical differences considered when 13. The aged biochars surface sulfur was increased by 12.7% (ABB) and 26.9% (APB) compared to BB and PB, respectively. The percentage of ash content in aged biochars decreased to 28.32% (ABB) and 21.56% (APB) compared to the control, respectively , which could be due to Fenton-like system agents incorporation onto the surface of the biochars, that was also confirmed by other studies25. Peaks at 2950\u20132860\u00a0cm\u22121 were the stretching of \u2013CH2\u2013 or \u2013CH3 in aliphatics or alicyclics (\u03bdC\u2013H) and showed a slight decrease in aged biochar after sorption of the 2,4,6-TCP , and showed a transmittance decrease in sorbed biochar, which indicated the decrease of carboxyl group. A decrease of stretching appeared in aromatic C\u2013O (\u03bdC\u2013O) at 1094\u00a0cm\u22121. Aliphatic CH2 deformation (868\u00a0cm\u22121) may be replaced by 2,4,6-TCP, which was decreased after sorption  was conducted to detect the functional groups in the biochars as shown in Fig.\u00a0s and O1s was done to check the changes , C\u2013O (286.1\u00a0eV), C=O (287.0\u00a0eV). The main four peaks for O1s were C=O at 532.5\u00a0eV, O\u2013H at 530.3\u00a0eV, O\u2013C=O at 532.8\u00a0eV, and C\u2013OO at 533.8\u00a0eV. The O\u2013H functional group was obviously found in aged biochar, which was less pronounced in the fresh biochar. Aliphatic stretching was found to decrease in O1s, the increased O groups was probably focused on aromatics13. Aging biochars showed adsorption peaks of C\u2212O, C=O and O\u2212C=O bonds due to surface oxidation. As a result of rapid oxidation of the surface of the biochar, excessive free radicals are formed at the earlier stage of aging, which initially leads to the formation of C\u2013O and O\u2013H bonds, and later some of them are converted to C=O and O\u2013C=O as the aging process continues. During the aging of biochars, the proportion of aliphatic C\u2212H increased relative to the aromatic C\u2212H, this may be due to the opening of the benzene ring or the volatilization of small molecules containing the benzene ring28. The FTIR and XPS results showed that aged biochars were equipped with more oxygen-containing functional groups27. The singlet oxygen (1O2) was the main reactive species for organic pollution degradation, which was confirmed in the modified biochar for methylene blue sorption29.Further analyses of C and O were conducted by using XPS technique Fig.\u00a0. Data frges Fig.\u00a0C. Three In brief, both FTIR and XPS showed the increase of C and O throughout the aging process, which was in accordance with the compositional analysis. This may be the result of ash, carbonate and few dissolvable carbons leaching which washed down or decomposed into inorganics, while persistent C like aromatics remained intact. The increased O was noticed in oxygen-containing functional groups in aromatic C structure from XPS and oxidation reaction, especially carboxyl aromatics.30. So, with aging the surface area and pore volume of biochars were also increased by 29.1% (APB), 23.5% (ABB) and 43.4% (APB), 32.4% (ABB) compared with control . When the solution pH was lower, the biochar adsorption capacity was higher, therefore, the better adsorption was at low pH in this study .The Freundlich sorption isotherm was fitted to study the effects of initial 2,4,6-TCP concentration on maximum adsorption capacity of biochars Table . The Fre13. Over 240\u00a0min, sorption was increased at a low rate with a fluctuating trend, as the exterior surface was hard to adsorb more ions while the interior sorption was still developing. The adsorption performance of aged biochars was superior to fresh biochar with higher adsorption rate. Different adsorption rates indicated that adsorption of 2,4,6-TCP onto biochars was not only a physical reaction, but also a chemical reaction, which was confirmed in the 2,4,6-TCP desorption and GC\u2013MS maps (Fig.\u00a0The adsorption capacity of fresh biochars and aged biochars increased rapidly with the increase in initial concentration of 2,4,6-TCP and then tended to keep stable with time longer than 120\u00a0min and biochar reaching the saturation Fig.\u00a0. Aged biaps Fig.\u00a0.Figure 5The 2,4,6-TCP adsorbed on the biochar was also desorbed with desorption solution Fig.\u00a0. The 2,4The 2,4,6-TCP degradation components, at the end of the degradation experiment, were analyzed by GC\u2013MS Fig.\u00a0. The 2,431. The micropore filling was the dominating adsorption mechanism; the micropore size distribution affected the adsorption mechanism of organic pollutants sorption. Hu et al.24 found that adsorption was the dominant sorption mechanism of phenanthrene by the oxidized biochars. The surface properties changed as increased oxygen-containing functional groups provided more adsorption sites, resulting in strong complexation of 2,4,6-TCP with aged biochars. The aging process played an active role in the 2,4,6-TCP sorption onto the biochars. So, the application of biochar in the wastewater could be a wise strategy for the remediation of organic contaminated water, which may increase the adsorption capacity over a longer period of time during natural aging process. Moreover, the disinfectant byproducts during chlorination promoted degradation to low molecular weight organic matter along with more active sites on the wheat straw biochar32. Kim and Hyun33 also found that the organic pollutions sorbed by Miscanthus derived biochar was greatly attributed to the effect of\u00a0\u03c0\u2013\u03c0 electron donor\u2013acceptor interaction and calcium\u00a0complexation. In short, it can be concluded from current study that the sorption of 2,4,6-TCP was mainly determined by the special biochars properties and microstructure that included\u00a0electron interaction and\u00a0the formation of carbon\u00a0bonds.The efficient adsorption of 2,4,6-TCP onto aged biochar was attributed to the improvement in the biochar surface properties and functional groupsFresh and aged biochars have significantly different physico-chemical and surface properties. Aged biochars have better ability to sorb 2,4,6-TCP efficiently compared to fresh biochars. Adsorption of 2,4,6-TCP onto aged biochars was influenced by solution pH, contact time, temperature, biochar\u2019s properties and strong interaction with active sites. The biochars sorption abilities for the 2,4,6-TCP were also influenced by types of feedstock. The mechanism of 2,4,6-TCP adsorption on the aged biochar was attributed to the acidic functional groups, amorphous carbon, and the complex micropore structure, which were different in aged biochar compared to fresh biochars. So, biochars could be used in the natural environment to remediate organic pollutants in wastewater for a longer time, as biochars with aging become more active and efficient.Supplementary information."}
+{"text": "Low levels of micronutrients have been associated with adverse clinical outcomes during viral infections. Therefore, to maximize the nutritional defense against infections, a daily allowance of vitamins and trace elements for malnourished patients at risk of or diagnosed with coronavirus disease 2019 (COVID-19) may be beneficial. Recent studies on COVID-19 patients have shown that vitamin D and selenium deficiencies are evident in patients with acute respiratory tract infections. Vitamin D improves the physical barrier against viruses and stimulates the production of antimicrobial peptides. It may prevent cytokine storms by decreasing the production of inflammatory cytokines. Selenium enhances the function of cytotoxic effector cells. Furthermore, selenium is important for maintaining T cell maturation and functions, as well as for T cell-dependent antibody production. Vitamin C is considered an antiviral agent as it increases immunity. Administration of vitamin C increased the survival rate of COVID-19 patients by attenuating excessive activation of the immune response. Vitamin C increases antiviral cytokines and free radical formation, decreasing viral yield. It also attenuates excessive inflammatory responses and hyperactivation of immune cells. In this mini-review, the roles of vitamin C, vitamin D, and selenium in the immune system are discussed in relation to COVID-19.  Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has spread rapidly across the world, with 39,944,882 confirmed cases and 1,111,998 deaths reported to the World Health Organization (WHO), as of 19 October 2020 . AlthougRecently, the European Society for Clinical Nutrition and Metabolism (ESPEN) proposed 10 practical recommendations for the management of COVID-19 patients . The rec6, B12, zinc, and selenium have been associated with adverse clinical outcomes during viral infections [1, B6, B12, D, folate, selenium, and zinc were determined in 50 patients with COVID-19. Seventy-six percent of the patients had vitamin D deficiency, and 42% had selenium deficiency [Low levels of micronutrients such as vitamins A, E, Bfections . A recenfections  demonstrfections . Serum lficiency ,11,12 anficiency . Howeverficiency ,15. Seleficiency , and a sficiency . Vitaminficiency ,19.This mini-review discusses the role of vitamin C, vitamin D, and selenium in immunity, and the beneficial effects of these micronutrients in reducing the risk of infectious diseases, particularly COVID-19.COVID-19 can develop into acute respiratory distress syndrome, secondary infection, and sepsis . An intrVitamin C treatment has antiviral effects. Clinical trials have shown that administration of high doses of vitamin C has beneficial effects against the common cold ,23. A hi2\u2212, NO3\u2212, C-reactive protein, and lactate dehydrogenase in blood, compared to healthy individuals. After oral or intravenous administration of vitamin C with methylene blue and a known antioxidant N-acetyl cysteine, the blood levels of NO3\u2212, methemoglobin, C-reactive protein, and lactate dehydrogenase were markedly decreased in four out of five patients [Vitamin C potentially attenuates excessive immune response in patients with COVID-19. Severe COVID-19 infection induces pulmonary and systemic inflammatory responses . The micpatients . This stpatients . In anotpatients . These s+ to synthesize poly(ADP-ribose) for DNA repair [+ is needed for GAPDH activity, depletion of NAD+ decreases the enzymatic activity of GAPDH. Thus, the inhibition of GAPDH by a high dose of vitamin C may reduce the activation of immune cells by decreasing adenosine triphosphate (ATP) production in the cells [Vitamin C may prevent the hyperactivation of immune cells by inhibiting glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The glycolytic enzyme GAPDH can regulate the rate of glycolysis in activated myeloid and lymphoid cells . VitaminA repair . As NAD+he cells .Clinical trials are needed to investigate the effect of vitamin C on COVID-19 infection. A clinical trial was conducted from 14 February, 2020 to 30 September, 2020 in Zhongnan Hospital of Wuhan University, China . In the Adequate vitamin D levels in the body can be achieved by sufficient vitamin D consumption and sun exposure. The risk factors for vitamin D deficiency are age, smoking, obesity, and chronic diseases such as diabetes and hypertension . 25-hydrStudies have investigated the relationship between vitamin D deficiency and COVID-19 ,43. As vVitamin D reduces the risk of viral infections. It improves the body\u2019s physical barrier by regulating the production of proteins for tight junctions , adherenVitamin D stimulates the production of antimicrobial peptides, such as cathelicidin and defensins  that havVitamin D modulates helper T cell responses. It reduces T helper type 1 (Th1) immune responses  and induVitamin D may prevent cytokine storms in patients with COVID-19. COVID-19 can lead to cytokine storms and immunogenic damage to the endothelium and alveolar membrane , which mRecent studies have shown that vitamin D deficiency is correlated with poor prognosis in COVID-19 patients. However, there was no association between blood concentration of vitamin D and the risk of COVID-19 in the UK biobank . TherefoSelenium deficiency may be a risk factor for COVID-19 mortality. A cross-sectional study conducted in Germany showed that the serum level of selenium was significantly higher in the surviving patients with COVID-19 compared to the deceased patients with COVID-19 . AnotherSelenium deficiency exacerbates virulence and progression of viral infections, such as influenza A ,72 and C+ T cell response. It increased CD4+ T cell activation, proliferation, and differentiation by maintaining the intracellular level of free thiol in mice fed with a high-selenium diet (1.0 mg/kg body weight) compared to a low-selenium diet (0.08 mg/kg body weight) or medium-selenium diet (0.25 mg/kg body weight) for 8 w [+ T cells isolated from mice fed with a high-selenium diet demonstrated increased T cell receptor (TCR) signaling and TCR-stimulated IL-2 expression. In addition, high consumption of selenium induced the Th1 phenotype with increased IFN-\u03b3 in CD4+ T cells [Selenium demonstrates antiviral effect by regulating CD4 for 8 w . CD4+ T  T cells . Seleniu T cells . Seleniu T cells . As IL-1+ T cells and natural killer (NK) cells. TNF-\u03b1 and IFN-\u03b3 have antiviral effects against influenza virus in CD8+ T cells [+ T cells was lower in the lungs of selenium-deficient mice than in selenium-sufficient mice [+ T cells by increasing the number of cells in the human peripheral blood lymphocyte population [Selenium is important for the function of cytotoxic effector cells, such as CD8 T cells . Seleniu T cells . The nument mice . Dietarypulation . Furtherpulation  and mouspulation . TherefoSelenium plays an important role in the production of antibodies. Selenoprotein deficiency induced impaired T cell maturation, functions, and T cell-dependent antibody response in mice . Selenop2 to prostacyclin I2 in rats [Blood coagulation may increase mortality in patients with COVID-19 . A low p in rats , inducin in rats . This miNutritional therapy should be a part of patient care for survival of this life-threatening disease (COVID-19), as well as for better and shorter recovery. Most importantly, checking malnutrition and providing optimal nutritional supplementation are critical steps for optimal functioning of the immune system in the human body. Patients with malnutrition are more likely to be from lower socioeconomic groups; thus, nutrition supplementation is important for the risk group as well as older adults who have a relatively weak immune system. In this review, we focused on the importance of vitamin C, vitamin D, and selenium for immunity enhancement. The immunomodulatory properties and the consequences of deficiencies or supplementation of these micronutrients against viral infectious diseases, including COVID-19, are summarized in"}
+{"text": "In the face of rising university tuition costs and a longstanding skills gap in the US workforce, a growing number of people access higher and continuing education programs via online platforms. There are serious concerns that online learning disadvantages members of underserved communities, thereby exacerbating social inequalities. However, it is hard to evaluate these concerns at scale partly due to selection effects. Policy responses to the COVID-19 pandemic, such as nonessential business closures, suddenly changed how people spent their time, which allowed us to estimate effects on demand for online learning and how it varies along socioeconomic dimensions. Unlike most prior studies that find education technology to maintain or amplify inequities, we present causal evidence for its potential democratizing effects. n = 277,425). Exploiting the staggered adoption of actions to mitigate the spread of COVID-19 across states, we identify the causal effect at the neighborhood level. The adoption of nonessential business closures led to a 38% increase in new users and a 6% increase in engagement among existing users. We find that these increases are proportional across higher- and lower-income neighborhoods and neighborhoods with a high or low share of Black residents. This demonstrates the potential for online platforms to democratize access to knowledge and skills that are in high demand, which supports job security and facilitates social mobility.The COVID-19 pandemic has changed peoples\u2019 lives in unexpected ways, especially how they allocate their time between work and other activities. Demand for online learning has surged during a period of mass layoffs and transition to remote work and schooling. Can this uptake in online learning help close longstanding skills gaps in the US workforce in a sustainable and equitable manner? We answer this question by analyzing individual engagement data of DataCamp users between October 2019 and September 2020 ( Productivity growth in the digital economy has expanded at roughly four times the rate of the overall economy, raising the demand for skilled knowledge workers , 2. As tThe ongoing coronavirus pandemic has accelerated these patterns, reducing the returns of a traditional college education after the transition away from in-person classes . StudentThis sudden shock led people to shift their allocation of time, including a substantial increase in investments in human capital accumulation. A leading online course provider, Coursera, saw a jump in its visitor numbers from 27 million to over 70 million between February and July 2020 . The incThis research investigates the pandemic's effect on the adoption and use of online learning and its implications for the future of education in the years ahead. Drawing on individual-level records from DataCamp , one of We exploit the staggered adoption of NBC across states because it provides plausibly exogenous  variation in the time that individuals allocate between employment and personal activities. Our identifying assumption is that unobserved shocks to the demand for online learning are uncorrelated with the adoption of NBC. Since these state policies are driven much more by political affiliation than by the number of local infections or other real factors , we arguOur results indicate that the passage of NBC is associated with a 38% increase in new DataCamp users and a 6% increase in engagement among existing users. This suggests that the pandemic brought people to start acquiring programming skills online (intensive margin effect) as well as it encouraged already active learners to study more (extensive margin effect). We observe little variation between lower- and higher-income regions and no variation based on racial composition. All demographic regions experienced a substantial increase in online learning following the adoption of these state policies. This suggests that, at least over the pandemic, the expansion of online learning through DataCamp has had a \u201cdemocratizing\u201d effect in the market for technical (programming) educational services.n = 277,425 and n = 69,942, respectively) across  zip code-based regions resemble the broader US population along several key dimensions . Although the regions represented in our samples are more populated  compared with all US regions , the median household income, percentage of college-educated individuals, and percentage of Black residents are only marginally higher than the population average. Moreover, the share of individuals in the retail trade, wholesale trade, and information services in our samples is not significantly different from the population. Our sample therefore adequately represents regions that were particularly hard hit by the pandemic: poorer and racially diverse regions with lower education levels and less digitally intensive workers . The effect estimate rises, rather than falls, to 14% after adding zip code-specific demographic characteristics (column 2), which suggests that the areas that were harder hit by the pandemic were more likely to experience an increase in new users. The effect estimate further increases to 37% after we control for week fixed effects (column 3), which eliminates variation from the fluctuations in aggregate demand for online learning that could be correlated with state pandemic responses.However, these estimates could still be biased due to the combination of time-varying or time-invariant state characteristics that are correlated with NBC adoption. To address these concerns, we control for the weekly number of coronavirus cases per capita and an index of compliance with social distancing policies (column 4). Our coefficient estimate on NBC remains the same, suggesting that our results do not reflect reverse causality stemming from the rise in the severity of coronavirus or other social distancing policies. Finally, we also add state fixed effects to observationally compare online learning interactions before vs. after the adoption of the NBC (column 5). Given that our coefficient estimates are statistically indistinguishable with and without state fixed effects, we conclude that selection effects\u2014the possibility that individuals in some states are simply more likely to start online learning than others\u2014play a limited role in accounting for the surge in online learning.For weekly exercises among existing users, we find a similar pattern of results. In particular, the raw correlation indicates that individuals in states that adopted NBC have 5% higher weekly engagement (column 1). The inclusion of zip code demographic characteristics does not alter the effect estimate (column 2). After we introduce week fixed effects, the estimate rises to 11% (column 3), consistent with the direction of bias present for the user registration outcome. Further adjusting for weekly coronavirus cases per capita and compliance with social distancing policies produces a statistically indistinguishable estimate (column 4). Finally, when we exploit within-state variation, we find that the adoption of NBC is associated with a 6% rise in weekly engagement (column 5), which is significant at the 10% level. Although the effect magnitude and statistical significance decline, we still find robust evidence that NBC led to increased adoption of online learning and allocation of time to online learning activities.SI Appendix, Table S2 has detailed estimates).Having examined average treatment effects, we next investigate sociodemographic heterogeneity and the effect of NBC on online learning. Specifically, we define subgroups in terms of household income and the share of residents who are Black, retail workers, and college educated. SI Appendix, Table S3 has detailed estimates).We find further evidence of a democratizing effect in terms of existing user engagement, shown in The coronavirus pandemic has been associated with a wide array of negative outcomes, ranging from mass unemployment to elevated suicide risk . This reThe effects of the pandemic have been more severe in disadvantaged communities, which also tend to benefit less from advances in EdTechs . For exaSI Appendix, Table S1), as DataCamp targets young professionals and university students, thus mitigating zip code-based discrepancies. Prior work showing sociodemographic inequalities in online courses has also been conducted at the zip code level and even national level , the race distribution, the age distribution , the education distribution , the poverty rate , and the industry and occupational distribution. We also recognize that, while there might be heterogeneity in how male and female users respond to NBC, there is not enough variation across zip codes in the share of males vs. females to tease out these effects. SI Appendix, Table S1.Finally, we obtain daily county data on coronavirus cases and deaths from the Johns Hopkins Coronavirus Resource Center and weekly county data on compliance with social distancing policies from Unacast . Their sWe focus our analysis on two different outcomes to distinguish between an effect on people deciding to start online learning (a measure of the extensive margin in online learning) and an effect on the level of engagement among existing online learners (a measure of the intensive margin). The first outcome measure is defined by the number of platform registrations per week. The second outcome measure is defined by the sum of exercises completed per week by existing users. Existing users are defined as having registered at least 22 wk prior to the start of the NBC in their state and as having completed at least one exercise during the 44-wk window surrounding the NBC. Both outcome measures are aggregated to the zip code level by summing over individuals.We use a number of covariate measures for regression adjustment and to estimate heterogeneous treatment effects. Since we do not have individual demographic characteristics, we use zip code characteristics as a proxy. For each zip code, we consider the following characteristics as covariates: total population (log transformed), median household income (log transformed), race distribution , age distribution , the percentage of individuals with at least a college degree, the poverty rate among White and Black individuals, and the percentage of workers in the retail trade sector.Additionally, we use two time-varying county-level variables for regression adjustment: the number of cumulative coronavirus cases per capita and a measure of social distancing compliance from Unacast. These measures help control for changes in the intensity of the pandemic and the adherence to the policies that could otherwise explain differences in the demand for online learning. For example, areas that were more adversely affected could experience an exodus of residents; controlling for these factors ensures that the comparison is between observationally equivalent areas.To analyze heterogeneity in the average treatment effects, we created four indicator variables to identify relatively more vulnerable populations in terms of income, race, employment sector, and level of education. Specifically, the indicators record if a user lives in a zip code with above median 1) log-transformed median household income, 2) share of the population that is Black, 3) share of workers in the retail trade sector, and 4) share of the population with a college or higher degree.Our analytic strategy is to use the fact that NBCs were implemented in some states sooner than others. Our identifying assumption is that unobserved shocks to the demand for online learning are uncorrelated with the time that states enacted their NBC, conditional on a set of observable covariates including coronavirus cases. Because states adopted NBC policies at different points in time, state fixed effects allow us to compare outcomes in states that adopted NBCs sooner with those that adopted them later. We acknowledge that the pandemic affected a range of industries asynchronously and led to sporadic closures across sectors by states, which make a perfect identification strategy difficult. To ensure our decision was meaningful, we studied two of the major policies by state, the SAHO and NBC. SAHO generally occurred 1 or 2 wk after NBC. We found that the change in our outcomes aligned more closely with NBC, namely that the increases in users and exercises aligned with this date for our sample. This makes intuitive sense as we consider the nature of our project, exploring change in online learning, which is likely associated more with individuals who would be affected by NBC.Our identification strategy yields robust causal estimates as long as unobserved determinants of the decision to start online learning with DataCamp are uncorrelated with the timing of NBC. A recent study showed that the timing and type of state policies that were adopted were strongly correlated with political affiliation, which is unlikely to be correlated with the demand for online learning . Still, We use the following regression specification to estimate causal effect of NBC on the demand for online learning:yilt denotes our measure of online learning  for individual i in zip code z and time t, Pst denotes an indicator for whether an NBC was in effect in state s at time t, g(Xzt, where Supplementary File"}
+{"text": "With a motivation to immerse students in engineering design, graphics communication, and computer aided design (CAD) skills early-on in the biomedical engineering curriculum, we launched a new 2-unit laboratory course on \u201cGraphics Design in BME\u201d in the Spring 2020 quarter for UC Davis sophomores. Due to the COVID-19 pandemic, the course met with the significant challenge of conversion to an online mode of teaching, instead of planned face-to-face instruction. Providing formative feedback was thought to be an important step to help students succeed in their final CAD project of the course. In the process of designing feedback, we found that the concept of feedback is still fragile in an online learning environment because online learning settings provide distinct pedagogical demands as compared to face-to-face settings. The situation is especially delicate in the context of contemporary higher education imparting engineering skills, where students attend large classes, with diminished opportunities to interact with the teaching staff. The challenge we faced was to provide meaningful dialogic feedback in an online environment, especially while teaching engineering graphics design to a large class. Here we addressed this challenge by focusing on the process of structuring\u00a0meaningful feedback. We designed a project assignment to be submitted as multiple deliverables to be submitted in two-stages. Then, we characterized its feedback with multiple notions, such as dialogic iterative cycles, personalized, goal-directed, immediate, in written format, and having a peer assessment component. The process of providing formative feedback online through the structure mentioned in this paper resulted in students\u2019 improved scores on the final project elements. It also helped us identify the common issues students are faltering in a graphics design class, and provide customized guidance, ideal examples of expected work, and more resources to motivate each student group to achieve mastery of course content.The online version contains supplementary material available at 10.1007/s43683-021-00046-z. The COVID-19 pandemic-based shift of decision to launch our new course on Graphics Design in BME in an online mode instead of face-to-face, raised a big challenge in terms of providing meaningful feedback to a large, online class of students for improving their problem solving, graphics communication, and design skills. Feedback was needed for reducing discrepancies between students\u2019 understandings and the goals of the assignment.23etc. are also reduced in an online environment, which would otherwise help and equip students with a better appreciation of what is expected of them, and develop their understandings of academic terms and appropriate practices as they begin to write project reports. This situation is especially delicate in the context of contemporary higher education imparting engineering skills, where students attend large classes with fewer opportunities for an individual student to interact with teaching staff.Many theoretical models describe the mechanism of feedback production, how it is received, and what effect it has on students.Most works in the past have treated feedback as a singular concept. Here we treated feedback as more than one notion and focused primarily on the needs of learning rather than the capacities of the teacher as suggested by Boud and Molly.The aim of this paper is to: (1) report how we structured formative feedback in an online transitioned course on Graphics Design in BME, in response to the COVID-19 pandemic, and (2) report the assessed effects of the formative feedback provided online, to enhance students\u2019 performance in a computer-aided bioreactor-design project.Apply spatial visualization and create orthographic and isometric views of objects.Construct 2D and 3D drawings, and assembly and sub-assembly structures.Follow engineering design procedures including problem identification, problem formulation, approaches, methodology, and solution.Use industry-standard software packages for the design of a 3D Computer-Aided Design prototype of a bioreactor.Work in a team to document and present the process of Computer-Aided Design of their product.Develop an ability to utilize online learning tools and collaborate online.The Learning Objectives (LOs) of this course were set such that at the end of the course, students would be able to:Specifically, an end-of-the-quarter bioreactor project was designed to teach students to (i) identify a biomedical graphics design challenge for creating a bioreactor that satisfies a given need, (ii) evaluate various parameters for the optimal design of the bioreactor based on literature search, (iii) Select specific parameters for bioreactor design using a weighted decision matrix, (iv) synthesize a preliminary concept of the prototype on paper and in CAD, and (v) communicate the model formation process and drawing in CAD accurately in the form a written report.All students had a common challenge for their project\u2014to create a bioreactor using CAD for enhancing the viability and proliferation of mesenchymal stem cells on porous silk-fibrin 3D scaffolds (project details are in Supplementary Information). The course had 98 students split into 24 teams. Each team designed a bioreactor using CAD. The project constituted 15% of the final grade, including a written project report (10%) and a CAD portfolio (5%).via a peer review process, where at least three different students graded a submission anonymously, and scores were averaged by a TA.The project was divided into several assignments due week 2 to 9. Final projects were due in week 10 of instruction. Rubrics and additional resources were provided to students for each assignment. Written formative feedback was given using Canvas LMS through weeks 2\u20136. We analyzed the pre and post-feedback performance of students after coding the data upon approval of the Institutional Review Board (IRB). The timeline of project deliverables is indicated in Table S1. The Project Need Statement, Goals & Objective, Decision Metrics, and Preliminary Concept were assessed by the instructor. Hand-drawn Sketches were assessed Research shows that viewing feedback more as dialogue than the transmission of information,Dialogic feedback suggests an interactive exchange in which interpretations are shared, meanings are negotiated and expectations are clarified.iterative cycles such as two\u2010stage assignments or multi-stage assignments that can involve feedback on the first stage, intended to enable the student to improve the quality of work for a second (or later) stage submission.5Personalization of the feedback allowed tailoring comments to students\u2019 learning needs.7peer feedback. Peer feedback involves students engaging in reflective criticism of other students\u2019 products.Another element that influences the potential for dialogic feedback is content is also important, where \u2018content\u2019 involves both the verification and elaboration aspects. The verification is a simple judgment of whether an answer is correct, and elaboration provides hints, cues, analogies, explanations, etc.function was therefore directive in nature.Feedback presentation\u00a0of our feedback components was immediate; provided within 1\u20132\u00a0weeks of students\u2019 assignment submissions (see Supplementary Table\u00a01). This decision was based on Mason and Bruning\u2019s framework for computer-based feedback.19Finally, the We provided feedback mainly in written format than oral format, because Chickering and Ehrmann\u2019sComment feature and the\u00a0Highlight and Free Draw\u00a0annotation types in the point annotations as shown in Canvas DocView in Figs.\u00a0Free Text, Strikeout\u00a0annotation types were also found useful. Students could view our DocView comments from the assignment Submission Details page. The Assignment Comments section  and 4 (Drawings using Solidworks) because some teams were still working upon their CAD parts at the time of feedback (week 9). To facilitate feedback provision on these deliverables in the future, we will shift the last two deliverables by a week earlier.via Canvas LMS. Detailed rubrics were provided to every student and each submission was analyzed per rubric by three students anonymously. We observed that the peer feedback process enhanced student engagement with the course content. Several students provided elaborate feedback with specific comments about positive and negative issues in a submission. Our experiences about peer feedback processes are in agreement with previous findings showing that the opportunities for dialogic peer formative feedback promote learning support and self-regulation.13One project element\u2014the Hand-Drawn Sketch warrants special discussion. The hand drawn-sketch assignment required anonymous peer feedback which was enabled via graphics/drawings; better interpretation of feedback by students than on the earlier assignments. We did not do any follow-up interviews or student surveys on this issue, but this will be worth doing in the future to know the underlying causes.While the feedback process significantly enhanced the students\u2019 final scores in all project deliverables, not all student teams reached an \u2018Accomplished\u2019 and above (Exemplary) level in the rubric, which essentially meant scoring more than 80% on a project deliverable. This is illustrated through data in Table\u00a0Reflecting deeper on the student performances in the first three assignments of the project, namely the Need Statement, the Goals and Objectives, and the Decision Metrics assignments, we realized a need to introduce another competency in this course and train students to interpret the given feedback. Studies such as that by WinstoneThe assessment of student project deliverables while constructing feedback also helped us spot the common issues that students were faltering upon in each project deliverable. These \u2018common issues\u2019 were mistakes found in submissions of about 5\u20136 teams (about 1/4th of a class) in their first iteration of the project deliverables. The early review of student-work helped us find these shortfalls, as listed in Fig.\u00a0Student\u2019s progress in submissions showed progressive evolution of drawings from simplified preliminary concept drawings to more concrete, practical bioreactor CAD designs. For example, images in Figs.\u00a0The written mode gave us an excellent means to provide more intimate and protected feedback to student teams. The written form of feedback was perhaps less intimidating than the demands of face-to-face communication. This\u00a0was illustrated by the fact that we received requests from 6 student teams (out of 24) for additional feedback on their iterations of the project, beyond the feedback cycle implemented that quarter. Furthermore, typed annotations of the student submissions eliminated any possible barrier of the illegibility of any written feedback. In Ferguson\u2019s study,One observation this quarter was that students, in general, lacked coordination efforts for the final assembly of their project and were not comfortable approaching teammates in difficulties since the pandemic began. In that vein, a teammate rating assignment and continuous feedback for improvement can be helpful, as shown in Supplementary Materials (S2). Another idea is to go over the team-work logistics with each team, such as how are they going to make decisions about process, leadership, deputization, and coordination.We identified feedback characterized by multiple notions, such as dialogic iterative cycles, personalized, goal-directed, immediate, in written format, and having a peer assessment component. The process of providing formative feedback online through the structure mentioned in this paper helped us identify the common issues students are faltering at in a graphics design class and gave us opportunities to provide customized feedback,\u00a0and ideal examples of expected work. The process of giving feedback inspired us with ideas and resources to teach innovatively and help students to achieve mastery of content in this course. It also possibly provided opportunities for metacognition to students by having them reflect on their previous assignment iteration.The formative feedback type we gave in the online environment resulted in students\u2019 improved scores on the final project elements. These results agree with many benefits of providing formative feedback reported in the literatureSupplementary material 1 (DOCX 51\u00a0kb)Below is the link to the electronic supplementary material."}
+{"text": "BackgroundAppendicitis is the most common indication for emergency surgery in the world. There is no one laboratory or radiological test that is used to diagnose it. Various routine and novel blood markers have been identified, however none have proved to be conclusive. The aim of this study was to combine routine blood markers to increase the sensitivity and specificity in diagnosing histologically confirmed appendicitis.MethodsWe retrospectively reviewed the theatre logs for the calendar year of 2015 to identify all of the appendectomies which were performed. We reviewed all of the admission bloods for the patients - including their white blood cell (WBC) count, their neutrophil count, and their C-Reactive protein (CRP) value. We also reviewed all of the histology to identify the inflamed appendices, and analysed all of this information together.ResultsThe neutrophil count is the most sensitive of the three blood markers with a score of 82%. It has a specificity of 63%. The CRP value is the most specific of the three blood markers with a value of 67% and a sensitivity of 76%. WBC has a sensitivity of 75% and a specificity of 63%. Combining all of the blood values (i.e. elevated white blood cell count or elevated neutrophil count or elevated CRP) demonstrates a sensitivity of 96% and a specificity of 45%.ConclusionCombining routine admission blood markers  can assist in diagnosing appendicitis in unwell patients with abdominal pain. Appendicitis is the most common indication for emergency surgery ,2. The aRatios of blood results have also been studied including white cell lymphocyte ratio, white cell neutrophil ratio, and neutrophil ratio  to diffeTherefore, acute appendicitis largely remains a clinical diagnosis. This can have many consequences. A late or missed diagnosis can result in complications such as perforation, abscess formation or, in extreme cases, death . It can Thus this study sought to analyse the usefulness of widely available inflammatory biomarkers in diagnosing acute appendicitis. Inflammatory biomarkers were also analysed in different combinations to establish if this would increase the sensitivity and specificity of diagnosing histologically inflamed acute appendicitis.We retrospectively reviewed the data from all patients who had appendectomies carried out in a regional Irish hospital in 2015. The hospital serves a large geographic area with a county population of approximately 150,000 people . Theatre log books were reviewed to identify every appendectomy that took place in the hospital. Data captured included patient demographics, serological results, radiological investigation results, type of operative technique, and histological findings.9/L) in the adult population. Reference ranges for normal WBC serum levels are 4 - 14.5 (x109/L) in the paediatric population (age <=12 years old). WBC <4 (x109/L) is considered low for the adult and paediatric population. WBC >11 (x109/L) is considered high for the adult population. WBC >14.5 (x109/L) is considered high for the paediatric population (age >12 years old). The normal reference range for the absolute neutrophil count (ANC) is 1.5 - 8 (x103/mL) for the adult and paediatric population. ANC is considered low if it is <1.5 (x103/mL). ANC is considered high if it is >8 (x103/mL).The blood results  and histology for all of these patients were reviewed and analysed using an Excel spreadsheet. A review of the ultrasound (US) imaging done on each of these patients was also carried out. Normal CRP levels are considered as <=5 mg/L - regardless of age of patient. CRP levels of >5 mg/L are considered high. Reference ranges for normal WBC serum levels are 4 - 11  for Mac (64-bit Intel).Two hundred forty-one appendectomies were performed between January 1, 2015 and December 31, 2015. One hundred seventy-five (73%) appendixes had histopathological findings of inflammation, 173 of these (98.8%) were reported as inflamed. One appendix was found to have a mucinous neoplasm, and one appendix contained a neuroendocrine carcinoid tumour. Sixty-six appendixes had normal histopathological findings - this includes one appendix that had a faecolith noted on analysis. The negative appendectomy rate was 27.4%.A breakdown of the demographics and operative method can be found in Table One hundred seventy-seven (73%) appendixes were removed laparoscopically, and 64 (27%) were removed as an open procedure. Of the 64 open appendectomies performed, 42 (66%) were performed on the paediatric population (<=18 years old) even though they represented 48% of the total number of appendectomies.CRP analysisA CRP was done on 173 of the 175 patients who had a histologically inflamed appendix, and 61 of the 66 normal appendixes. The CRP was normal in 41 24%) inflamed appendixes, and elevated in 132 (76%) inflamed appendixes. Out of the 66 non-inflamed appendixes, CRP was normal in 41 (67%) patients, and was elevated in 20 (33%) patients. The mean CRP values for the inflamed appendixes was 59.2, and it was 22.5 in the normal appendixes. The results are summarised in Table 4% inflamThe sensitivity of using an elevated CRP to diagnose a histologically inflamed appendix was 76%. The specificity was 67%. The receiver operating characteristic (ROC) area was 0.72. The positive predictive value (PPV) of an elevated CRP was 87%. The negative predictive value (NPV) was 50%. The area under the curve (AUC) is displayed in Figure WBC analysisA full blood count (FBC) was done on 174 of the 175 patients who had a histologically inflamed appendix, and 64 of the 66 normal appendixes. The WBC was normal for 43 (25%) inflamed appendixes, low for two (1%) of them and elevated in 129 (74%) inflamed appendixes. Out of the 64 non-inflamed appendixes, WBC was normal in 40 (63%) patients and elevated in 24 (37%) patients. The mean WBC count for the inflamed appendixes was 15.1 and 10.2 for the normal appendixes. The results are summarised in Table The sensitivity of using a high or low WBC to diagnose a histologically inflamed appendix was 75%. The specificity was 63%. The ROC area was 0.69. The positive predictive value of a high or low WBC was 85%. The negative predictive value was 48%. The AUC is displayed in Figure Neutrophil analysisThe neutrophil count was normal for 31 (18%) inflamed appendixes, and elevated in 143 (82%) inflamed appendixes. Out of the 64 non-inflamed appendixes where an FBC was done, neutrophil count was normal in 40 (63%) patients and elevated in 24 (37%) patients. The mean neutrophil count for the inflamed appendixes was 12.4 and 7.1 for the normal appendixes. The results are summarised in Table The sensitivity of using a high neutrophil count to diagnose a histologically inflamed appendix was 82%. The specificity was 63%. The ROC area was 0.72. The positive predictive value of a high neutrophil count was 86%. The negative predictive value was 56%. The AUC is displayed in Figure Histology and combined blood panel resultsOut of the 175 histologically inflamed appendixes, 174 had either an FBC or CRP taken. Ninety-nine (57%) patients had a combination of elevated CRP, leukocytosis (or leukopenia), and neutrophilia. Seven patients with histologically confirmed appendicitis had a normal CRP, WBC count and neutrophil count. Five of these seven patients were under the age of 18. The sensitivity of either a high CRP, leukocytosis or neutrophilia was 96%. The specificity was 45%. The ROC area was 0.71. The positive predictive value of abnormal blood results was 83%. The negative predictive value was 81%. These results are summarised in Table A summary of all of the blood results are found in Table A routine blood panel is taken on all patients that present to the emergency department (ED) with any infective or inflammatory symptoms. This includes an FBC, CRP and a metabolic/renal profile. At present, a single blood or urine marker for acute appendicitis does not exist, and the existing blood markers used are not sensitive or specific enough for it . Our resA meta-analysis by Kabir et al. has demonstrated that a WBC count in isolation is not a good marker to use for diagnosing appendicitis as it can be elevated in response to any inflammatory condition . ShogileNeutrophil count can also be used to help identify an inflamed appendix ,8\u00a0althouCRP also has a variable role in the literature when it comes to appendicitis. It has been noted in the systematic review by Kabir et al. that CRP is better for detecting complicated or late-stage appendicitis as it is a lag indicator and is less useful for early-stage appendicitis . Other pSome research has been done in the field of combining serological markers to increase sensitivity and specificity for diagnosing acute appendicitis. Birchley demonstrates that combining WBC count, neutrophil count, and CRP gives an odds ratio of 18 when trying to identify acute appendicitis - although it should be noted that this is only applicable when all of those tests are abnormal . FarooquOur findings when the three different blood results were combined were promising. It is worth emphasizing that we combined any abnormal WBC count, neutrophil count, or CRP value - we did not specify that all three results had to be abnormal. They demonstrated a sensitivity for detecting a histologically confirmed appendicitis of 96% - this is encouraging as it demonstrates that if an appendix is histologically inflamed, it is highly likely to be reflected in at least one of the blood results. Therefore if WBC count, neutrophil count, and CRP are all normal, then it is likely that the appendix is normal - if clinical suspicion persists, then either imaging or a diagnostic laparoscopy should be performed. Although the specificity was only 44% - this is not surprising as these blood markers could be raised due to any inflammatory or infective condition. This is similar to findings published by Andersson who found that a combination of WBC >10 and a CRP >8 gave an accuracy of 0.96 (area under the curve) for diagnosing appendicitis .The US sensitivity (59%) and specificity (85%) results in our study were not encouraging - but they are similar to a study by Yuan et al. who reported figures of 50% and 98.5% \u00a0and D\u2019SoThis study has several limitations. These include the fact that it was a retrospective study. No clinical information was captured about any patient. No complications for any patient were included. No information was recorded about the use of CT scans for patients in this study. We would recommend a more detailed future study which would include clinical details for the patient prompted the decision to bring the patient to theatre.Appendicitis is the most common surgical emergency in the world. There is no one diagnostic blood test or radiographical scan which has been shown to categorically prove appendicitis. The gold standard for diagnosing it is to examine the histology post-operative removal. Several standard blood tests have been examined and multiple novel blood tests have been examined to try and identify a better way of diagnosing it. The existing literature suggests combining routinely available blood tests may be a way forward. Our research builds on that premise and demonstrates that while elevated WBC, neutrophil count, and CRP in isolation can be helpful in aiding the clinical diagnosis of acute appendicitis, they are more useful when combined."}
+{"text": "The SARS-CoV-2 coronavirus is wreaking havoc globally, yet, as a novel pathogen, knowledge of its biology is still emerging. Climate and seasonality influence the distributions of many diseases, and studies suggest at least some link between SARS-CoV-2 and weather. One such study, building species distribution models (SDMs), predicted SARS-CoV-2 risk may remain concentrated in the Northern Hemisphere, shifting northward in summer months. Others have highlighted issues with SARS-CoV-2 SDMs, notably: the primary niche of the virus is the host it infects, climate may be a weak distributional predictor, global prevalence data have issues, and the virus is not in population equilibrium. While these issues should be considered, we believe climate\u2019s relationship with SARS-CoV-2 is still worth exploring, as it may have some impact on the distribution of cases. To further examine if there is a link to climate, we build model projections with raw SARS-CoV-2 case data and population-scaled case data in the USA. The case data were from across March 2020, before large travel restrictions and public health policies were impacting cases across the country. We show that SDMs built from population-scaled case data cannot be distinguished from control models (built from raw human population data), while SDMs built on raw case data fail to predict the known distribution of cases in the U.S. from March. The population-scaled analyses indicate that climate did not play a central role in early U.S. viral distribution and that human population density was likely the primary driver. We do find slightly more population-scaled viral cases in cooler areas. Ultimately, the temporal and geographic constraints on this study mean that we cannot rule out climate as a partial driver of the SARS-CoV-2 distribution. Climate\u2019s role on SARS-CoV-2 should continue to be cautiously examined, but at this time we should assume that SARS-CoV-2 will continue to spread anywhere in the U.S. where governmental policy does not prevent spread. In March 2020, the USA had the most reported cases of COVID-19, the disease caused by the coronavirus SARS-CoV-2 . While wOrganisms are distributed within their environment based on both direct and indirect interactions with biotic and abiotic variables \u2014SARS-CoVModeling the SARS-CoV-2 viral distribution from the early part of the outbreak in relation to climate and other abiotic variables could help refine our understanding of its spread, hopefully adding to the knowledge gleaned from studies on human transmission dynamics , which aEarly studies suggest that transmission of SARS-CoV-2 may have, at a minimum, a loose association with climatic features. There have been numerous reports showing a correlation of case incidence with cool temperatures and low humidity . FurtherBiologists often employ species distribution models  to predict geographic ranges of species. SDMs employ environmental data  to predict if geographic space is suitable for a given species or population . These mResearchers early in the pandemic created SDMs of SARS-CoV-2 using climatic variables . These sHowever, a research group put forth a strong rebuttal  to some Still, the climate-based SDMs for SARS-CoV-2 presented recently may, to at least some extent, reflect the climatic preferences of their host. Careful consideration of host availability (human population density) and pathogen ecologies (abiotic variables related to transmission) may be necessary to frame analyses modeling global distributions , and mayCOVID-19 was first detected in the U.S. on 20 January 2020 , has sinHere we develop a suite of data visualizations, SDMs and comparative niche overlap analyses using both climate and human population data to determine whether the effect of climate can be appropriately disentangled from other drivers of SARS-CoV-2 transmission, given early records for the virus in the U.S. We feel that examining a relatively early time-point of the pandemic in the U.S. is useful, as it in part (probably largely) predates the impact of major shifts in public health policy for the U.S.https://github.com/rsh249/cv19_enm/releases/tag/v0.0.5). All analysis code was written using R 3.6.2 (The code and results developed in this study are deposited under a CC-BY-NC 4.0 License on GitHub ( R 3.6.2 . Plots f R 3.6.2 .https://www.geonames.org/). While exact virus case geolocations or even town-level data would provide finer scale resolution to our analyses, these data are likely the best curated dataset for the U.S. for this time period.SARS-CoV-2 case data for U.S. counties  were collected from the New York Times database  on 31 MaInterpolated climate data averaged from 1970 to 2000 for the month of March were accessed through the WorldClim v2.1 database . The sevTotal cumulative positive case data for SARS-CoV-2 in each reporting county on 30 March 2020, were used to extract data for the seven abiotic variables listed above. Probability density distributions for SARS-CoV-2 were produced to characterize the likelihood of case occurrence given the available range of climate values. These distributions are calculated from case occurrences and corresponding climate data using a Gaussian Kernel Density Estimator and standard bandwidth estimation. These distributions can be interpreted similarly to histograms, but are normalized to be comparable between different sample sizes. Raw case data  were scaled to reflect virus cases in each county unit by dividing total cases by the county population from the 2010 U.S. Census. These population-scaled viral cases (cases/population) were used as probability density weightings, and resulting curves were standardized to an area of one. Probability densities were also calculated with raw case count data and county-level population as weightings. Probability density estimation and visualization was done with the R \u201cggplot2\u201d package . To testOccurrence data for raw  SDMs were generated by expanding each county climate record by multiplying that occurrence by the total SARS-CoV-2 case count for each county. Occurrence data for population-scaled SDMs were generated the same way as for the raw SDMs except that county climate records were multiplied by the total case count divided by the county population. Then population-scaled values were multiplied by an expansion factor of 100,000 so that all counties with at least one case were represented. More explicitly: raw data = total viral cases; population-scaled data = total viral cases/human population \u00d7 100,000 (the expansion factor for our data). Viral host availability  was modeled to serve as a null distribution to be used as a control comparison for viral SDMs.Maxent, an algorithm for presence-only distribution models , parametGiven recent critiques, it has become clear that when using SDMs in relation to SARS-CoV-2, it is worth documenting how close a study may come to the gold standards set for this type of modeling . The modNiche overlap and similarity tests were conducted with the \u201cecospat\u201d library  to compaThe human population climate curves are visually similar compared to the population-scaled SARS-CoV-2 climate curves for all seven abiotic variables for March 30 . There dr = 0.75, p < 2E\u221212); between the number of humans in a county and the number of viral cases; however, the population-scaled viral cases do not have a strong relationship  with human populations . Population-scaled case and human population models built using these optimal model parameters appear to be highly similar, with areas of high suitability predicted for much of the West Coast and most of the eastern half of the U.S. . There aModel testing for the SARS-CoV-2 raw case dataset identified a Maxent model with linear and quadratic feature classes and a regularization multiplier of 1.5 as the optimal model with a high model fit (Avg. Test AUC = 0.88), but with a slightly lower model transferability evidenced by higher AUC variance (Test AUC variance = 0.007) than the population-scaled model. The SDMs of the raw virus cases  fail to p < 0.01) than would be expected by chance (p = 0.02), while the overlap test was insignificant (p = 0.20), as seen in The niche overlap and similarity tests both find that the models of SARS-CoV-2  and human population have significantly higher overlap  Issue: the virus is spreading and the population was not in equilibrium with climate or any other putative niche dimension in March, let alone today. Our thoughts: it was not in equilibrium and that is imperfect; however, SDMs have been useful for a number of non-equilibrium systems, like species invasions during their spreading phase  IssIt has now been argued that SDMs of SARS-CoV-2 can help to predict where the virus may generally be found now and in the future , as wellThere are individuals who regularly go undetected for SARS-CoV-2 \u2014currently estimated at up to 25% . This isOur results are based on data for a single, large country. While conducting an analysis on global data would have been ideal, we avoided this because this type of global analysis is known to have certain issues and inconsistencies . While ihttps://github.com/rsh249/cv19_enm/releases/tag/v0.0.5). We believe it is imperative for all pipelines and scripts to be made available for any SARS-CoV-2 research to ensure that models can be improved upon and any errors can be more quickly uncovered and resolved.We believe the New York Times  providinThere are many avenues to pursue regarding SARS-CoV-2 modeling and predictions. We are excited to see researchers from a variety of fields extending their toolkits towards understanding this virus. We hope that ecological studies like this and others can play a role without overcomplicating the research efforts put forth by epidemiologists. Still, studies should familiarize themselves with current critiques of SDMs for SARS-CoV-2 modeling and be cautious of their inputs and conclusions.With improving data, we feel that future studies should better be able to examine the system globally while considering human populations and public policy efforts at curbing the virus. We also believe that it will at some point, in the U.S. and elsewhere, be worth examining death rates across different areas, as it would be helpful to know if climate or other abiotic variables might impact this statistic. Coupling regularly updated data with automated online resources would also be particularly helpful in learning how this virus may spread.Species distribution models from SARS-CoV-2 population-scaled cases did not appear to be distinguishable from human population density for an early point in the pandemic for the U.S. Future studies looking at climate\u2019s impact on this virus should, wherever possible, take into account human population density in any analyses.10.7717/peerj.10140/supp-1Supplemental Information 1Probability densities of SARS-CoV-2 coronavirus cases  compared to the probability densities of human populations (curves in blue) in each US county for each of seven climate variables. Probability density curves are standardized to an area of one.Click here for additional data file.10.7717/peerj.10140/supp-2Supplemental Information 2(A) Species distribution model of the SARS-CoV-2 coronavirus (using raw data) for March 30, 2020. (B) Human population distribution model for the US from 2010.Click here for additional data file.10.7717/peerj.10140/supp-3Supplemental Information 3p-values correspond to greater niche overlap or similarity than expected by random models.(A) Niche Overlap and (B) similarity tests for Maxent species distribution models built with SARS-CoV-2 coronavirus case data compared to one built with human population density as occurrence data; actual model overlap indicated by a red marker in both plots. Significant Click here for additional data file."}
+{"text": "Mediator of Paramutation1 (Mop1), a gene encoding a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on  MuDR element, it is essential for preventing heat-induced, stable and heritable changes in both H3K9me2 and H3K27me3 at this element, and for concomitant changes in transcriptional activity. These finding suggest that RdDM acts to buffer the effects of heat on silenced transposable elements, and that a loss of DNA methylation under conditions of stress can have profound and long-lasting effects on epigenetic silencing in maize.Most plant genomes are mostly transposable elements (TEs), most of which are held in check by modifications of both DNA and histones. The bulk of silenced TEs are associated with methylated DNA and histone H3 lysine 9 dimethylation (H3K9me2). In contrast, epigenetically silenced genes are often associated with histone lysine 27 trimethylation (H3K27me3). Although stress can affect each of these modifications, plants are generally competent to rapidly reset them following that stress. Here we demonstrate that although DNA methylation is not required to maintain silencing of the  Transposable elements (TEs) are a ubiquitous feature of all genomes. They survive in large measure because they can out-replicate the rest of the genome . As a coIn plants, heritable epigenetic silencing of TEs is almost invariably associated with DNA methylation \u201312. The de novo methylation of newly synthesized DNA from previously methylated DNA sequences is thought to be transcription by RNA POLYMERASE IV (POLIV) of short transcripts from previously methylated templates We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Best regards, OrtrunOrtrun Mittelsten ScheidAssociate EditorPLOS GeneticsWendy BickmoreSection Editor: EpigeneticsPLOS GeneticsAs you will see from the detailed comments of the reviewers, they agree that your data about the cooperation of epigenetic factors at a specific transposon under different genetic and environmental conditions revealed interesting and novel insight into the complexity of transposon silencing. The MuDR element is documented as an excellent and well-defined model. It is to be hoped that the findings will prove valid for other elements, but evidence for this and a broader analysis is certainly future work.However, the reviewers have suggestions and concern that require revising the current manuscript thoroughly. They request full data availability, state-of-the-art quantification of gene expression and bisulfite conversion control, and access to internal controls for some of the assays. They recommend providing more and better references to previous work on the maize RdDM mutants and the role of the polycomb complex beyond FLC. An important point raised repeatedly is the striking difference of heat stress activation between younger and older developmental stages, which should be explained or at least discussed. The different kinetics of transgenerational methylation adaptation could be strengthened as suggested by data for the intermediate generation.Text and Figures need several modifications, additions, and careful editing to ensure congruency and correctness.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1:\u00a0The manuscript by Guo et al., \u201cRNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays\u201d shows how MuDR transposon silencing is maintained by overlapping epigenetic mechanisms: DNA methylation, H3K9 and K27 dimethylation (me2) as well as H3K27me3. Several findings presented the manuscript are novel and significant for the field of epigenetics and transposon regulation:- A single transposable element (TE) displays different epigenetic regulation on its 5\u2019 and 3\u2019 ends (TIRA and TIRB respectively) through histone modifications, H3K9me2 and H3K27me2 on TIRA, and H3K27me3 on TIRB. Although those marks are well associated with transcriptional gene silencing, they have been shown to play different roles . Here, the authors illustrate a very clear example of all those marks cooperating to maintain a functional TE under control.- The authors also show how loss of RdDM and DNA methylation, present at both TIRs together with histone modifications, does not result in the immediate transcriptional reactivation and transposition of MuDR. However, in the absence of DNA methylation MuDR reactivates upon heat stress. Many studies in Arabidopsis have shown that loss of RdDM is largely inconsequential to plant development or genome instability caused by TE reactivation under laboratory standard growth conditions. Here, it is shown how DNA methylation is not only required for the transgenerational stability of the TE silencing, but to prevent the mobilization of MuDR under environmental stresses that might represent a more natural situation.The manuscript is well written, methods are well described, and the claims are properly contextualized in light of current literature. There are only minor corrections that would improve the quality of the manuscript that are listed below:- As stated in the data availability policy: \"numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information\". I couldn't find any spreadsheet with data relative to qPCR and phenotyping graphs while the authors claim that \"all data ara fully available without restrictions\". Authors should check with editors whether the raw data should be provided for those figures to be on the safe side.- The introduction does a very good job at explaining the reader the biological model and working system. However, I have the impression it fails to introduce the biological question that the authors want to address. This is found within the first three sentences of the results section (lines 202-204).- In lines 208-210, the authors state that \u201cin control plants \u2026 all cytosines in TIRA were unmethylated \u2026 indicated that bisulfite conversion was efficient\u201d. I disagree, as bisulfite conversion is performed independently for each DNA sample, the only way to address conversion efficiency is to investigate DNA methylation levels from sequences with known methylation (no DNA methylation) within the same DNA sample or spiked unmethylathed DNA before conversion (such as plasmid or lambda DNA). Authors might want to rephrase or eliminate the statement.- In several instances  expression of MuDR is investigated by RT-PCR. qRT-PCR might have been more appropriated as means to properly investigate subtle effects on MuDR expression.- Given that the authors have used internal controls for the ChIP-qPCR it would be advisable to add those in the supplementary data as means for the reader to see the intrinsic variability of their ChIP experiments.Reviewer #2:\u00a0RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.Guo et al. provides a highly detailed study of the epigenetic regulation related to the activity of a single Mu element in wild type and mop1 mutant maize plants. The work describes the relations of DNA methylation and histone modifications of the element's terminal inverted repeats to transcriptional silencing and reactivation by the Muk element and heat, respectively. The latter relations are determined further in the context of transgenerational inheritance.A strength of the work is the clearly defined genetic test system. By combining it with RT-PCR, clonal bisulfite sequencing, and ChIP analyses, the authors convincingly demonstrate that loss of DNA methylation in mop1 mutants does not prevent heritable silencing. Instead, the stable silencing in mop1 mutants is associated with the increase of different histone modifications at the two terminal inverted repeats of the Mu element, which mediate promotor function for the respective genes. For TIRB H3K27me3 is newly attributed to transposon silencing in maize.As the main result the authors demonstrate that in contrast to wild type, missing DNA methylation in mop1 mutants allows for reactivation of the TE by heat treatment, which is associated with a reduction of the respective repressive histone modifications. This result indicates DNA methylation in functioning as a buffer against the effects of heat stress.The reactivation was seen in leaves of young, 14 day-old, but not in leaves of older, 28 day-old, plants. Given the relatively short time interval between the sampling and the similarity of the tissues, this result is somehow unexpected. Do the older plants respond to the heat treatment as tested by hsp90 expression in the younger ones?In the analyses of transgenerational stability of the reactivated state for TIRA both MuDR/-; +/+ active and H5 reactivated samples show extreme, fully demethylated DNA. In contrast the heat stressed (H2 reactivated) samples are strongly methylated in all contexts as the silenced MuDR*/-;mop1/+ are. For TIRB similar results were obtained but only related to asymmetric DNA methylation. These results are discussed as an adaption of the methylation state to the activity state of the locus, only after multiple generations. To strengthen this point it would be required to analyze DNA methylation in the generations in between i.e. H3 and H4.Although this work is a mainly descriptive work on a specific example of a single transposon type exemplar, the indications of this work that DNA methylation can rather respond to than determine the activity of a locus and that the balance of DNA methylation and histone modifications is dependent on transcriptional activity, adds important information on the variety of mechanisms involved in transgenerational silencing of transposable elements in maize. Especially, it points to new unexplored mechanistic relations and independence respectively between DNA methylation and histone modifications in this context. The main statement that RdDM is responsible for the prevention of the transposon silencing reversal is solely built on the involvement of MOP1 in RdDM. The analysis of non-coding RNA could have contributed to a mechanistic understanding of the findings.Minor comments:How do the author explain the loss of methylation at the 5' end of the TIRB in MuDR*/-; mop1/+ silenced plants in the generation following Muk exposure?Line 35: dimethylation instead of demethylationThat Aat is a housekeeping gene that was used as a positive expression control should be mentioned already in the legend of figure 1.In figure 3A, right scheme L7 instead of L3 was collected according to the main text.Sentence in line 322 needs rewordingThat MuDR~ indicates a reactivated MuDR element should be mentioned already in the legend of figure 4.In figure 7 B sequence orientation of TIRB is reversed compared to figure 1.Line 810: Ear ears (doubling)In the legend of figure 6 description of C is missingReviewer #3:\u00a0In this work, Guo et al. use the maize Mutator system for studying the epigenetic silencing mediated by the RdDM pathway. For doing this, they produced maize plant families segregating for a single silenced MuDR element and homozygous or heterozygous for mop1 . In brief, by analyzing MuDR expression, DNA methylation and specific histone modifications (H3K9 and H3K27di-tri methylation) at TIRs of the Mu elements, they observed that loss of methylation does not result in a restoration of that transposon activity; instead, histone modifications are responsible for heritable maintenance of silencing. In addition, in mop1 mutants a short exposure to high temperature rapidly reverses both transcriptional silencing and histone modifications in a heritable way. Based on these observations, they concluded that DNA methylation is not necessary to maintain silencing although, they suggest, it is required to buffer the effects ofthe environmental stress on transposable elements.In general, I find the manuscript not accurately and not clearly written; it contains many typing errors that complicate the reading. There are also some incongruences in the Figures and in particular, some explanations regarding the gels are incomplete.I also have other concerns on the manuscript content.When describing the mop1 mutant (lines 91-99) the authors use the definition \u201clargely phenotypically normal\u201d.This definition is quite ambiguous, especially because the mutant plants are used in heat stress experiments and developmental/genetic defects might indirectly interfere with stress response.Moreover, much research work has been done and published on maize RdDM pathway and other maize epiregulator mutants, which can be either cited or at least discussed here. They also could be tested for validating the results of this work in different epigenetic background. In particular, the relationship between H3K9 methylation and DNA methylation is not sufficiently described, although this is fundamental to discuss the results of the work presented in the manuscript.Similarly, I find that citing the peculiar example of AtFLC locus, which is regulated by vernalization and many other epigenetic marks at its chromatin, for describing the role of H3K27me3 modification in gene transcriptional regulation does not give an appropriate and exhaustive information on what is known on this modification on different genomic context.Regarding the results of the work, my principal and general concern is that the authors investigated the variation in methylation and a few histone modification patterns specifically and only at the MuDr sequences; this approach is quite limiting in studying the regulation of transposons expression by pathways that are known to have a genome-wide role. I think that \u201ccontrol sequences\u201d must be used in the experiments to validate the results obtained for MuDr. Although many genotypes and progenies were characterized for transcriptional activation, DNA methylation and histone modifications, no information on the effect of the MuDr integration on the mopi1 genetic background or of the stress treatments at are genomic loci are reported in the manuscript.The observation that the combination of heat stress and mop1 mutant reactivated the transposon transcription is quite interesting and very similar to the cited example of Onsen in Arabidopsis. However, the authors observed that the reactivation of MuDR occurred only in young maize plant leaves, while older plants behave differently. No explanations or discussion on these findings were provided by the authors.After reading the results of this work, we can be convinced that a couple of histone modifications have a principal role in controlling MuDr activation than DNA methylation in maize; however, we miss any evidence and sustainable hypothesis on the epigenomic context and on mechanisms which mediate this activation.Minor points:Grammatical errors in lines:41, 47, 48, 76, 95, 101, 109, 137, 173, 188, 233, 264, 321, 380, 395, 397, 409, 411, 427, 510, 517In all the gel images please explain the lane and use a ladder. In general, the quality of the figures must be improved.Fig 3.A MuDR*/- Fig 4 is a repetition of Fig3Reviewer #4:\u00a0Review of Guo et al., 2020 - RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea maysREVIEWER SUMMARY:In this manuscript, the authors investigate the relationship between the loss of CHH methylation due to mutation in Mop1 and transcriptional activation of the stably silenced Mutator DNA transposon, MuDR, in maize. They found that loss of mop1 function and CHH methylation at MuDR TIRs does not alleviate silencing of MuDR but results in an increase in H3K9me2 at the 5\u2019 TIR (TIRA) and an increase in H3K27me3 at the 3\u2019 TIR (TIRB) which suggests that these marks are required to maintain silencing. In addition to differences in histone modifications between TIRs, the mop1-dependent DNA methylation pattern differs between TIRs where TIRA loses methylation in all sequence contexts while TIRB only loses CHH methylation suggesting that MuDR is regulated by different epigenetic pathways despite high sequence similarity between TIRs. Stably silenced MuDR (MuDR*) plants homozygous for a mop1 loss of function mutation display reactivation of MuDR upon exposure to a 4-hour heat treatment at the 14-day seedling stage that is associated with reduction of H3K9me2 at TIRA and H3K27me3 at TIRB, with a concomitant increase in H3K4me3. This reactivation is constrained to a specific developmental window and dependent on loss of mop1 function because reactivation is not observed in mop1/+ heterozygotes, +/+ wildtype, or at the 28-day seedling stage in the mop1/- background. The authors show that the heat-dependent activation of MuDR is somatically heritable in the absence of the inducing signal by analyzing leaf 10 and tissue from the tassel which are derived from primordial cells generated well after the stress was applied. This reactivation is also inherited transgenerationally for at least 5 generations in the presence of functional mop1 (mop1/+) and MuDR TIRs maintain lower levels of H3K9me2 and H3K27me3 and high levels of H3K4me3. The authors show that methylation patterns progressively resemble stably active MuDR elements over 4 generations in the presence of functional Mop1, with TIRA progressively losing all methylation and TIRB gaining methylation in all sequence contexts.SIGNIFICANCE OF FINDINGS:These results are significant because they dissect a well-characterized epigenetically regulated locus to show that mop1  functions to buffer MuDR silencing from alterations in chromatin modifications (H3K9me2 and H3K27me3) during heat stress. Loss of DNA methylation has been shown to alleviate silencing at many different TEs but the authors clearly show here that MuDR relies on two epigenetic silencing pathways that may have different requirements for DNA methylation. Although this work is focused on a single locus, it is novel because it describes the relationship between loss of methylation and transgenerational inheritance of heat-stress altered chromatin states and transcriptional activation of MuDR. The fact that RdDM is required for preventing heat-induced transgenerational epigenetic changes is fascinating and will require further investigation to determine the generality of these results. This work is important because it increases our understanding of the role of RdDM and builds on previous work investigating stress-responsive transposon activation and the function of Mop1 in maintaining silencing during stress.OVERALL QUALITY AND PRESENTATION OF THE WORK:The research methods described are certainly of sufficient quality to draw the authors\u2019 conclusions. However, the presentation of the work, especially the figures, require correction and appear to be incomplete (see reviewer comments). Although the rationale and approach are clear and justified, the manuscript should be carefully edited for sentence structure and brevity before publication.SPECIFIC COMMENTS:Abstract/Introduction The authors refer to Mop1 as Modifier of paramutation but the commonly used name is Mediator of paramutation.Figure 2 Lines under the genotypes indicating silenced versus active are not positioned correctly. Figure 4 Add to legend that red text is heat stressed and black text is control.Figure 6 Figure caption is not correct. No caption for 6D, 6C caption incorrect.Figure 8 Lines under genotypes indicating silences versus active are not positioned correctly.Figure S3 It is important to show HSP90 expression in tissue-matched non-stressed control plants.74-78 Pol IV and V are DNA-dependent RNA-polymerases, not DNA polymerases.Introduction/Discussion The authors need to include discussion of mop1/RdDM in stress response. Although mop1 does not dramatically alter transcription under normal conditions, mop1 has strong effects on ABA-responsive transcriptional programs  and loss of rmr6 (Pol IV) also shows defective response to drought . The authors should also discuss Mikula, Genetics, 1995 which showed that heat-stress alters epigenetic inheritance of R paramutation in maize.General Reviewer Comments It would be interesting to test the heat-dependent MuDR* reactivation in MuDR*/-;MuKiller/-;mop1/- plants. This would be in the presence of the inducing factor and could provide information about how heat and loss of RdDM affects the establishment of silencing. This is not critical to the relevance of this manuscript.386-390 The author states that the increase of methylation at TIRA in the active MuDR/-; Mop1 WT lines is associated with the decrease in H3K9me2 but Figure 7 shows that TIRA is unmethylated in active wildtype MuDR lines and is not consistent with the previous paragraph.Discussion The authors do not discuss the heritable increase in H3K4me3 and how this mark, and the active transcription of the genes associated with it, are relevant to maintaining that activation even in the presence of RdDM and differences in methylation patterns of TIRA and TIRB.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the No:\u00a0As stated in the data availability policy: \"numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information\". I couldn't find any spreadsheet with data relative to qPCR and phenotyping graphs while the authors claim that \"all data ara fully available without restrictions\". Authors should check with editors whether the raw data should be provided for those figures to be on the safe side.Reviewer #1:\u00a0Reviewer #2:\u00a0YesReviewer #3:\u00a0NoneReviewer #4:\u00a0Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article , there is only one point that needs another revision: reviewer 2 found some discrepancy between the new data added and the interpretation in the text. We therefore ask you to modify the manuscript accordingly.\u00a0In addition we ask that you:1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.archive. 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You will be contacted if needed following the screening process.PLOS has incorporated\u00a0To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Ortrun Mittelsten ScheidAssociate EditorPLOS GeneticsWendy BickmoreSection Editor: EpigeneticsPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1:\u00a0The authors have answered to all my comments and I'm satisfied with the revised version of the manuscript. Therefore, I recommend it for publication.Reviewer #2:\u00a0Guo et al. provides a detailed study of the epigenetic regulation related to the activity of a single Mu element in wild type and mop1 mutant maize plants. The indications of this work that DNA methylation can rather respond to than determine the activity of a locus and that the balance of DNA methylation and histone modifications is dependent on transcriptional activity, adds important information on the variety of mechanisms involved in transgenerational silencing of transposable elements in maize. Especially, it points to new unexplored mechanistic relations and independence respectively between DNA methylation and histone modifications in this context.In the revised version my comments are adequately addressed, except the concerns raised in the context of the transgenerational adaption of the methylation states to the activity states after stress induced reactivation:In the analyses of transgenerational stability of the reactivated state for TIRA both MuDR/-; +/+ active and H5 reactivated samples show extreme, fully demethylated DNA. In contrast the heat stressed (H2 reactivated) samples are strongly methylated in all contexts as the silenced MuDR*/-;mop1/+ are. For TIRB similar results were obtained but only related to asymmetric DNA methylation. These results are discussed as an adaption of the methylation state to the activity state of the locus, only after multiple generations. To strengthen this point it would be required to analyze DNA methylation in the generations in between i.e. H3 and H4.The authors now provide additional data for TIRA and TIRB on H4, which show the same full demethylation as in H5. The text is not changed accordingly (Line 377) to mention the analysis of the H4 generation neither the conclusion that multiple rounds of meiosis are likely required to adapt the methylation profile to the active state. Clearly this conclusion is weakened by the new results, but can be resolved only by analysis of H3 and the heat-stressed, reactivated H1, which the authors do not provide. The new results however indicate that a gradual loss of methylation in adaption to the activity state of the elements over multiple generations is less likely and it is questionable whether meiosis is required or related to the phenomenon at all. Alternatively, the adaption of the methylation state might be independent of meiosis and started already in H1. Indeed, although not quantitatively analyzed, the methylation profiles of reactivated H2 show demethylation compared to the silenced H1 generation. The new results should be described in the text and the interpretations adjusted.Examples of remaining grammatical or typing errors:Line 386 \"latter\" instead of \"later\"Line 332 \"could\" is doubledLine 485 \"in maize\" instead of \"is maize\"Reviewer #3:\u00a0The authors have taken into consideration all concerns raised by my first report at theoretical level. I think that both introduction and discussion were greatly improved.I understand that the study of the their system/model in a different genomic background is not that simple as well as it is not the validation of the results for MuDr at histone modification level. However, I am convinced that these validations are essential to confirm their observations.Reviewer #4:\u00a0All the major issues are addressed, minor issues are listed belowLine numbers Comment47 Change out-replicated to out-replicate145-147 Sentence is confusing, please clarify.543 Change understanding to understand566 Remove \u201cfor\u201d or \u201cin\u201d595 Spelling error for collected**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1:\u00a0YesReviewer #2:\u00a0NoneReviewer #3:\u00a0YesReviewer #4:\u00a0Yes**********what does this mean?). 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PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  10 Jun 2021PGENETICS-D-20-01828R2 RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea\u00a0mays. Dear Dr Lisch, We are pleased to inform you that your manuscript entitled \"RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea\u00a0mays.\" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. 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+{"text": "In general, the MRF, IBF, CCOS and shallow DCE combined technique brought much help to the enhancement of laser damage resistance of fused silica, and could be used as a process route in the manufacturing process of fused silica.The enhancement of laser damage resistance of fused silica optics was a hotspot in scientific research. At present, a variety of modern processes have been produced to improve the laser induced damage threshold (LIDT) of fused silica optics. They included pre-treatment processes represented by flexible computer controlled optical surfacing (CCOS), magnetorheological finishing (MRF), ion beam finishing (IBF), and post-treatment processes represented by dynamic chemical etching (DCE). These have achieved remarkable results. However, there are still some problems that need to be solved urgently, such as excessive material removal, surface accuracy fluctuation in the DCE process, and the pollution in MRF process, etc. In view of above problems, an MRF, CCOS, IBF and shallow DCE combined technique was used to process fused silica optics. The surface morphology could be greatly controlled and chemical etching depth was reduced, while the LIDT increased steadily. After processing by this combined technique, the LIDT increased to 12.1 J/cm J. Bude found particle pollution on the surface limited the LIDT improvement of fused silica optics. After advanced migration process (AMP) 3.0 process, the particles were obviously restricted, and the surface was free from damage under 351 nm, 3 ns (FWHM), 9.5 J/cm2 laser irradiation [As a material with excellent optical properties, fused silica was widely used in the manufacturing process of key optics in high-energy laser systems, such as National Ignition Facility (NIF) in the United States, SG-III laser facility in China and Laser Megajoule system in France, etc. ,2,3. ForEven so, the laser damage resistance of fused silica optics still could not satisfy the service requirements, so scholars considered that the existence of micro defects limited the further improvement of LIDT of fused silica optics, including small-scale mechanical defects in the sub-surface damage (SSD) layer ,7, conta62\u2212, which was not conducive improving the damage threshold further [Scholars decided to examine the micro defects issue further. Shao Ting found that a deep DCE technique could remove chemical structure defects (oxygen deficit center (ODC)/non-bridging oxygen hole center (NBOHC) formed in RIE process . Sun Lai further ,14,15,162 [2, which was related to the residual metal elements such as Fe. The existence of metallic pollution would increase the absorption of laser energy and result in laser damage [Ion beam finishing (IBF) was a non-contact flexible technique which could achieve material removal by high-energy ion beam . During 2 . Magneto2 . Howeverr damage . For theIn this work, the MRF, IBF, computer controlled optical surfacing (CCOS) (this technique was used to control the medium-high frequency errors introduced in MRF process), shallow DCE technique was used to fabricate fused silica optics, and the changes of surface morphology, photo-thermal absorption, and LIDT were explored. In We had prepared four square optics , serial number: 1\u20134, damage layer depth: 1\u20132 \u03bcm) with the size of 35 mm \u00d7 35 mm \u00d7 10 mm, produced by the same vendor with conventional grinding and polishing process.Pre-treatment process: Sample 1 was blank and used as control. Sample 2 was processed by MRF   was used for pre-cleaning (time: 80 min), to remove the pollution on the surface introduced by pre-treatment and transportation process; (2) deionized water  was used to remove the residual inorganic acid, after which the samples were dried by air; (3) Etchant   was used to etch the surface materials and the etching rate was determined to be 0.1 \u03bcm/min. Sample 1\u20134 was etched for 1 min; (4) after the HF-based etching process, the samples were cleaned by deionized water. Steps 1\u20134 were carried out in a Class 100 clean room.Post-treatment process: After pre-treatment processing, sample 1\u20134 was processed by the DCE technique including inorganic acid cleaning process and hydrogen fluoride (HF)-based etching process. The DCE process was carried out under megasonic conditions which were produced by a Teflon-lined, multi-frequency megasonic transducer , 1.3 MHz), and the whole DCE process was divided into four steps: (1) inorganic acid  analysis method was carried out, and the valid region of the PSD curve was chosen to analyze the changes in manufacturing process. The PSD curve showed the correlation between amplitude and specific frequency, and could be used for the medium-high frequency error analysis of the optical surface.The absorptivity of optics was detected by a large aperture photo-thermal absorption platform . When the surface was irradiated by laser, some areas absorbed energy, and resulted in a rise in temperature, which would cause the changes of refractive index or other parameters. This minor difference would be captured by the detector, and then the absorptivity could be calculated. During the test, the inspection caliber was set at 35 mm \u00d7 35 mm, step length 1 mm, pump power 2 W, pulse repetition frequency (PRF) 50 kHz, polarimetric whitening filter (PWF) 950, integral time 300 ms, test wavelength 355 nm. The measurement method was transmission and the test sensitivity was better than 0.05 ppm.The LIDT test was carried out with 1-on-1 mode on the LIDT test platform equipped with a frequency-tripled Neodymium (Nd): Yttrium Aluminum Garnet (YAG) laser. The laser could produce 5 ns (FWHM) pulse in a single-longitudinal mode with the repetition rate of 1 Hz. The wavelength of the laser was operated at 355 nm and the spot diameter of laser beam was about 1.2 mm. During the test, ten testing sites with a certain laser flux were chosen on the surface and the distance between the two sites was 3 mm. The laser damage spot was observed by a long-focal-distance camera. The damage probability was obtained by calculating the number of the laser damage spot at each gradient.The damage morphology was detected by high-resolution microscope . The magnification was operated at 500 times. Through the test, the morphology characteristics of laser damage could be obtained.The roughness of sample 1\u20134 after pre-treatment technique was detected by MicroProf WLI . Nine spots were chosen and a single region was set at 0.47 mm \u00d7 0.35 mm. The roughness was taken from the average value of the nine spots.To accurately grasp the changes of the medium-high frequency of optical surface, we extracted the PSD information of sample 3\u20134, and the valid region was chosen to be analyzed.In In order to clarify the absorption characteristics, a large aperture photo-thermal absorption platform was used to detect the absorptivity level of sample 1\u20134 (processed by pre-treatment technique).In After that, we detected the absorption level of sample 1\u20134 after the DCE post-treatment process.According to In order to explore the changes of anti-laser damage characteristics of optics under different techniques, an LIDT platform was used to detect the damage threshold of sample 1\u20134. The 0 probability damage threshold and 100% damage threshold results are shown in 2, and 24.64 J/cm2, respectively. Compared with other samples, its anti-laser damage characteristics were the best. The 0 probability and 100% probability damage threshold of sample 1 were 12.54 J/cm2 and 22.33 J/cm2, which were close to that of sample 2. For sample 3, its damage threshold was the lowest, and the values were 8.58 J/cm2 and 12.65 J/cm2, respectively. Through the results, it was determined that the trend of 0 probability damage threshold of different samples was the same as that of the 100% probability damage threshold, namely 4 > 1 = 2 > 3.In In order to clarify the difference of damage threshold between 1\u20134 further, we fitted the threshold test results. 2. After processing by MRF, CCOS, IBF and the shallow DCE combined technique, the laser damage resistance of the optical surface was significantly improved. The fitting value of sample 2 was 10.9 J/cm2, which was a little higher than that of sample 1 (10.8 J/cm2). The fitting value of sample 3 was 9.1 J/cm2, which was lower than those of the other three samples.In From In this part, we detected the laser damage morphology after LIDT test by high-resolution microscope, and the results are shown in In As shown in the results in Though many scholars had already confirmed that MRF and IBF improved the surface quality of fused silica optics, a few questions were still worth discussing in this work, such as (1) sample 1 had lower absorptivity than sample 4, but its LIDT was lower than sample 4. What were the reasons that led to mismatching between photo-thermal absorption and LIDT; (2) after processing by the CCOS technique, the medium-high frequency errors of sample 3 were controlled a, but itFor question 1, the sub-surface damage layer was thought to be the major factor, its existence led to the mismatching between photo-thermal absorption and LIDT. In the traditional polishing process, material removal was mainly achieved by a scratching effect between the water-based abrasive and the optical surface, and a sub-surface damage layer with a certain depth about hundreds nanometers to a few microns would form under the surface. In the sub-surface damage layer, a small amount of impurities and scratches existed, and they would cause intense energy absorption in the LIDT test, which would cause laser-induced damage.After the traditional polishing process, no obvious defects appeared on the near surface. In a photo-thermal test, the relatively smooth near surface would not cause excess energy absorption when irradiated by the low-power pump of the absorption platform and the absorptivity would remain at a lower level. In contrast, the negative effects of the sub-surface damage layer were prominent in the LIDT test; the defects in the damaged layer would strongly absorb the laser energy and cause laser damage. In For question 2, in a previous study , we had In order to explore other factors further, we detected the roughness of sample 1\u20134 after the pre-treatment process.According to For sample 3, the increasing of absorption c also miThrough the absorption results and damage morphology results, we verified the negative effect of the hydrolysis layer further. Due to the existence of the hydrolysis layer, not only did the photo-thermal absorption decrease, but the LIDT of sample 3 had also been affected. For question 2, we thought that the influence of medium-high frequency control was weaker than the disadvantages of roughness deterioration and the existence of hydrolysis together, which led to the LIDT decreasing in sample 3.For question 3, the absorptivity fluctuation of sample 4 after the IBF process was related to the existence of a sputtering damage layer with different depth.N was the atomic density of material. \u03b3 was a mass related constant. m was a constant related to incident ion beam energy. \u03b5 was ion beam energy. mC was a constant related to interatomic potential. d(\u03b5) was the sputtering distance.In the IBF process, high-energy ions would invade the surface for a certain distance driven by an ion beam. According to Lindhand Scharff and Schiott (LSS) range theory and Sigmund sputtering theory, the sputtering distance was related to ion beam energy:a was sputtering distance. \u03b8 was incident angle of ion beam. \u03c3a and \u03bca were the deposition width of ion beam on the incident direction and perpendicular to the incident direction. sd was the sputtering damage layer depth. When the incident angle was 0 degree, Equation (2) could be changed to Equation (3):In Equation (1), the ion sputtering distance showed a positive correlation with ion beam energy. During the ion beam sputtering process, lattice dislocation, holes and other micro structural defects would be introduced along with the invasion of ions. The structural defects would cause a small amount of energy absorption. To clarify the relation between sputtering distance and ion beam energy, we established a model .(2)ds=aAccording to Equations (1)\u2013(3), the sputtering damage layer depth showed a positive correlation with the sputtering distance. When the energy beam increased, the ion beam sputtering distance increased and a deeper sputtering damage layer formed.Due to the micro fluctuation of the surface, the ion beam energy in different areas was different, the principle of which is shown in In In summary, the MRF, CCOS, IBF and shallow DCE combined techniques could remove the surface materials effectively along with keeping the surface quality stable, and the LIDT of fused silica optics could be improved steadily. Meanwhile, the sub-surface damage layer and the hydrolysis layer needed to be removed in order to inhibit the laser damage.2. From the results, it can be seen that absorption presents a strong correlation with LIDT, specifically that lower absorption could a obtain higher LIDT.As an important optical material, fused silica had been applied to the manufacturing process of optics used in high-flux laser systems, and its manufacturing level had a vital influence on the performance of the laser systems. After years of effort, modern optical processing techniques represented by MRF, IBF, and DCE had been developed to manufacture fused silica and to improve its laser induced damage threshold. In this work, MRF, IBF, CCOS and the shallow DCE combined technique was chosen to process fused silica optics, and the changes of photo-thermal absorption and LIDT in the manufacturing process were also explored. After processing by combined technique, the photo-thermal absorption of the optic decreased to 0.085 ppm while LIDT increased to 12.1 J/cmMeanwhile, we had found that some factors would affect the absorption properties and laser damage resistance of fused silica to varying degrees, including medium-high frequency errors, roughness, and the hydrolysis layer. Compared with other factors, the hydrolysis layer and the sub-surface damage layer had a great impact on LIDT. When irradiated by laser, the absorptive pollutants in the hydrolysis layer would cause complex laser damage. For the hydrolysis layer and the sub-surface damage layer, it could be removed by the MRF and IBF technique, which made the LIDT increase.In conclusion, MRF, CCOS, IBF, and the shallow DCE combined technique could effectively control the photo-thermal absorption and improve the laser damage resistance as well as keeping the surface quality stable. This process could lay a foundation for the manufacturing process of fused silica optics with high laser damage resistance and ensure the development of laser systems."}
+{"text": "Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 \u03bcM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice\u2019s na\u00efve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-IThe  The image of the scorpion has long been connected to human history, being represented in cults, legends, philosophy and arts, as it is one of the oldest animals on the planet. Dating from the Silurian period, more than 400 million years ago, scorpions are organisms that have long intrigued human beings ,2.Androctonus and Leiurus (North Africa and Middle East), Centruroides (Mexico and the United States) and Tityus (South America and Trinidad) -Proctolin -Proctolin [Interestingly, both hypotensins were able to increase the catalytic activity of ACE, but in different ways. While TsHpt-I activated ACE by 64%, TsHpt-II increased by 46% the hydrolysis of the substrate Abz-RGFK-EDDnp. In fact, in studies on the determination of the hypotensive mechanism of TsHpt-I, ACE activation can also be observed; however, the results were not discussed . Studiesroctolin , which mAs hypotensins demonstrated new activities in vitro, cytotoxicity and possible pro- or anti-inflammatory actions were investigated in order to increase our knowledge of these molecules. Both hypotensins have immunomodulatory potential, with pro-inflammatory effects on murine peritoneal macrophages, when used at a concentration of 100 \u00b5g/mL. Interestingly, at this concentration, both hypotensins did not exert cytotoxic activity on the tested cells, which makes the two molecules even more interesting, due to their pharmacological potential for the long-term development of new immunostimulants and/or adjuvants. Pucca and collaborators  demonstrT. serrulatus venom, based on its ability to reduce neutrophil infiltration and TNF production in a murine model of sponge implant-induced inflammation. On the other hand, increased macrophage infiltration was observed in this model, indicating a pro-inflammatory role, which demonstrates the need for further studies on the mechanisms of action of TsHpt-I [Cassini-Vieira and colleagues suggested an anti-inflammatory role for TsHpt-I from  TsHpt-I .Tityus stigmurus scorpion, were recently described and confirmed [Interestingly, both treatments with both peptides promoted a significant increase in the phagocytic index, demonstrating that the pro-inflammatory action of these peptides also affects the macrophages\u2019 biological function. This phenomenon is interesting, considering the possible development of immunomodulators. It is known that adjuvant and/or immunostimulant molecules generally induce a pro-inflammatory environment that favors the activation of antigen-presenting cells and, consequently, the development of adaptive immunity against specific antigens. Increased macrophages\u2019 phagocytic capacity by hypotensins may reflect increased microbicidal activity and/or antigen presentation. The anti-candida and anti-biofilm activities of TistH, a hypotensin present in the venom of the onfirmed , but funTityus serrulatus venom, new studies aiming at drug development should be very carefully carried out in order to minimize unexpected effects.Despite the biotechnological potential of hypotensins, the activities already described for these peptides, and the new results showed in the present work, indicate that both molecules do not have a specific target or mechanisms of action. Considering that they are multifunctional toxins present in the Despite the great similarity between the primary structures of hypotensins, different levels of interactions with the vasopeptidases ACE and NEP were observed. Hypotensins increase ACE activity at different levels while TsHpt-I is a non-competitive inhibitor of NEP, suggesting other hypotensive mechanisms for this peptide in addition to those already described. Furthermore, the release of some interleukins may suggest a role for these peptides in the inflammatory response induced by the venom. Hypotensins are multifunctional toxins, and further studies are needed to clarify the potential of these molecules for biotechnological use.E. coli 0127:B8, Trypan Blue, Giemsa stain and glutaraldehyde solution were purchased from Sigma-Aldrich . Neprilysin and the Fluorescence Resonance Energy Transfer (FRET) substrates, Abz-FRK (Dnp) P-OH and Abz-RGFK (Dnp)-OH, were provided by Prof. Adriana Carmona, from the Department of Biophysics of UNIFESP-EPM, S\u00e3o Paulo, SP, Brazil. Acetonitrile and TFA used in RP-HPLC were acquired from J. T. Baker . Fetal cow serum (FCS) and penicillin and streptomycin antibiotics were purchased from Cultilab . Tetrazolium salt 3--2,5- diphenyltetrazolium bromide (MTT) was purchased from Invitrogen . DMSO was purchased from Merck . BD Cytometric Bead Array Mouse Inflammation Kit was purchased from BD Biosciences . The Saccharomyces cerevisiae suspension was obtained from washing and adjusting the concentration of bread yeast  in RPMI.The synthetic peptides TsHpt-I and TsHpt-II were obtained by the solid-phase peptide synthesis method, and purchased from GenOne Biotechnologies . Angiotensin Converting Enzyme (ACE) from rabbit lung, RPMI 1640 medium, LPS from 2 10 \u00b5M, pH 7.0 buffer. For NEP, 10 \u00b5M of TsHpt-I or -II was incubated with 1.5 ng of peptidase, 3.5 \u00b5M Abz-RGFK (Dnp)-OH in Tris HCl 50 mM, pH 7.5 buffer. All reactions occurred at 37 \u00b0C, in a final volume of 100 \u00b5L, in a Victor 3 fluorimeter  adjusted for excitation and emission readings at 320 and 420 nm, respectively, for 15 min (one reader per minute). Results were obtained in triplicate and analyzed using GraFit 5 .For ACE assays, 10 \u00b5M of each peptide was incubated with 3.0 ng of peptidase and 10 \u00b5M of Abz-FRK (Dnp) P-OH substrate, in Tris HCl 100 mM, NaCl 50 mM and ZnCl2 10 \u00b5M, pH 7.0 buffer, and NEP (1.5 ng), in Tris HCl 50 mM, pH 7.5 buffer, at 37 \u00b0C for 4 h. Samples containing only the synthetic peptides were used as the negative control. After incubation, samples were analyzed by reverse phase chromatography on RP-HPLC , using a Restek Ultra C-18 column . Solvents used were 0.1% TFA in water (solvent A), and acetonitrile plus solvent A (9:1) as solvent B. Separations were performed at a flow rate of 1 mL/min and a 20\u201360% gradient of solvent B over 20 min. In all cases, elution was followed by the measurement of ultraviolet absorption (214 nm).The synthetic peptides TsHpt-I and TsHpt-II (30 \u00b5M) were incubated with ACE (3.0 ng), in Tris HCl 100 mM, NaCl 50 mM and ZnClTo determine the inhibition constant (Ki) of TsHpt-I over NEP, four concentrations of Abz-RGFK (Dnp)-OH  and 3 \u00b5M and 4 \u00b5M of TsHpt-I were tested using 1.5 ng of peptidase in 100 \u00b5L of final volume of Tris HCl pH 7.5. The Km value of the substrate used was determined to be 14 \u00b5M . Control2 chamber. All experimental procedures involving animals were in accordance with the ethical principles in animal research adopted by the Brazilian Society of Animal Science and the National Brazilian Legislation no.11.794/08. Animal care and experimental procedures were approved by the Institutional Committee for the Care and Use of Laboratory Animals from Butantan Institute .Male young, between 8 and 12 weeks of age and weighing between 20 and 22 g, BALB/c mice adults were used. Mice were obtained from the Central Animal Facility of the Butantan Institute and housed in the Laboratory of Immunochemistry bioterium, Butantan Institute. The mice were kept in boxes lined with shavings, containing 3 animals per box, under natural light, full-time ventilation and exhaustion, filtered water and commercial feed ad libitum. After a period of 2 to 3 days of acclimatization of the animals, they were euthanized in a COn = 6) were collected by two washes with 5 mL of RPMI medium. The cells obtained were washed twice with RMPI medium, at 400 g, for 10 min, at 18 \u00b0C, and resuspended in R10 medium . After counting in a Neubauer chamber, in the presence of Trypan blue, the cell concentration was adjusted to 2 \u00d7 106 cells/mL. Cells were distributed into 96-well culture plates (2 \u00d7 105 cells/100 \u00b5L/well) and incubated for 2 h at 37 \u00b0C and 5% CO2. After incubation, non-adherent cells were discarded, and adherent cells (macrophages) were resuspended in R10 medium containing the respective tested stimuli, at different concentrations, in triplicates, in a final volume of 200 \u00b5L/well. Cells resuspended in R10 medium were used as a negative control, and cells stimulated with LPS (5 \u00b5g/mL) as a positive control. Cells were then incubated for 24 h at 37 \u00b0C and 5% CO2. After this period, the culture supernatants were collected and stored at \u221280 \u00b0C, for subsequent dosage of cytokines, and cell viability was determined by MTT assay.Peritoneal exudate cells , MTT assays were performed, consisting of the addition of 100 \u00b5L of R10 containing 0.5 mg/mL of tetrazolium salt 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in all wells, followed by a new incubation, under the same conditions, for 4 h. Then, the supernatants were discarded, and 100 \u00b5L/well of DMSO were added to dissolve the formed crystals. Absorbance was determined in a spectrophotometer at 540 nm. Cell viability was calculated based on the absorbances of the samples and the negative control (cells cultivated only with R10).The concentration of pro- and anti-inflammatory cytokines  in the samples incubated with both hypotensins  was determined by the CBA method (BD Cytometric Bead Array Mouse Inflammation Kit), according to the manufacturer\u2019s instructions. The samples were acquired in a BD FACSCanto II flow cytometer, and the data were analyzed using BD FCAP Array version 3.0 software.n = 6) were obtained as described above (item 5.5.1). Cells were distributed in 24-well culture plates (5 \u00d7 105 cells/500 \u00b5L/well), with glass coverslips inside, and incubated for 2 h at 37 \u00b0C and 5% CO2. After incubation, non-adherent cells were discarded, and adherent cells (macrophages) were stimulated in vitro with hypotensins, in duplicate, at a concentration of 100 \u00b5g/mL, for 24 h at 37 \u00b0C and 5% CO2. After this period, the culture supernatants were discarded, and the cells were incubated for 1 h with a suspension of Saccharomyces cerevisiae at a concentration of 1.5 \u00d7 106 yeasts/mL/well. Coverslips were washed 10 times with PBS, fixed with 0.5% glutaraldehyde and stained by Giemsa. Phagocytosis was evaluated by immersion optical microscopy (1000\u00d7) and quantified by counting approximately 200 cells per cover slip. The percentage of phagocytic cells and the average number of internalized yeasts per phagocytic cell were calculated. The phagocytic index was obtained by multiplying the two values (percentage \u00d7 mean) and represents the total phagocytic capacity of each cell population.Peritoneal macrophages from na\u00efve BALB/c mice (p-value < 0.05 were considered significant.The results were statistically analyzed using the GraphPad Prism 5 program, using the one-way ANOVA test followed by Tukey\u2019s post-test. Results with a"}
+{"text": "Findings also showed that 14% of the participants exhibited compulsive buying behavior. This study provides sufficient proof of the reliability and validity of RCBS-TC and CHRS. Their relationship was explored based on two sets of samples from different regions in Asia, which contributes more applicability in a cross-cultural context.There is no previous research that has explored the correlation between compulsive buying and hoarding in the Chinese population. This study aims to determine the relationship between compulsive buying and hoarding in a sample of the Chinese population comprising participants from mainland China (emerging economy) and Hong Kong (developed economy). Self-reported measures consisting of demographic questions, the Chinese version of the Hoarding Rating Scale (CHRS), and Richmond Compulsive Buying Scale-Traditional Chinese (RCBS-TC) were administered to participants. After data collection, common method biases were precluded. The RCBS-TC and CHRS were validated by confirmatory factor analysis and found correlated by Pearson correlation coefficient. The RCBS-TC and CHRS demonstrated satisfactory levels of internal consistency . A three-factor model, including hoarding, obsessive-compulsive, and impulse control disorders, was obtained through Confirmatory Factor Analysis (CFA) with the satisfactory fit for the total sample from Hong Kong and mainland China. A significant correlation was found between RCBS-TC and CHRS ( Compulsive buying refers to the chronic, repetitive purchasing behavior in response to negative events and/or feelings , 1992. SPrevious studies on compulsive buying have mainly been conducted in developed countries or regions e.g., . In receJust as profound as compulsive buying, hoarding has aroused the interest of psychiatrists in the past decades. Hoarding was first regarded as a measure to cope with shortages and scarcity during the 1970s in the United States . McKinnoThe prevalence of hoarding has been estimated through samples in different contexts. In a German population-based sample, the prevalence of hoarding was around 4.6% without significant age and gender differences . A low lA common belief is that compulsive buying is associated with hoarding because nearly all hoarders suffer from compulsive buying, while not all compulsive buyers are hoarders , 2009. ICompulsive buying and hoarding are associated with OCD , 2002 anBased on the above discussion, issues in compulsive buying, hoarding, and the relationship between the two concepts need to be addressed. To our knowledge, no studies have been conducted in the Chinese population-based sample from both mainland China and Hong Kong. Therefore, for determining the relationship between compulsive buying and hoarding, it is necessary to carry out a study on compulsive buying and hoarding where the subjects are selected from a Chinese population-based sample.In the present study, we investigated compulsive buying and hoarding in a Chinese population-based sample using self-administered questionnaires. The purpose of the study was to explore the association between compulsive buying and hoarding based on a sample of Chinese from mainland China and Hong Kong. It could also create an opportunity to explore the relationship between compulsive buying, hoarding, and stages of economic development as Hong Kong is a developed region and mainland China is an emerging economy.This study employed a cross-sectional, multi-center, and correlational design.via an online survey. A survey was conducted in mainland China through Sojump, a popular online consumer panel survey company. Sojump developed a database of more than 2.6 million consumers from different cities in China. Participants received points as a reward for participation in the survey, and the points could be redeemed for various gifts. The online survey generated 632 valid responses. In Hong Kong, we created a website questionnaire. The link to the webpage was posted on Facebook and an online forum in Hong Kong. The online site collected 642 valid responses.All data were obtained The questionnaires consisted of three parts, including demographics, the Chinese Hoarding Rating Scale (CHRS), and Richmond Compulsive Buying Scale-Traditional Chinese (RCBS-TC). The demographic data included gender, age, marital status, education, and income level. Five questions were based on hoarding dimensions, including clutter, difficulty discarding, excessive acquisition, distress, and impairment, and six items that were about compulsive buying were divided into OCD and ICD dimensions.N = 1028)  was first developed by  = 1028) . Based o = 1028) . The con = 1028) . CHRS waThe Richmond Compulsive Buying Scale-Traditional Chinese (RCBS-TC)  is the tt-test, chi-square, and Cronbach\u2019s \u03b1), was conducted with SPSS software, version 22.0 for Windows. The Linear Structural Relations software, version 8.70 for Windows was used for the CFA. Common method bias was checked to confirm the distinct construct of two scales in three dimensions  and inferential statistics  and married containing cohabiting . Around 46.2% (N = 589) of the participants had a bachelor\u2019s or higher degree and 47.9% (N = 610) were under the age of 30. Although the level of income in mainland China is generally lower than that in Hong Kong, the sample size in the original interval of monthly income, namely, lower than HK$3000, HK$3001\u20135000, and HK$5001\u201310,000, was insufficient to determine the income differences between participants from Hong Kong and mainland China. As a result, these three options were merged into one, and we found that the monthly income of 55.8% (N = 710) of the participants was lower than HK$10,000.A total of 1,274 valid samples comprised 664 women and 610 men, taking up 47.9 and 52.1% shares, respectively. However, since some of the original options regarding age, marital status, education, and income level were lacking in sufficient samples, we combined several options. Marital status was re-grouped into two categories in which the marital status of all the participants fell in the option of single, including being single again after marriage (N = 642) and mainland China (N = 632) differed in age, marital status, education, and income. On average, the participants from mainland China were younger compared to participants from Hong Kong, but the latter were wealthier as expected. The education level among mainland Chinese participants was not balanced, with more participants having degrees but a lesser number holding high school certificates. A huge disparity in gender was not seen.As shown in Common method bias , which is usually caused by single measurement and data from the same source, can be ascribed to the measurement method rather than to the constructs . ParticuCommon method bias should be reviewed prior to the main study . In our The Cronbach\u2019s \u03b1 of RCBS-TC was 0.872, with corrected item-total correlation coefficients of 0.592\u20130.763. Both coefficients indicated that the scale had satisfactory internal consistency. The Cronbach\u2019s \u03b1 of CHRS was 0.828 with corrected item-total correlation coefficients of 0.560\u20130.712, which indicates satisfactory internal consistency.X2 = 299.9, p < 0.1, Non-Normed Fit Index (NNFI) = 0.97, CFI = 0.98, Standardized Root Mean square Residual (SRMR) = 0.036, RMSEA = 0.07; Hong Kong sample: X2 = 329.3, p < 0.1, NNFI = 0.96, CFI = 0.97, SRMR = 0.051, RMSEA = 0.105; mainland Chinese sample: X2 = 106.16, p < 0.1, NNFI = 0.98, CFI = 0.98, SRMR = 0.039, RMSEA = 0.05).Starting with one latent variable, CFA was conducted three times with one latent variable added each time. p < 0.1).All five items of CHRS and six items of RCBS-TC have a factor loading at or above 0.5 (as shown in The average variance extracted (AVE) of the hoarding dimension was 0.4981, which was lower than 0.5, but maybe acceptable because composite reliability (0.8292) was higher than 0.6, and hence, the convergent validity of the construct was still adequate . The AVEr = 0.473, p < 0.001). Considering the interrelations among the subscales of compulsive buying and hoarding, unlike the findings of r = 0.430, p < 0.01), but not the acquisition subscale. The weakest association was observed between the difficulty discarding subscale and RCBS-TC , which was consistent with the results of Mueller\u2019s research, but weaker.Pearson correlations were used between RS-TC and RCBS-TC to explore the relationship between compulsive buying and hoarding. The CHRS scores correlated significantly with the RCBS-TC scores but were not very strong  of compulsive buying, the CFA results were consistent with the findings of Significant correlations were observed between the measures of hoarding and compulsive buying. The associations between the two scales and their subscales were weaker than what was found by preceding scholars . In the Limitations should also be considered. On one hand, the sample we utilized had not been identified as clinically compulsive buyers and hoarders or found seeking treatment before we conducted the research. Hence, this limitation could affect the prevalence rate of compulsive buying and hoarding, and we also lost the opportunity to find out the appropriate cut-off point for Chinese hoarders. On the other hand, the use of self-report instruments could lead to response biases despite CHRS, which was adapted from the original semi-structured instruments and RCBS-TC, which was well validated.To sum up, the present research is the first to investigate the association between compulsive buying and hoarding in a Chinese population-based sample with participants from Hong Kong (developed economy) and mainland China (developing economy). Through a comparison of the compulsive buying and hoarding behaviors of two sets of participants, we can further understand their discrepancies, and targeted treatments can be developed in the future.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The study procedures were carried out in accordance with the Declaration of Helsinki. The Institutional Review Board of the University Research Centre, The Open University of Hong Kong (HE20Jul2017-S&T2017/01). The patients/participants provided their written informed consent to participate in this study.HH and SL were involved in the development of the idea, project supervision, and data collection. HH, MZ, and SL carried out data analysis, literature review, draft of the manuscript, and review of the manuscript. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Rho-GTPases control a variety of cellular functions mainly by regulating microtubule and actin dynamics, affecting the cytoskeleton, and are important regulators of the structural plasticity of dendrites and spines. Members of the Rho-GTPase family include Ras-related C3 botulinum toxin substrate 1 (Rac1), RhoA (Ras homologous), and cell division control protein 42 (Cdc42). Cdc42 is involved in the regulation of a variety of tumor and non-tumor diseases through a cascade of multiple signaling pathways. Active Cdc42 can regulate intercellular adhesion, cytoskeleton formation, and cell cycle, thus affecting cell proliferation, transformation, and dynamic balance as well as migration and invasion of tumor cells by regulating the expression of effector proteins. Here we discuss the role of Cdc42 in promoting metastasis, invasion, epithelial-mesenchymal transformation and angiogenesis in malignant tumors. The significant role of Cdc42 in non-tumor diseases is also discussed. Since Cdc42 plays a central role in the development of various diseases, small molecule inhibitors targeting Cdc42 have important clinical significance in the prevention and treatment of these diseases. Rho-GTPases are a family of small GTP-binding proteins in the Ras superfamily, and their relative molecular weight is approximately 20-25 KD. Rho is found in all eukaryotes from plants to humans. Twenty members of the Rho family are divided into classic and non-classic groups. The peptide chain length of a member of the classic family is approximately 3-4 times that of a member of the non-classic family. The classic Rho-GTPase group includes RhoA (Ras homologous), Ras-related C3 botulinum toxin substrate 1 (Rac1), and cell division control protein 42 (Cdc42), which are modulated by the effects of Rho-specific guanine nucleotide exchange factor (GEF) and GTPase activating proteins (GAPs) and guanosine nucleotide dissociation inhibitors (GDIs). Rho-GEF regulates the exchange of GTP with GDP, thereby activating Rho-GTPases. Simultaneously, GAPs accelerates GTP hydrolysis and restores these proteins to an inactive state. Post-translational modification and the GTP/GDP cycle together regulate the biological activity of Rho-GTPases Saccharomyces cerevisiaeCdc42 is a member of the Rho family with a molecular weight of 21 KD and is first discovered in yeast, Cdc42 is particularly important in yeast germination Cdc42 regulates microtubule cytoskeleton through microtubule reorganization, including asymmetric localization of the spindle. In addition, Cdc42 facilitates the development of the pancreas, blood cells, eyes, and skin Active Cdc42 can regulate the expression of downstream effectors, and enhance cell proliferation, polarity, adhesion, and migration, as well as dynamic changes of the cytoskeleton PAKs are downstream proteins of Cdc42 and are known for their roles in the cytoskeleton and are critical in the dynamic recombination of F-actin and tubulin. The PAKs family contains six proteins: PAK-1(\u03b1-PAK), PAK-2 (\u03b3-PAK), PAK-3 (\u03b2-PAK) (group \u2160), PAK-4, PAK-5, and PAK-6 (group \u2161). Group \u2160 PAKs have an autoinhibitory domain and can interact with the kinase domain in a cis-autoinhibitory interaction. Binding of GTPase to PAKs disrupts this autoinhibition, leading to dimerization and trans-autophosphorylation of the two PAK monomers 2+ to form a complex, which binds to Cdc42 in an initial and transient manner. The C-terminal region of the ACK contributes to the overall binding affinity through several scattered residues ACK is a non-receptor tyrosine kinase and functions downstream of growth factor receptors and has been implicated in survival, neuronal signaling, and androgen receptor activation through identification of its phosphorylation targets. The conformation of ACK binding to Cdc42 is significantly different from that of other CRIB effector proteins, all of which form a short segment of intermolecular \u03b2-sheet with Cdc42. Leu449, Ser-450 and Ala-451 residues in ACK are the key factors for binding to Cdc42 . PI3K I has three homologous isoforms: PI3KCA, PI3KCB, and PI3KCD. PI3KCA mutations have been found in colon and non-small-cell lung cancer PI3K and PI5K are the core effector proteins of Cdc42 /Rac. Cdc42 /Rac and PI3K are mainly present in the mammalian system. PI5K and PI3K can produce PIP2 and act as a second messenger to regulate the dynamic balance of the cytoskeletonIsoleucine-glutamine-motif containing GTPase-activating proteins (IQGAPs) promotes cell metastasis and invasion by binding to the activated Cdc42-GTP. IQGAPs are downstream effectors of Cdc42 and consist of two key regions, including a calmodulin binding IQ-motif and a GTPase activating protein related domain. Upregulated IQGAPs bind to the activated Cdc42-GTP Drosophila melanogaster, the gene encoding the formin protein Diaphanous is involved in cytokinesis Caenorhabditis elegans, loss of the formin protein Cyk-1 function causes polar-body extrusion in embryonic mitosis Formins are defined by a unique and highly conserved C-terminal formin homology 2 (FH2) domain and an N-terminal proline-rich formin homology 1 (FH1) domain N-WASP is composed of GTPase-binding domain, CRIB and a VCA domain. Cdc42 interacts with and activates N-WASP. N-WASP then binds and activates the actin-associated protein 2/3 (Arp2/3) complex, leading to actin polymerization DrosophilaDrosophila neuroectoderm and neuroblast development and mammalian epithelial cell apical-basal polarity and axon-dendrite polarity of neuron The PAR protein family contains from PAR1 to PAR6. PAR3, PAR6 and atypical protein kinase C (aPKC) form a ternary complex named PARs complex that localizes asymmetrically in polarized cells, while PAR1 localizes to complementary domains. PAR1 phosphorylates PAR3 to block PAR3 binding to aPKC and inhibits cortical association of PAR3/PAR6/aPKC in MLK3 is a member of the mixed spectrum kinase family (MAPK). The MAPK family is composed of an amino N-terminal SH3 domain, a catalytic domain, leucine/isoleucine zipper motifs, and a CRIB motif. Activated Cdc42 interacts with MLK3 through CRIB motif and promotes the catalytic activity of MLK3. In a Cdc42-mediated MLK3 activation model, Cdc42 binds to the CRIB domain of MLK3 and blocks the association between the N-terminus of MLK3 and proline 495, thereby reducing the autoinhibition of MLK3 and forming an open structure that promotes autophosphorylation MAPK signaling pathway consists of MAPKKK, MAPKK and MAPK, and they undergo phosphorylation in turn in response to internal and external stimuli. When these three kinases are activated, phosphorylated MAPK (ERK) will migrate to the nucleus and phosphorylate nuclear transcription factors to sequentially phosphorylate of specific transcription factors and then initiate transcription process EphA regulates actin cytoskeletal dynamics through Rho family GTPases. The Rho-GEF ephexin, associates with EphA receptors can activate Cdc42. After EphA binding, ephexin becomes phosphorylated and increased Rho activation, which leads to a destabilization of the actin cytoskeleton. A number of Rho family GAPs and GEFs are activated by EphA forward signaling. The GAP \u03b12-chimaerin is the major GAP downstream of EphA4 in midline guidance processes. \u03b12-chimaerin binds to the activated EphA4 receptor Although Cdc42 is first found in yeast, where it plays an important role, it is equally important in the occurrence and development of various human malignant tumors. In most human normal tissue cells, Cdc42 expression is low/medium both at the mRNA and protein level. However, in tumor cells, the high expression of activated Cdc42, abnormal ubiquitin ligase, and EGFR degradation inhibition result in malignant tumor progression , and fatty acid synthase, supporting breast cancer cells continuous growth Cdc42 is generally upregulated and involved in the activation of cell signaling pathways, including PAK and N-WASP, which are closely related to the malignant proliferation of tumor cells Xenopus laevis, indicating that Rhov, Rac1, Cdc42, and their downstream effectors PAK-1 was involved in EMT EMT is a major driver of cancer cells invasion and metastasis. EMT allows cancer cells to metastasize and break the adhesion between cells. EMT plays an important role in physiological and pathological processes, including the promotion of wound healing, tissue fibrosis, and cancer progression. EMT and the formation of new blood vessels are the basis of tumor metastasis. EMT is a complex cellular process characterized by the loss of E-cadherin and adhesion complex, and is regulated by EMT-induced transcription factors  During colorectal cancer progression, there are two patterns of metastasis including mesenchymal metastasis and amoeba metastasis. The switching of tumor cell metastasis patterns depends on the response to Rho-GTPases We previously reported that polyploid giant cell cancer cells (PGCCs) could be induced and purified by cobalt chloride. PGCCs were found to be in a dynamic equilibrium with diploid cancer cells and could be formed through endoreduplication or cell fusion Hypoxic inducible factor (HIF), cyclin D1, IL-6, IL-8, fibroblast growth factor (FGF), and integrins, are associated with the process of angiogenesis. HIF induces gene transcription of vascular endothelial growth factor, nitric oxide synthase, platelet-derived growth factor-\u03b2, and angiopoietin 2 during EMT Cdc42 participates in the regulation of a variety of biological processes. The increased expression of Cdc42 can promote the proliferation, invasion, and migration of cancer cells, and accelerate malignant progression of cancer. Overexpression or loss of Cdc42 also contributes to the development of some benign diseases through the same or similar signaling pathways as malignant tumors, such as insulin secretion, insulin resistance, airway inflammation and neurodegenerative diseases Fig..The constitutive activity of Cdc42 can interfere with the differentiation of \u03b2 cells, inducing the rise of blood glucose sometimes leading to hyperglycemia. In normal human cells, Cdc42 regulates the formation of normal islet morphology associated with \u03b2 cell function and proliferation +Foxp3+ reduces regulatory T cells, inhibits the development of thymus cells, and suppresses the differentiation of Th1/Th17 cells + T cells. In contrast, Cdc42 regulates clonal expansion but not activation in CD8+ cells Signal transducer and activator of transcription 6 (STAT6) can be activated by IL-4 and IL-13. GATA3 has been demonstrated to play important roles in innate lymphoid cells and to regulate T cell development, proliferation, and maintenance, in addition to controlling Th2 differentiation Endo, M., J.E. et al. confirmed that Cdc42 could stimulate the activity of mTOR complex 1, thereby up-regulating the transcription factors required for the formation of neural precursor cells in normal brain tissue. The brain-specific Cdc42 isoform, Cdc42b, is essential for promoting the transformation of neural progenitor cells into neurons. Cdc42b and activated ACK jointly down-regulated mTOR expression and promoted neuronal differentiation in vivo and in vitro in pre-clinical settings. Commonly used small molecule inhibitors of Cdc42 are listed in Table-There are many small molecule inhibitors of Cdc42. However, there are few clinical trials about Cdc42 inhibitors and most studies of cdc42 inhibitors are performed Most of the current inhibitors have been developed interact with GEF and bind nucleotides to inhibit Cdc42. Azathioprine (AZA) 1 and AZA197 are GEF interaction inhibitors. AZA1 can effectively inhibit the activation of Cdc42 and Rac1, reduce the signal transduction of the PAK pathway, and inhibit tumor growth. The main mechanism of AZA197 anti-tumor effect is through the inhibition of cell proliferation and induction of apoptosis. AZA197 destroys the interaction between Cdc42 and GEF and specifically inhibits the activity of Cdc42. AZA197 inhibits the proliferation, migration, and invasion of cancer cells in colon cancer and prostate cancer Casin controls GEF activity by blocking PIP2 on the Cdc42/RhoGDI complex and inhibiting Cdc42 nucleotide exchange and further targeted regulation of Cdc42 silico, MBQ167 is shown to bind to the Asn 39 side chain of Cdc42 and Rac to form an H bond, which is thought to inactivate the GEF binding region MBQ167 is an effective dual inhibitor of Rac and Cdc42. In in vitro and inhibits the interaction between Cdc42 and intersectin in lung cancer A549 cell lysates. ZCL278 is also a GEF interaction inhibitor. It can destroy the interaction of ITSN with Cdc42 and on the surface of the Cdc42 Phe56 wide-ranging combination, inhibiting migration in the PC-3 cell line without destroying cell vitality and suppressing cellular invasion and migration in pancreatic cancer cell lines ZCL compounds inhibit cell proliferation and cell cycle progression by inhibiting the EGFR-kRas signaling pathway. Study has shown that ZCL278 plays an important role in inhibiting the invasion and metastasis of pancreatic cancer cells ML141, is a competitive inhibitor of Cdc42 activity. It is a type of nucleotide inhibitor and non-steroidal anti-inflammatory drug (NSAID)-related compound, which can specifically block the GTP binding domain in vitro, which is stimulated by PIP2 and mediated by the Cdc42/Toca-1/N-WASP/Arp2/3 signaling pathway Secramine inhibits actin assembly In this review, we described the structure and functions of Cdc42 and its family, analyzing its major regulatory effectors, and summarizing the drugs that specifically target Cdc42 in disease treatment. Cdc42 is widely involved in the regulation of human malignant and non-neoplastic diseases, and plays an important role in the invasion and metastasis of tumors, cell proliferation, and cell polarity. Furthermore, the potential regulatory role of Cdc42 in non-tumor diseases and malignant diseases is discussed. So far, although some inhibitors of Cdc42 protein have been developed, few drugs can be used in clinical treatment, and the regulatory mechanisms of Cdc42 in a variety of diseases need to be further explored in order to achieve effective treatment."}
+{"text": "Background: Fear of progression (FoP), or fear of cancer recurrence (FCR), is characterized by worries or concerns about negative illness-related future events. Actually, to worry is a common cognitive process that, in its non-pathological form, belongs to daily life. However, worry can also become pathological appearing as a symptom of mental disorders. This study aimed at investigating the associations among daily worry, pathological worry, and FoP in patients with cancer.Methods: This is a cross-sectional study that includes 328 hospitalized patients with cancer. Patients filled out the FoP Questionnaire (FoP-Q), the Worry Domains Questionnaire (WDQ) for the assessment of daily worry, and the Penn State Worry Questionnaire (PSWQ) for the assessment of pathological worry. Depressive, anxiety, and somatic symptoms were measured with modules of the Patient Health Questionnaire . Furthermore, a structured clinical interview was conducted for the assessment of anxiety disorders. The hierarchical multiple linear regression analysis was used to identify factors independently associated with FoP.Results: Mean age of the participants was M = 58.5 years (SD = 12.8), and 64.6% were men. FoP and worry were significantly intercorrelated (r = 0.58\u20130.78). The level of FoP was most strongly associated with daily worry , followed by pathological worry . Further significant determinants were younger age and depressive and anxiety symptoms. Clinical variables were not independently associated with FoP. The final model explained 74% of the variance.Discussion: Fear of progression is strongly associated with daily worry and pathological worry. These results bring up the question of whether FoP is an expression of a general tendency to worry. Whether a general tendency to worry, in fact, represents an independent vulnerability factor for experiencing FCR/FoP needs to be investigated in a longitudinal research design. Many people experience recurrent thoughts about possible risks and threats. To think repetitively about such future uncertainties and dangers is quite common. In a study with community-dwelling elderly people, Golden et al.  found thRegarding specific worry topics, people tend to worry about interpersonal relationships, self, work, future events, finances, and mostly health . Furthermore, clinical FCR was more frequent at higher levels of pathological worry  by Herschbach et al.  was usedTwo measures were applied for the assessment of worry.\u03b1 = 0.95.The WDQ  and \u03b1 = 0.80 (GAD-2).Symptoms of depression and anxiety were assessed using the ultra-short screening versions of the Patient Health Questionnaire (PHQ), i.e., PHQ-2 and GAD-2. Both modules comprise two items, which are rated on a 4-point scale from 0  to 3 (nearly every day) , cancer site , comorbidity (none/present), and disease status . For additional analyses, Pearson's correlations were used to assess intercorrelations between worry and FoP, and differences in worry mean scores between groups of clinical versus non-clinical FoP were investigated using the independent sample t-test. Effect sizes (Cohen's d) are reported for these group differences. Alpha level was set as p < 0.05. All analyses were conducted using SPSS/PC software package version 24 .Descriptive statistics were computed for the study variables. The research question was investigated using the hierarchical multiple linear regression analysis. In order to control for known and potential covariates, sociodemographic characteristics were entered in the first step, clinical variables in the second step, and mental health variables in the third step. As our main study  than those patients who agreed (p < 0.001). Of those who agreed, 15 patients did not provide the data on FoP, leaving 328 patients available for the analysis. In light of the low number of patients who were excluded from the analysis, we refrained from conducting a drop-out analysis.Of 529 patients who were approached for participation, 49 patients met the exclusion criteria. Thus, 480 patients were available for study participation. A total of 343 patients (71.5%) agreed to take part. Patients who declined participation did not differ from the study participants with regard to sex or cancer site, but patients who declined were older . The majority of them (64.6%) were men. A total of 60.7% of the patients suffered from gastrointestinal cancer  and 39.3% suffered from hematological malignancy . For most of the patients, this was the first occurrence of the disease (69.0%), and the mean time since the first cancer diagnosis was 27.1 months . Further sociodemographic and clinical characteristics are presented in The mean age of the patients was r = 0.78 with daily worry (WDQ) and r = 0.64 with pathological worry (PSWQ). The two worry measures correlated r = 0.58 .Descriptives of the continuous variables are given in n = 66) showed significantly higher (p < .001) daily worry . The final regression model revealed that younger age, current partnership, higher anxiety, depressive, and somatic symptoms, absence of an anxiety disorder, and higher worry were significant determinants of FoP , and the significance of this finding remains unclear.According to several reviews of FCR/FoP (Crist and Grunfeld, Finally, our results also support some current psycho-oncological interventions addressing FCR/FoP. A recent systematic review and meta-analysis showed that psycho-oncological interventions are effective in reducing FCR/FoP (Tauber et al., This study has some strengths, such as the detailed assessment of worries in patients with cancer, the reasonable sample size, and the inclusion of a set of covariates. But, clearly, it also has some limitations. First, the cross-sectional design precludes inferences about longitudinal associations between worry and FoP. Then, there is a sampling bias as patients who declined participation were older than those who agreed to take part. Furthermore, we did not assess other psychological variables that have proven relevant as possible control variables, especially those that are regarded as important in current theoretical conceptualizations of FCR/FoP, e.g., meta-cognitive beliefs. Moreover, we did not assess the whole spectrum of mental disorders but restricted our assessment on the anxiety disorders. Finally, there might be a bias due to shared method variance. We applied three self-reporting measures focusing on different aspects of worry that were moderately to highly intercorrelated. Thus, the results of this study should be replicated using different assessment approaches.This study has shown that worry represents an independent determinant of FoP. As such, the results support current theoretical conceptualizations of non-clinical and clinical FCR/FoP (Fardell et al., a.dinkel@tum.de.The datasets presented in this article are not readily available because participants did not provide consent for public availability of the data. Requests to access the datasets should be directed to Andreas Dinkel, The studies involving human participants were reviewed and approved by Ethics committee of the Faculty of Medicine, Technical University of Munich. The patients/participants provided their written informed consent to participate in this study.AD designed the study. KK was responsible for data acquisition. KK and BM-M prepared and analyzed the data. AD wrote the first draft. All authors contributed significantly to the interpretation of the data and the final version of the manuscript and gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Animal welfare is a growing public concern that has the potential to undermine the social license of zoos and aquariums. The lack of consensus on how animal welfare is defined across such a diverse sector combined with and a widespread belief that commercial priorities such as entertaining visitors conflicts with animal welfare, hinders efforts to effectively address this fundamental issue for the sector. Data derived from an audit of habitats across a major North American wildlife attraction revealed that holistic animal welfare assessments undertaken by animal carers embracing three principal constructs of animal welfare, correlated strongly with visitor perceptions of animal happiness. Visitor assessments of animal happiness also correlated with animal carer assessments of social, behavioural\u00a0and locomotor opportunities and inversely with the prevalence of stereotypic behaviours, supporting the proposition that folk conceptions of animal welfare are more accurate than may have previously been considered to be the case. However, the holistic animal welfare assessment inversely correlated with assessments of a habitat's capacity to safeguard welfare as determined by the facility's veterinary staff, supporting the proposition that tensions exist between physical and psychological components of captive animal welfare provisioning. This further underlines the importance of clarity on how animal welfare is conceived when developing institutional animal welfare strategies. Finally, the data also showed that both holistic animal welfare assessments and visitor perceptions of animal happiness strongly correlated with the level of enjoyment experienced by visitors, challenging the belief that animal welfare competes with the commercial priorities of zoos and aquariums. The audit supports the case that maintaining high animal welfare is a commercial imperative as well as a moral obligation for zoos and aquariums and underlines the necessity to utilize conceptions of animal welfare that acknowledge the centrality of the affective states of animals in maintaining those standards. A holistic welfare audit of habitats within a large wildlife attraction correlated with visitor perceptions of animal happiness and visitor enjoyment but inversely correlated with a veterinary\u2010based welfare audit. These findings underline the significance of differing welfare conceptions in establishing institutional animal welfare management priorities and the potential tensions between physical and psychological priorities in captive animal care. Most zoo animal welfare stakeholder groups do not have publicly available animal welfare definitions and those that do, differ from the reported scientific consensus. For wildlife attractions, this ambiguity has the potential to create confusion, misalignment of effort, conflict, and an erosion of trust with the general public. An animal welfare audit undertaken across a large wildlife attraction encompassing a range of stakeholder perspectives and welfare conceptions revealed differing alignments; the performance of habitats from a veterinary management perspective inversely correlated with a holistic animal welfare assessment undertaken by animal care staff utilising a suite of broad\u2010based criteria, encompassing three principle animal welfare constructs relating to natural living, effective states and health/biological functioning. This holistic assessment correlated with visitor perceptions of animal happiness as well as their enjoyment of those habitats. Visitor assessments of welfare also correlated with animal carer assessments of social, behavioural and locomotor opportunities and with the prevalence of stereotypic behaviours. The findings of this audit support the case that tensions can exist between veterinary and holistic animal welfare\u2010based priorities in achieving peak captive animal welfare and support the case for a consensus in defining animal welfare centred around the affective states of animals. Finally, the audit suggests folk understandings of animal welfare are more accurate than may have been considered to be the case,\u00a0 and that welfare is important in shaping visitor experiences of wildlife attractions, illustrating the strategic as well as moral importance of animal welfare to wildlife attractions. As the name implies, the HolisticWI is intended to encapsulate the all\u2010round welfare requirements of the animals recognizing that their welfare is dependent upon their affective experiences .1.Are the animals in the habitat healthy?2.Are the animals in the habitat happy?3.Did you enjoy viewing the habitat?The final welfare perspective was derived from visitors who provided scores on a habitat wide basis regardless of the number of species within each habitat and was collected using touch screens adjacent to habitats. The touch screens offered visitors a series of five emojis with which they ranked their opinions on the performance of habitats ranging from a positive response represented by an open\u2010mouthed smiling face, a neutral response represented by a face that is neither smiling nor sad/angry, through to a negative response represented by a visibly angry face. Following an extensive trial, three mechanisms were put in place to eliminate potentially spurious data from visitors interacting with the touch screens without properly engaging in the content. Firstly, two questions were included relating to each habitat being considered that needed to be answered correctly before the input was included in the analysis. Secondly, if responses to questions were given in a time deemed shorter than it would be possible to read the question and respond, that input was also excluded. Finally, the order in which the emoji\u2010based ranking was presented from positive to negative was alternately reversed along the horizontal axis, to preclude respondents simply tapping the screen at a specific location and potentially creating a false correlation between separate questions. The questions asked of the visitors in this format were:Of the three criteria making up the visitor assessment, the first two criteria relating to health and happiness are collectively referred to as the visitor welfare index (VisitorWI). Since visitors to a wildlife attraction are arguably more likely to approve of such facilities in providing appropriate environments for resident animals than those that would actively avoid them, the proportion of visitors approving of habitats was considered as more relevant than an average rank score across the surveys completed. An approval index was therefore calculated based on the percentage of visitors providing a positive ranking; the higher two of the five options available to visitors for each criterion represented by the closed and open\u2010mouthed smiling emojis.In all three assessments, the one to five rankings were set out such that the lower the score, the poorer the welfare performance is likely to be; so for example, a rank of one, respectively, represented the categories including the least happy, most stereotypic, least able to locomote, most predisposed to physical/physiological challenges, least able to support the provision of emergency care and so on.3In total, animal care staff undertook 1220 assessments across 133 habitats yielding an average of 9.2 assessments for each habitat. Visitors undertook a total of 2958 assessments across 29 habitats of which, 1337 assessments (45.2%) passed the validation filters, with each habitat subsequently yielding an average of 46.1 validated visitor assessments. The veterinary team collectively assessed 108 habitats.\u03c72\u2009=\u2009170.165 , p\u2009<\u2009.00001) with a Nemenyi post\u2010hoc test revealing assessments of stereotypies and abnormal behaviours were significantly different from all other assessment criteria at the same level of significance , p\u2009<\u2009.0005) with a Nemenyi post\u2010hoc test revealing ease of emergency care was significantly different from morbidity and mortality , p\u2009<\u2009.01) with a Nemenyi post\u2010hoc test revealing assessments of animal happiness were significantly different from assessments of visitor enjoyment and assessments of animal health at the same level of significance (p\u2009<\u2009.05), see Figure\u00a0To determine whether the criteria making up each of the three stakeholder group welfare assessments were not simply mirroring each other, Friedman rank\u2010sum tests were carried on each. For the five metrics that make up the HolisticWI; there was found to be a statistically significant difference between the metrics \u2009=\u2009\u2212.27514, p (two\u2010tailed)\u2009=\u2009.00413, see Figure\u00a0rs (32)\u2009=\u2009.45254, p (two\u2010tailed)\u2009=\u2009.00931), the visitors' approval rating of the happiness of resident animals in each habitat \u2009=\u2009.63474, p (two\u2010tailed)\u2009=\u2009.0001) and the visitors' enjoyment approval rating of each habitat \u2009=\u2009.37468, p (two\u2010tailed)\u2009=\u2009.03462, see Figure\u00a0rs (32)\u2009=\u2009.14445, p (two\u2010tailed)\u2009=\u2009.43026). Among\u00a0the criteria within the HolisticWI, the animal carers' perceived prevalence of stereotypies and/or abnormal behaviours within each habitat, significantly correlated with their assessments of the extent to which habitats failed to cater for resident animal's social requirements \u2009=\u2009.83431, p (two\u2010tailed)\u2009=\u2009.000), curtailed behavioural freedoms \u2009=\u2009.79575, p (two\u2010tailed)\u2009=\u2009.000), predisposed resident animals to physiological and or physical challenges =\u2009.74258, p (two\u2010tailed)\u2009=\u2009.000), and curtailed their locomotor opportunities \u2009=\u2009.67523, p (two\u2010tailed)\u2009=\u2009.000, see Figure\u00a0The HolisticWI was the most inclusive and broad\u2010based of the three assessments comprising elements relating to health/biological functioning and natural living, together with insights into the affective states of resident animals by considering the prevalence of stereotypic and other abnormal behaviors Fraser,\u00a0 and as srs (27)\u2009=\u2009\u2212.00682, p (two\u2010tailed)\u2009=\u2009.97307), the visitors' perception of animal health \u2009=\u2009.01739, p (two\u2010tailed)\u2009=\u2009.93138) nor the extent to which animal carers' believed habitats predisposed\u00a0resident animals to physiological and/or physical challenges \u2009=\u2009\u2212.1683, p (two\u2010tailed)\u2009=\u2009.08167). Ease of emergency care correlated with ease of preventative care \u2009=\u2009.32497, p (two\u2010tailed)\u2009=\u2009.00064, see Figure\u00a0rs (107)\u2009=\u2009\u2212.0864, p (two\u2010tailed)\u2009=\u2009.37392), nor with ease of emergency care \u2009=\u2009\u2212.06211, p (two\u2010tailed)\u2009=\u2009.52308) or preventative care independently \u2009=\u2009.39248). The veterinary team's assessment of morbidity and mortality did not correlate with the animal carers' assessment of the predisposition of animals to physical and physiological challenges \u2009=\u2009\u2212.01498, p (two\u2010tailed)\u2009=\u2009.87774), but the veterinary team's combined assessment of the ease of emergency and preventative care inversely correlated with the animal carers' assessment of the predisposition of animals to physical and physiological challenges \u2009=\u2009\u2212.19513, p (two\u2010tailed)\u2009=\u2009.04299, see Figure\u00a0As veterinary staff are highly influential in matters pertaining to animal welfare within zoos and aquariums but inevitably have a health\u2010centred focus, it is important to understand how the animal welfare conceptions of this stakeholder group related to that of the welfare conceptions/perceptions of visitors, and other more holistic animal welfare conceptions such as the one set out here. In addition to the inverse correlation with the HolisticWI \u2009=\u2009\u00a0.50018, p (two\u2010tailed)\u2009=\u2009.00355), the infrequency with which animal carers perceived animals expressed stereotypies and/or abnormal behaviours within each habitat \u2009=\u2009.46791, p (two\u2010tailed)\u2009=\u2009.00692)\u00a0and the extent to which animal carers perceived habitats to cater for the social requirements of resident animals \u2009=\u2009.3996, p (two\u2010tailed)\u2009=\u2009.02346, see Figure\u00a0rs (32)\u2009=\u2009.30097, p (two\u2010tailed)\u2009=\u2009.09415) or curtailed the locomotor opportunities of a habitat's resident animals \u2009=\u2009.32036, p (two\u2010tailed)\u2009=\u2009.07384). There was a significant correlation between the visitors' approval rating of the happiness of animals with their approval rating of their health \u2009=\u2009.51306, p (two\u2010tailed)\u2009=\u2009.00268) and the extent to which visitors enjoyed habitats \u2009=\u2009.55462, p (two\u2010tailed)\u2009=\u2009.00179, see Figure\u00a0rs (29)\u2009=\u2009.18065, p (two\u2010tailed)\u2009=\u2009.34835).Finally, the visitors' assessments enable us to understand how \u2018folk' conceptions of health and happiness relate to more evidenced\u2010based conceptions of animal welfare and management as well as the visitors' experiences of habitats. While VisitorWI correlated with the HolisticWI  in the British Isles and captive cetaceans around the world appear to support this proposition. Despite growing public concern for captive polar bear welfare and the publication of three critical reports on the welfare of British and Irish polar bears during the 1980s and 1990s (Ames,\u00a0The contrasting histories of the social acceptability of captive polar bears (0s Ames,\u00a0, the rec0s Ames,\u00a0, is argu0s Ames,\u00a0 when ambper se, rather attributing human\u2010like drivers for happiness/positive animal welfare would be if they were not based upon sound reasoning or a solid evidential base. The benefit of applying such a filter to animal welfare is it better reflects the growing concerns of many western societies and makes it impossible to disregard the affective states of animals, which are currently all too frequently subservient to the more quantifiable components of physical wellbeing (Veasey,\u00a0To diminish this ambiguity, which has been identified as a source of public concern and skepticism in respect of animal based attractions (Marinova & Fox,\u00a0 Veasey,\u00a0. To keep Veasey,\u00a0,\u00a02010.Such a conception subscribes to the belief that animal welfare is all to do with the feelings of animals (see Duncan & Petherick,\u00a04.3In addition to illustrating the value in establishing consensus on a definition of animal welfare that prioritizes the affective states of animals, the strong correlation between visitors' perceptions of animal happiness, the HolisticWI and visitors' enjoyment of those habitats (see Figures\u00a0Furthermore, the commercial impacts of welfare controversies on a wide array of wildlife attractions has been shown to be devastating, (see,\u00a0e.g.,\u00a0CBC,\u00a0While improvements in animal welfare provisioning over the last half\u2010century within most accredited \u2018western' zoos, and likely many more besides would be hard to dispute (see Kitchener & MacDonald,\u00a0As this audit is representative of a single large North American wildlife attraction, care must be taken extrapolating findings relating to the welfare conceptions of visitors and the veterinary teams to different locations. However, the HolisticWI is intended to reflect the needs of the animals themselves independent of any non\u2010scientific conception of welfare or cultural emphasis and so is more universally applicable. Further research exploring regional variations in stakeholder conceptions of welfare benchmarked against the HolisticWI is clearly warranted. Notwithstanding the potential cultural specificity of these findings, the audit suggest visitors to North American zoos and aquariums are probably more cognizant of animal welfare than they have been given credit for, and that they are potentially more sensitive to the affective states of animals than they are to the health status of animals on display. And so, the failure to meet the psychological needs of animals should be considered as an existential threat to the sector,\u00a0and to address this moral and strategic priority, zoos and aquariums are encouraged to assess the welfare status of their facilities and animals with a particular emphasis on the affective states of animals as reflected in the HolisticWI, and subsequently develop ongoing strategies to address those needs."}
+{"text": "In Australia, animal welfare protection is a state and territory responsibility. Having eight separate state and territories results in eight separate animal welfare legal frameworks, all which contain a primary piece of legislation and secondary or subordinate forms of legislation. These subordinate forms are known as regulations and codes of practice and are lower-ranking laws that are given powers under the primary legislation. Subordinate laws contain crucial details that govern everyday human\u2013animal interactions and industry practices, for example companion animals used for breeding. There has been no review of the animal welfare-focused subordinate laws in Australia. This study has assembled each animal welfare regulation and code of practice in force in Australia as a reference for practitioners working in specific animal-related areas to easily identify relevant documents pertaining to welfare and understand the nature of their responsibilities in terms of compliance with these documents. A total of 201 pieces of subordinate legislation were identified and presented through the following utility categories of animals: companion, production, wild/exotic, entertainment. The benefits of housing the information identified from this study on an online database for animal industries to use are discussed.The state-based approach to regulating animal welfare in Australia is thought to create national dis-uniformity in that each state and territory legislates and operates inconsistently. The animal welfare legal framework in each of the eight Australian jurisdictions is made up of a primary statute and subordinate legislation, where subordinate animal welfare legislation, in the forms of regulations and codes of practices, are lower-ranking laws that are given power under the jurisdiction\u2019s specific animal welfare statute. Since a review of animal welfare statutes identified broad patterns between the jurisdictions, this study is intended to be complementary by collating the subordinate legislation to provide a more comprehensive understanding of animal welfare laws in Australia. Using targeted search strategies stemming from the eight enabling animal welfare statutes, this study identified 201 pieces of subordinate legislation in force between 28 March 2022 and 5 April 2022. The scope of subordinate legislation is depicted through the following utility categories of animals: companion, production, wild/exotic, entertainment. Whilst subordinate legislation differed between the jurisdictions, it was common for similar welfare concerns or topic areas to be protected in higher-order legislation (statutes or regulations). Additionally, many jurisdictions were found to have similar shortcomings, all which likely could be managed through a mechanism of national data collection. Commonwealth of Australia Constitution Act 1901 (\u2018Constitution\u2019) [National animal welfare legislation is restricted in scope in Australia, as the tution\u2019) ,3,4,5,6.tution\u2019) , makes ntution\u2019) ,8 as in Subordinate laws are lower-ranking laws, meaning they are enabled by a statute. They are often referred to as \u2018delegated legislation\u2019, as Parliament \u2018delegates\u2019 the authority of creating such legislation  to the executive arm of government . AlthougCodes of practices (\u2018codes\u2019) are another form of subordinate legislation. They are known as \u2018quasi-delegated legislation\u2019 or \u2018soft law\u2019 , a partiWhilst regulations sit directly under their enabling statute , codes are found lower in the hierarchy . This meSubordinate legislation plays an important part in modern government in all common law countries, yet in a similar vein to animal welfare statutes, subordinate laws often come under scrutiny for their contribution to the inconsistent and contradictory framework of animal welfare legislation ,13,16,17Hence, this paper aims to assemble all subordinate laws given authority under the eight state and territory-based animal welfare statutes to understand the extent of the cross-jurisdictional differences. This study follows on the previous statutory comparison , by collSubordinate legislation in the form of regulations and codes of practice was identified directly from the eight Australian state and territory-based animal welfare statutes (enabling acts) in force between 28 March 2022 and 5 April 2022 . FederalAll identified subordinate legislation adopted or prescribed (incorporated under a provision in the enabling statute) under the eight state and territory-based animal welfare enabling acts  was acceDue to the breadth of subordinate legislation, this paper does not review the entirety of each regulation and code; rather, it focuses on the scope or area of protection it awards to animals. Regulations were manually reviewed to extract the type of animal use it pertains to  or the process it controls . Codes were manually reviewed similarly. However, in some cases, this information was not publicly available. In this case, the title of the code and the accompanying explanatory statement with the relevant Minister\u2019s signature at the time were used to confirm the codes scope of protection. Each Australian jurisdiction operates differently; therefore, when areas of animal use are not included in this paper, it should be assumed that their protection does not fall under animal welfare statute rather than that they do not receive any protection whatsoever.Using the search strategies stemming from the eight enabling acts, this study identified 18 regulations , 79 compThe use of subordinate legislation for companion animal protection differs between each jurisdiction . Whilst Subordinate legislation specific to production animals was generally consistent between the jurisdictions , as mostAnimal welfare subordinate legislation used for the protection of wild animals or exotic species kept in captivity differs substantially throughout the jurisdictions . The mosSubordinate legislation used to protect animals used for entertainment purposes shows a lack of consistency across the jurisdictions . HoweverPrevention of Cruelty to Animals Regulation 2012 (NSW), this offence does not apply to the compulsory codes listed under reg 33 as those are only admissible in court, meaning they can only be used as evidence in the circumstances where a defendant was charged with an offence under the enabling animal welfare statute. Similarly, WA compulsory codes are only admissible in court and there are no offences included for noncompliance. Finally, the statutory wording of the Victorian Prevention of Cruelty to Animals Act 1986 (Vic) suggests that compliance with codes (both compulsory and voluntary) can be used as a defence to an animal welfare offence; thus, there are no direct offences for noncompliance with a code.All jurisdictions list their offences for failure to comply with a compulsory code in statute except for SA, which uses regulations . Each ofEvery jurisdiction has discretion when deciding which codes are compulsory or voluntary, resulting in differences between the frameworks in each state and territory . Each juOf all codes included for analysis, those related to production animals were most common (58.3%). Within those production animal codes, the majority were adopted as voluntary (59.8%), whilst the remainder were compulsory (40.2%). Companion animal codes  were evenly split between compulsory (53.6%) and voluntary (46.4%), whilst wild/exotic animal codes equated to 16.6% of all codes , and codes used for animals in entertainment amounted to 9.2% of all codes .The four currently available animal welfare standards as of 6 September 2022 , cattle This paper outlines the scope of animal welfare protection detailed in subordinate legislation enabled under the state-based animal welfare statutes in Australia. Following from our previous cross-jurisdictional comparison of animal welfare statutes , this anAlthough this paper has identified some cross-jurisdictional differences between the areas of welfare protection covered by subordinate legislation, there were also striking similarities. For example, the states and territories are fairly consistent in giving the most legal weight  to similar issues of welfare concern. This was observed for the use of electrical devices and the performance of surgical procedures in companion animals, pig and poultry farming and livestock transportation. Whilst we cannot know the reasoning behind legislators\u2019 and government officials\u2019 decisions to place these provisions in higher-order legislative instruments, we can reason that it may relate to perception of threat to welfare, thus requiring increased enforceability. It is noteworthy that these are often the areas that have come under public scrutiny both domestically and globally, such as the welfare movement to ban battery cages for poultry farming ,52,53,54Australia is a vast and diverse country, with environmental, economic and social conditions varying across the jurisdictions . Animal Conversely, given this observation in low livestock rearing states, it is somewhat surprising that QLD and VIC have the highest proportion of voluntary codes considering they are some of the highest livestock producers. This leaves the details of husbandry and management in these industries outside the bounds of subordinate legislation and in the realms of judicial interpretation via interpretations of \u2018cruelty\u2019 as expounded in animal welfare statutes. Thus, although the locality can cause justified inconsistencies based on the state\u2019s locality, there still appears to be a dis-uniform approach to some areas that locality cannot explain, and perhaps political decisions are at play. However, it should be noted that both QLD and VIC are in the processes of reforming their animal welfare legislation, with both jurisdictions proposing to improve the welfare standards of production animals . As tAn area not depicted in the results of this study was the approach the jurisdictions have taken to regulating the welfare of animals used for scientific purposes. Each jurisdiction has uniformly adopted the latest edition of the Australian Code for the Care and Use of Animals for Scientific Purposes (\u2018Research Code\u2019) . The incAnimal Welfare Act 1985 (SA) s 19(2)(f) \u2018The Minister may impose conditions requiring the holder of the license to comply with such provisions of the Code as may be specified in the conditions\u2019. Whilst it is suspected that in reality most issued licenses refer to the Code in its entirety, it is clear that there is at least the potential for dis-uniformity in Code usage across the states and territories. Research institutions monitor compliance with the code internally through the nomination of an animal ethics committee that approves animal research in accordance with the Research Code. Any research carried out in breach of the Research Code will result in the potential loss of license by the institution and attract higher penalties, sometimes upwards of $10,000 [Code compliance is generally a license condition rather than a direct provision laid out within the Act. State-based statutes authorize Ministers to issue licenses to institutions for the use of animals in research. In general, these licenses are expected to refer to compliance with the code as being a license condition but noting that some states have maintained some discretionary power in this regard through the act wording, for example in the  $10,000 .There is also the niggling question of whether all aspects of the Code are actually enforceable in some states due to lack of coverage in the overarching enabling statute. For example, the Code covers all living vertebrates except human beings and includes fish. However, if fish are not considered within the definition of animals in the primary act enabling the Code, do they fall outside of the scope of protection? Alternatively, by referring to the Code in license provisions, perhaps they can legitimately be protected in research since any Code breach is then technically a license breach, signaling an act breach. This question likely needs further legal examination and judicial interpretation but again serves to highlight issues of dis-uniformity across the states.Animal Research Act 1985 (NSW). This raises questions around the interpretation of \u2018uniformity\u2019 within this context, as although the animal research code is uniformly functioning with its intended purpose in each jurisdiction (at least based on what the legislation implies), the framework applied is still dis-uniform. This is a common occurrence within animal welfare legislation, as often the scope of animal welfare protection is balanced against other legislative frameworks involving animals.Additionally, whilst there may be almost uniform adoption of the Code across the jurisdictions, the mode of adoption does differ. For example, seven jurisdictions  incorporate compliance with the Research Code through licensing under their animal welfare statute, however NSW differs as they have incorporated it under the The nature of human\u2013animal interactions is diverse and often based on individual and species-based considerations. They vary from our relationships with companion animals, which are often intrinsic in nature ,71,72, tThe interpretation that a uniform framework will result in uniform function is too simplistic, as previous research has established that even with uniform statute application dis-uniformity may remain though differences in enforcement . That isOne of the key features of subordinate legislation is how easily and quickly it can be amended to keep in line with public concerns and scientific advancements ,13. To bFurthermore, subordinate laws often date further back than statutes. For example, a majority of the animal welfare codes still in force are underpinned by the Model Codes of Practice that were introduced in the 1980s, whilst the last two decades have seen somewhat frequent amendments to animal welfare statutes throughout the jurisdictions ,80,81,82A point briefly touched on above is the need for balance between the protection of animal welfare and the profitability of animal-related industries. This is an area that subordinate laws neatly fall within as the Ministers who are delegated authority to create subordinate laws are often those involved in government agricultural departments. This creates a conflict of interest between promoting the profitability of the livestock industry and regulating it for animal welfare protection ,76. The On a similar note, the consultation processes involved in the development of codes of practice are also alleged to conflict with animal welfare protection. Ideally, the consultation process brings together all stakeholders involved within the relevant animal industry (often livestock industries) to understand each viewpoint and achieve balance between ethical views and practical working arrangements . HoweverWhilst a national uniform approach to animal welfare might be beneficial, it is likely not feasible within the current legal framework given the constitutional restrictions . This isIn lieu of a uniform approach, some form of federal database should be implemented in the interim. As previously discussed, national data collection will confirm Morton et al.\u2019s  hypothesAdditionally, such a database should be readily available and accessible to animal-related industries, as this will allow industries to understand their expectations in relation to animal welfare, as well as use the subordinate legislation (as many codes of practice were not readily available online during the data collection stage of this research). The database should contain information similar to this paper but hold further in-depth details on each regulation and code of practice written in lay language with the accompanying formal subordinate law attached. Ideally this would have search functionality to easily source and access these data. This tool could assist in improving the value of subordinate laws for informing and educating persons on their obligations to animals rather than just being used to punish noncompliance or providing defences to cruelty offences.Animal welfare subordinate legislation in Australia, in the forms of regulations and codes of practices, is much more extensive than some may think, making up the vast majority of all sources of laws within the animal welfare legislative framework. These documents house details on the range of human\u2013animal interactions that occur in everyday life. Given that these laws contain provisions relating to everyday husbandry practices and procedures, it is concerning that a majority were often decades old, such as the Model Codes of Practice, and hence, likely not reflecting the current societally accepted standards for animal welfare, and benchmarks for animal welfare science. However, in terms of uniformity, it was identified that each jurisdiction took a fairly consistent approach by giving the most legal weight to similar topics. This was achieved through incorporation in the regulations. Additionally, it is purported that dis-uniformity is likely caused by local societal pressures and circumstances, a concept that is encouraged through the concept of federalism, as well as overlapping animal-related legislative frameworks. Some form of federal data collation is required to confirm such hypotheses and provide some form of accountability in subordinate law-making in animal law in Australia. Such a database is recommended to be available online and written in lay language to enable animal-related industries to better understand their expectations in relation to animal welfare. Coupled with good evidence-based drafting and consultation, subordinate laws may even provide insight into goals for animal caretakers to strive towards for enhanced welfare protection, to shift from a reactive to a proactive approach to animal welfare."}
+{"text": "In developing countries, animal welfare concerns do not receive the same recognition as they do in higher-income countries, from policy and law, through to consumer awareness and purchasing options.While traditional farmers often have close bonds with their animals, knowledge and action gaps often limit more animal-friendly production.In some developing countries, livestock production has already largely commercialized and intensified. In these countries, citizens are becoming increasingly aware and sensitive to animal welfare issues, but animal welfare does not yet affect purchasing decisions.Future scenarios with higher animal welfare are possible, but will require joint efforts by various stakeholders in the livestock sector.Overall, much more research on animal welfare perceptions in developing countries is needed.Livestock make several important contributions to healthy diets and livelihoods. This is especially true in developing countries where otherwise nutritious food options are limited. To ensure the sustainability of food systems, production of animal-source foods must also align with societal views regarding farm animal welfare . Even thThe lack of attention given to animal welfare concerns in this context may be explained by the fact that per capita consumption of animal-source foods is relatively low in developing countries, although there are some exceptions such as Mongolia, Argentina, or Brazil . AdditioYet, livestock production in developing countries is not free of farm animal welfare problems. Production of animal-source food in developing countries is gradually converging with high-income countries. Even though extensive and subsistence livestock production is still common in various countries, capital intensity and mechanization of animal-source food production increase in most places . With thUp till now, however, the body of scientific evidence in this area remains very thin. Animal welfare perceptions were recently reported to have gained attention in some developing countries in South America, as well as in China or Mexico , but relPublic discussions on farm animal welfare emerged in high-income countries during the second half of the 20th century. Since then, meaning and interpretation of the term have continuously evolved and exhibit significant regional differences. The meaning and interpretation of what constitutes good animal welfare and the relevance that is assigned to it are not static parameters, but complex and multifaceted issues shaped by cultural, social, economic, ethical, religious, and political dynamics.In this article, we define farm animal welfare as a state of both physical and mental health of farm animals. It consists of three pillars: animal health, natural living, and affective states . Farm anThis definition of animal welfare based on the three pillars does not have a direct pendant in many societies of developing countries . In certThe lack of harmonization with regards to animal welfare definitions and perceptions poses a challenge when contexts are highly diverse. While attitudes toward animal welfare and according animal welfare legislations also vary between and within industrialized countries, much more research has been done in these regions. Perceptions toward animal welfare in developing countries are highly understudied, which complicates an assessment.Science can and needs to contribute to comprehensive definitions and standardized measurements of farm animal welfare, for example, through further development of methods that objectively measure and verify certain animal welfare aspects. That said, there is a risk that discussions on animal welfare in developing countries solely use \u201cWestern\u201d definitions of animal welfare, which may not always be appropriate to the cultural and religious contexts in developing countries . For exaIssues for the welfare of farm animals can occur at several different stages of production. These include on-farm treatment, loading and unloading of animals into transport vehicles, and the transport itself. Mental and physical harm can also occur shortly after birth through separation of the mother/siblings, during lairage, on livestock markets, or during stunning and slaughter. While a comprehensive overview of species and supply chain-specific animal welfare issues go beyond the scope of this article, it is clear that potential animal welfare issues depend on level of intensification of production systems, the human\u2013animal relation, and value chain characteristics such as transportation of live animals . UnderlyAt the same time, the social attitudes toward animal welfare not only depend on the actual conditions of animals, but also on the interpretations and importance attributed to different animal welfare conditions. Someone who places more weight on the biological functioning aspects of animal welfare may, for example, deem the relative behavioral restrictions of confined and modern pig production as acceptable because of the higher biosecurity and disease control opportunities. Another person, who places more weight on opportunities for natural behavior may be critical of such productions because they can restrict behavioral opportunities such as foraging, or social and maternal behaviors . Such anAnimal welfare concerns in scientific and public debates mostly relate to commercial intensive production systems . Such syIn high-income countries, production of animal-source foods coming from pigs and poultry is almost entirely intensive. This is not (yet) the case for several developing countries, where backyard systems, as those shown in The welfare of animals kept in extensive systems is oftentimes perceived as better compared to those of animals produced in intensive systems, especially with regards to affective states and natural living . Yet, exThe complexity and multidimensionality of animal welfare are rarely accounted for in traditional livestock systems in developing countries . That saStill, traditional livestock keepers in developing countries rarely view animal welfare as a goal in itself. When associated positively with animal productivity and food quality, good animal welfare is viewed as acceptable and achievable. Improvements in animal welfare become particularly difficult when trade-offs between productivity and welfare goals exist . While aFor consumers, animal welfare conditions may oftentimes not be easily observed or verified. Asymmetric information regarding animal welfare can, therefore, lead to a market failure when people want to purchase meat or dairy products with high levels of animal welfare . HoweverEven though extensive and smallholder farming systems are still common in most developing countries, the production of animal-source food intensifies in most places. Much of the growth in animal-source food production that has taken place in the last decades see  has beenIntensive systems for pig and poultry production now clearly dominate other production systems in some regions, most notably South America and China . Such coIn commercial intensive systems, farmers/animal caretakers typically do not have the same connectedness to their livestock compared to traditional farmers . We hypoIn those developing countries where intensive animal production systems are very common, animal welfare is a publicly discussed issue that concerns consumers, producers, and regulatory institutions. Even in developing countries where the topic of animal welfare is slowly moving on the radar of consumers, animal welfare considerations so far have minimal impacts on actual purchasing decisions. In Brazil, for example, most people agree that animal welfare is generally a relevant topic, yet less than 4% of interviewed consumers consider animal welfare during meat purchases . The disReligious beliefs and cultural traditions can be critical for animal welfare perceptions. The idea that animals should not be used for food or any purpose, for example, is a widespread view of Jains, Buddhists, and many Hindus . DevelopIn order to minimize unnecessary pain, the responsibility of slaughtering for such types of slaughter is typically given to skilled people such as elders or well-respected community members. At the same time, animals\u2019 bellowing during traditional sacrifices is an important indicator that the sacrifice has been successful. Stunning, which supposedly reduces stress and pain for the animal before and during slaughter, is therefore often not compatible with traditional beliefs . AlthougAnimal welfare perceptions can vary between rural and urban citizens . Even thLivestock production systems, cultures, and ethical viewpoints are diverse across and within developing countries. Still, there is an urgent need for animal welfare to increase in many places. In societies where animal welfare concerns are part of public discussions, consumers are generally supportive of approaches toward more animal welfare. In various South American countries or China, for example, consumers are in favor of animal welfare being part of school curricula and that policies and regulations to improve farm animal welfare are appropriate .As of now, legislature regarding animal rights in most developing countries is poor or does not exist at all. With some exceptions such as India, Malaysia, or Mexico, animal welfare legislation in developing countries lags behind those of most developed countries. Protection of farm animals in particular is even less enshrined in legislature . Animal Relatively higher animal welfare standards in developed countries can also have an influence on animal welfare in developing countries through requirements for exports . Yet, ifNGOs have been instrumental in putting animal welfare onto the agenda in developing countries in many ways; working equid charities, including those of the International Coalition of Working Equids, have a significant presence on animal welfare across developing countries. Animal welfare NGOs contributed to efforts to get animal welfare recognized at the United Nations Environmental Agency . They haPerceptions of animal welfare are important for the sustainability of food systems. While farm animal welfare has received considerable attention among developed countries, much less is known from the perspective of developing countries. In this article, we provided an overview over animal welfare concerns in the developing\u00a0world.We showed that the meaning of what constitutes animal welfare is not harmonized on a global level and therefore varies across different contexts. Furthermore, empirical data on animal welfare concerns among consumers and producers in developing countries are limited. Traditional livestock systems are often characterized by close bonds between farmers and their animals, but these systems are not per se free of animal welfare issues. In these systems, knowledge and action gaps tend to hamper the development toward more animal-friendly production. The commercial intensification of livestock production that has and is occurring in several developing countries creates new animal welfare issues that can be exacerbated by limited consumer expectations and legislative controls. In countries with largely intensified production, such as China, Mexico, or Brazil, citizens are becoming increasingly aware and sensitive to animal welfare issues. However, even in these countries, animal welfare plays a small role when it comes to actual purchasing decisions. Given the various animal welfare issues that exist in most parts of the developing world, improvements in animal welfare are needed. Yet, the pathways to such scenarios are difficult. Future scenarios with higher animal welfare will require joint efforts by various stakeholders in the livestock production\u00a0sector.Generally, data on the dynamics of animal welfare concerns in developing countries are still very thin, especially for least developed countries. Most of the insights presented in this article come from case studies and therefore may not be able to fully account for potential regional differences and nuances. Segmentation of animal welfare perceptions has shown to be important for citizens in South America , similar"}
+{"text": "The detection depth of current borehole acoustic reflection imaging is only tens of meters without high resolution. This considerably limits its wide application in the identification and fine description of unconventional reservoirs and in the optimization of drilling trajectories. Increasing the directional energy from the transmitter to a geological structure is an excellent way to solve this issue. In this study, a plasma source with a parabolic reflector was introduced during borehole acoustic reflection imaging. First, an experimental system was built for testing the plasma source. Next, the acoustic-electrical characteristics and directional radiation of the source were studied using experiments and a numerical simulation. Finally, the advantages, disadvantages, and feasibility of the plasma-transmitting source were analyzed; some suggestions for further work on the source and its logging application were proposed. The experimental and simulation results show that the use of a plasma source with a parabolic reflector can increase the detection depth of borehole acoustic reflection imaging to hundreds of meters with high resolution. This is crucial in imaging the geological structures near boreholes and enhancing oil\u2013gas exploration and development. Obtaining accurate formation information in large three-dimensional 3D) spaces around boreholes will aid in improving the efficiency of oil\u2013gas exploration and development. Borehole acoustic reflection imaging is a rapidly developing technology that can detect geological structures within tens of meters or more of a borehole. It can fill the gap between the shallow detection of conventional acoustic logging and the low resolution of seismic exploration. This logging method has the obvious advantage of describing the lateral inhomogeneity in carbonate reservoirs, the attitudes and distributions of fractures, and small geological structures near boreholes ,2. ThereD spaces However, current tools work at low detection depths and cannot provide satisfactory resolutions simultaneously, which seriously affects their application performance. This is attributed to the transmitter power, receiver sensitivity, formation attenuation, and other factors. Increasing the directional energy from the transmitter to a geological structure is an effective way to address this limitation. Currently, acoustic transducers based on the piezoelectric effect are used in various fields ,6,7. ThrThese three sources have certain advantages and disadvantages regarding source power, borehole effect, radiation directivity, formation attenuation, and radial resolution. A piezoelectric monopole source generally works in the range of 10\u201320 kHz and generates a wavefield without directivity in a homogeneous medium. It has high power, and its sound-pressure level (SPL) 1 m from the source in water can reach ~210 dB . CurrentHowever, it cannot satisfy the demand of the next-generation tool regarding high-resolution imaging at a detection depth of hundreds of meters, particularly in directional energy and frequency coverage. Therefore, developing a new downhole transmitting source with high power, wide band, and excellent directional radiation is necessary to achieve deep detection with high resolution in borehole acoustic reflection imaging.The plasma is composed of ions, electrons and unionized neutral particles. It is widely present in gas, metal, semiconductor and electrolyte solutions. There are two kinds of plasma: high-temperature plasma and low-temperature plasma. Chandra et al. used an quantum hydrodynamic model to study the quantum plasma based on a Hometopy Assisted Symbolic Simulation technique and INSAT-FORK code ,21,22. ABased on the abovementioned work, a plasma-transmitting source was introduced in borehole acoustic reflection imaging in this study. An experimental system was built to evaluate the plasma source. The acoustic\u2013electrical characteristics and the directional radiation of the source were studied through experiments and numerical simulations. The feasibility of the plasma-transmitting source was analyzed and some suggestions for further work on the source and its logging application are proposed.An experimental system was built to provide working conditions for the plasma source and measure its output. The voltage-regulation-and-boosting module comprises a regulator and a booster. The 220-Volt AC input is transformed into an adjustable signal from 0 to 250 V via the regulator. The booster increases this signal to thousands of volts, outputs it to the excitation module and monitors its change.The excitation module includes a charging, a discharging, and a release submodule. The electrode module was designed as a cylinder with several acoustic windows to adapt to the borehole environment and acoustic logging tool. The metal electrode is at the center of the cylinder. A high-voltage pulse is output to the electrode immediately after the discharge switch is turned on, causing high field strength in the electrode gap and generating an electric arc. Additionally, the arc expands rapidly and produces a shock wave that spreads outward because of the weak liquid compressibility. This is the working mechanism of a plasma source and the foundation of the design of the experimental system.The system can output 0\u201325 kV in adjustable voltage to the electrode. The measuring range of the high-voltage probe and current clamp are 0\u201340 kV and 0\u201325 kA, respectively. The pressure transducer can measure up to 300 MPa with a sensitivity of 0.73 mV/kPa.Based on the experimental system, the acoustic and electrical characteristics of a plasma source were tested according to the following procedure. First, turn on the charging switch to charge the storage capacitor. Next, turn on the discharge switch to output a high voltage to the electrode to generate a shock wave. Finally, use the release switch to release the residual energy. The electrical characteristics of the plasma source were evaluated by measuring the electrode voltage and current changes. Its acoustic characteristics was evaluated using the acoustic signal received by the pressure transducer.3 and a P-wave velocity of 1500 m/s. The reflector and its position can be described by z2 = 0.14 (r + 0.035), where z represents the well-axis direction and r is its radial direction with \u22120.035 m \u2264 r \u2264 0.035 m. The source is a Ricker wavelet with a dominant frequency of 10 kHz. The sandstone in the formation has a density, a P-wave velocity and an S-wave velocity of 2650 kg/m3, 3800 m/s, and 2300 m/s, respectively.A parabolic reflector can be used to improve the directional radiation of a plasma source in borehole acoustic reflection logging. p are the liquid density, velocity and pressure, respectively. The wave equation in the solid is shown in Equation (2), where u, A pressure\u2013acoustics interface was used for the borehole zone and the solid mechanics interface was used for the formation . The wavt = 0.4 ms. The energy is concentrated at a narrow angle (~60\u00b0) near the reflector\u2019s symmetry axis. This is much better than the case in which the source does not use a reflector and has no evident radiation directivity. The application of a plasma-transmitting source in borehole acoustic reflection logging was analyzed from the following two perspectives: (1) application feasibility and (2) further work on the source and its logging application.This research demonstrates that a plasma source with a parabolic reflector can release significant energy, generate strong wide-band shock waves, and achieve good directional radiation. These results indicate that plasma sources might play a crucial role in borehole acoustic reflection imaging.The feasibility of using the plasma source as a logging transmitter was compared with the current sources in (1)The influencing factors and control methods for the acoustic and electrical characteristics of the plasma source. It is necessary to further investigate the influence of different electrical parameters , electrode systems  and working environments  on the source characteristics. In particular, the source performance in a logging environment with high temperature (>125 \u00b0C), high pressure (>100 MPa) and liquid-filled borehole needs to be studied.(2)The directional radiation of the source. The near-field characteristics of the source must be investigated. It is necessary to manufacture parabolic reflectors with different parameters and evaluate them experimentally according to the numerical simulation. A small-sized controllable rotating parabolic reflector should be considered to achieve directional and scanning radiation. This could not only enhance the directional energy towards the target but also improve the azimuthal resolution of the tool based on the source.(3)The acoustic reflection imaging response of a geological structure. The logging response of a geological structure with different parameters  must be considered to provide a foundation for tool design and logging inversion based on plasma sources.(4)Downhole realization. A small downhole plasma source should be developed to prevent the energy losses caused by wireline transmission. Considerable work must be conducted, e.g., optimizing the electrode structure to improve its stability, selecting high-temperature-resistant components and determining a suitable level of directional-radiation energy to ensure deep detection without damaging the borehole.(5)Data acquisition and processing based on the source. In terms of data acquisition, two points need to be considered: (1) increase the acquisition time of the full waveform to correspond with the increased detection depth and (2) separate the detection process for the near- and far-borehole zones into two data acquisitions. This helps to improve the signal-to-noise ratio of the reflection wave. In terms of data processing, the extraction of the weak reflection wave and corresponding high-resolution imaging need further research.Although feasible and promising, borehole acoustic reflection imaging based on a plasma source is still in its early stage. Considerable research with theoretical analyses, numerical simulations, experimental tests and field applications is required to introduce this source into logging applications, including the following aspects:Based on the investigation of the existing sources, a plasma source was introduced into borehole acoustic reflection imaging. An experimental system was built for evaluating the plasma source, and the source\u2019s acoustic\u2013electrical characteristics and directional radiation were studied through experiments and a numerical simulation. The feasibility of the plasma transmitting source was analyzed and some suggestions for further work on the source and its logging application were proposed. The following conclusions can be drawn.(1)The current monopole, dipole and phased-array sources cannot satisfy the demand of next-generation tools regarding high-resolution imaging at detection depths of hundreds of meters, particularly with respect to the effective energy and frequency coverage.(2)A plasma source with a parabolic reflector can release high levels of energy, generate strong wide-band shock waves and achieve good directional radiation. The development of borehole acoustic reflection imaging based on this source is feasible and promising. This would considerably increase the detection depth with high resolution to improve the performance of oil\u2013gas exploration and development.(3)Borehole acoustic reflection imaging based on plasma sources is still in the early stage and requires further research in terms of theoretical analyses, numerical simulation, experimental testing and field applications."}
+{"text": "Influenza virus circulation virtually ceased in Canada during the COVID-19 pandemic, re-emerging with the relaxation of restrictions in spring 2022. Using a test-negative design, the Canadian Sentinel Practitioner Surveillance Network reports 2021/22 vaccine effectiveness of 36% (95%\u202fCI: \u221238 to 71) against late-season illness due to influenza A(H3N2) clade 3C.2a1b.2a.2 viruses, considered antigenically distinct from the 3C.2a1b.2a.1 vaccine strain. Findings reinforce the World Health Organization\u2019s decision to update the 2022/23 northern hemisphere vaccine to a more representative A(H3N2) clade 3C.2a1b.2a.2 strain. In Canada, as elsewhere, public health measures such as physical distancing, masking requirements and vaccine passports were implemented early in the coronavirus disease COVID-19) pandemic to control transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [9 pandemiUsing a test-negative design (TND), the Canadian Sentinel Practitioner Surveillance Network (SPSN) assessed 2021/22 vaccine effectiveness (VE) against late-season AH3N2) illness from March to July of 2022. With the incorporation of SARS-CoV-2 surveillance into SPSN activities, we also explored confounding due to potentially correlated influenza and COVID-19 vaccination behaviours, recently suggested by others as influential on influenza VE estimates [N2 illnesNasal or nasopharyngeal specimens and epidemiological data included in VE analyses were collected from consenting patients presenting within 7 days of acute respiratory symptom onset to community-based clinics or COVID-19 assessment sites in the provinces of Alberta, British Columbia and Ontario. Influenza VE analyses were restricted as per usual to those with influenza-like illness (ILI) defined by acute onset of fever and cough, and at least one other symptom including sore throat, myalgia, arthralgia or prostration . Fever wInfluenza and COVID-19 vaccination status was based on participant self-report of 2021/22 vaccine receipt\u2009\u2265\u20092 weeks before symptom onset; patients vaccinated\u2009less than\u20092 weeks before symptom onset or with unknown vaccination status/timing were excluded. Virtually all of the 2021/22 influenza vaccines available in contributing Canadian provinces were egg-based and inactivated products. The 2021/22 influenza vaccine contained an A/Cambodia/e0826360/2020 (H3N2)-like virus belonging to clade 3C.2a1b.2a.1 .VE was calculated as 1\u2009\u2212\u2009odds ratios (OR)\u202f\u00d7\u202f100%. ORs were derived by comparing influenza test positivity between vaccinated and unvaccinated participants using logistic regression, adjusting for potential confounders as specified. In exploratory analyses we excluded COVID-19 cases (NAAT-confirmed for SARS-CoV-2) from influenza virus test-negative controls , and addOf 327 eligible specimens collected during the analysis period, 42 (13%) tested positive for influenza virus and all were A(H3N2) . Of the Supplementary Table S1). Of these 77 participants, 72 (93%) were vaccinated against COVID-19. Compared with the SARS-CoV-2-negative controls shown in Supplementary Table S1, SARS-CoV-2-positive and inconclusive controls were older  (p<0.001), more often with comorbidity (62/208 (30%) and 32/77 (42%), respectively) (p = 0.061), and vaccinated against influenza (108/208 (52%) and 47/77 (61%), respectively) (p = 0.170).In exploratory analyses excluding the 77 (27%) of 285 influenza virus test-negative controls that were NAAT positive (n\u202f=\u202f76) or inconclusive (n\u202f=\u202f1) for SARS-CoV-2, participant characteristics were similar (for the detailed results on this see Supplementary Table S1). Variation by month was significant for the likelihood of test positivity but not for being vaccinated. In primary analysis of fully adjusted VE we included age and comorbidity as well as province and month for consistency with previous SPSN analyses [Overall, 54% of negative controls and 36% of influenza cases were vaccinated (p\u2009=\u20090.024) . Age andanalyses . For refCrude VE against influenza A(H3N2) was 53% (95% CI: 9\u201376). Adjustment for age and comorbidity had the greatest individual impacts in lowering VE estimates. In primary analysis, fully adjusted VE was 36% (95% CI: \u221238 to 71) . AdjusteIn the wake of COVID-19-related restrictions beginning March 2020, community-level influenza virus circulation essentially halted in Canada [Confidence intervals around our VE estimates are wide and we cannot rule out an interpretation of no protection. However, point estimates remain the most likely findings and, despite a longer interval since vaccination, are comparable to VE estimates against influenza A(H3N2) recently reported from the United States (US)  for the period spanning October 2021 to April 2022  and fromThe current immuno-epidemiological context may be further complicated because circulation of influenza (and other respiratory viruses) virtually ceased during the pandemic, although insofar as that context was shared between vaccinated and unvaccinated individuals, it should not have influenced VE estimates. Excluding COVID-19 cases from the influenza virus test-negative controls lowered our influenza VE estimates (from about one third to one quarter), contrary to the direction of effect Doll et al. theorised due to positive collinearity between influenza and COVID-19 vaccination . UnderpiWith respect to limitations, our results should be interpreted with caution given small sample size and wide confidence intervals. Residual bias and confounding cannot be ruled out. Generalisation to other jurisdictions where the mix of circulating viruses and other context differ should be undertaken cautiously.After a 2-year hiatus, influenza A(H3N2) viruses belonging to clade 3C.2a1b.2a.2 contributed to an atypical late spring 2022 wave in Canada. Despite an unusually long interval since vaccination, the mismatched 2021/22 vaccine reduced the risk of influenza A(H3N2) illness by about one-third, comparable to previous seasons. The findings reinforce the World Health Organization\u2019s decision to switch to a more representative clade 3C.2a1b.2a.2 strain for the northern hemisphere 2022/23 A(H3N2) vaccine component."}
+{"text": "This research aimed to evaluate the impacts of transfusing packed red blood cells (pRBCs), fresh frozen plasma (FFP), or platelet concentrate (PC) on postoperative mechanical ventilation time (MVT) in patients with acute Stanford type A aortic dissection (ATAAD) undergoing after total arch replacement (TAR).The clinical data of 384 patients with ATAAD after TAR were retrospectively collected from December 2015 to October 2017 to verify whether pRBCs, FFP, or PC transfusion volumes were associated with postoperative MVT. The logistic regression was used to assess whether blood products were risk factors for prolonged mechanical ventilation (PMV) in all three endpoints .ORPMV\u226524) = 1.045, p = 0.005; ORPMV\u226548 = 1.060, p = 0.002; ORPMV\u226572 = 1.051, p = 0.011]. pRBC transfusion  and PC transfusion  were independent risk factors for PMV. FFP had no noticeable effect on PMV .The mean age of 384 patients was 47.6 \u00b1 10.689 years, and 301 (78.39%) patients were men. Median MVT was 29.5 (4\u2013574) h (h), and 213 (55.47%), 136 (35.42%), and 96 (25.00%) patients had PMV \u226524 h, \u226548 h, and \u226572 h, respectively. A total of 36 (9.38%) patients did not have any blood product transfusion, the number of patients with transfusion of pRBCs, FFP, and PC were 334 (86.98%), 286 (74.48%), and 189 (49.22%), respectively. According to the multivariate logistic regression of three PMV time-endpoints, age was a risk factor [PMV \u2265 24 h odds ratio (In patients with ATAAD after TAR, the incidence of PMV was very high. Blood products transfusion was closely related to postoperative mechanical ventilation time. pRBC and PC transfusions and age increased the incidence of PMV at all three endpoints. Acute Stanford type A aorta dissection (ATAAD) is a fatal cardiovascular disease. Due to the widespread lesion, the mortality with immediate surgical intervention is still as high as 30% and drug treatment is as high as 58% , 2. TotaA total of 384 consecutive patients (47.6 \u00b1 10.689 years) with ATAAD undergoing TAR were enrolled at Beijing Anzhen Hospital of Capital Medical University from December 2015 to October 2017. The inclusion criteria were as follows: patients who had been diagnosed as Stanford type A aortic dissection by aortic enhanced CT and who underwent surgical treatment; those with the onset of disease at \u226414 days; and those aged \u226518 years. Patients with traumatic aortic dissection, those with aortic dissection during pregnancy, those with the onset of disease at more than 14 days, and those aged <18 were excluded. All the clinical data were retrospectively recorded from the medical records; 33 cases had been eliminated with insufficient data. This retrospective study was approved by the Medical Ethical Committee of the Beijing Anzhen Hospital of Capital Medical University (2020100X) and waived the need for individual patient consent.2 > 90% or PaO2 > 60 mmHg  or more , 10. In  60 mmHg , 12. TheThe respective totality of packed red blood cells (pRBCs), fresh frozen plasma (FFP), or platelet concentrate (PC) that patients transfused intraoperatively and postoperatively were recorded. The total number of pRBCs units (U) did not include autologous blood stored intraoperatively and postoperatively. Indications for pRBCs transfusion included the hemoglobin level of \u22649.0 g/dl on the surgery day or \u22647.0 g/dl postoperatively. The PC transfusion indications included life-threatening active bleeding, the platelet count \u2264 50,000/ml, or the platelet count <100,000/ml with bleeding. The FFP was transfused when the prothrombin time was >1.5 times the upper limit of normal with a prolonged cardiopulmonary bypass (CPB) time or clinical signs of bleeding \u201315.via the perfusion limb of the four-branch graft. SCP was discontinued after anastomosis of the left common carotid artery, the left subclavian artery, and the innominate artery. Then, CPB flow was gradually resumed normal and began to warm.All procedures were median sternotomy, and total CPB with selective antegrade cerebral perfusion (SCP) was performed in the right axillary artery and the right atrium. The arterial hemoperfusion consisted of right axillary artery perfusion and one branch of a four-branch aortic graft. The ascending aorta was clamped during cooling, and a cold-blood cardioplegic solution was antegrade infused into the coronary ostia by a longitudinal incision in the proximal ascending aorta. An anastomosis of the four-branch aortic graft to the aortic root was finished in the cooling phase; the circulatory anastomosis was arrested after the nasopharyngeal temperature reached 25\u00b0C. After the brachiocephalic arteries were clamped, unilateral SCP was started through the right axillary artery. The anastomosis of the stent graft in the aortic arch to the four-branch graft distal end was completed, and the four-branch graft was clamped. The lower body got blood perfusion t-test and non-parametric tests were used to compare continuous variables. The categorical variables were compared by the \u03c72 analysis or Fisher's exact correction. The p-value of <0.05 was considered significant. Then, a stepwise multivariable logistic regression analysis was used to determine factors independently associated with PMV. Statistical Package for the Social Sciences Version 26.0  was used to analyze data. Prism 9  was used for drawing figures.Descriptive statistics were expressed as means \u00b1 standard deviations or median (interquartile range) for continuous variables and percentages and frequencies for categorical variables. Independent-samples U, respectively. Median MVT was 29.50 (14.00\u201371.75) h, the number of cases of PMV \u226524 h, \u226548 h, and \u226572 h was 213 (55.47%), 136 (35.42%), and 96 (25.00%), respectively. More perioperative baseline data and main complications characteristics were shown in The age (mean \u00b1 standard deviation) of all 384 patients was 47.64 \u00b1 10.69 years, 301 (78.39%) patients were men, and time  from related symptom onset to TAR was 24 (13.00\u201348.00) h. The median CPB time was 204.50 (180.00\u2013234.00) min (min), and 18 (4.70%) patients had the median CPB time of over 300 min A total of 27 (7.00%) patients received recombinant factor VIIa (rFVIIa) infusion in-hospital, and the median volume of the total intraoperative and postoperative of pRBCs, FFP, and PC was 8.00 (4.00\u201314.00) U, 400.00 (0\u2013800.00) ml, and 0 (0\u20132.00) p = 0.001). In different periods of PMV , the respective transfusion volume of pRBCs, FFP, or PC was statistically different (As shown in ifferent .p = 0.003), but not on PMV \u226524 h or \u226572 h. PMV \u226524 h and \u226548 h were affected  by renal artery involved dissection, but not PMV \u226572 h. Left ventricular end-diastolic dimension showed a statistical significance (p = 0.032) only in PMV \u226572 h. The platelet (PLT) count was different  both in PMV \u226524 h and \u226548 h. When the CPB was \u2265300 min, the difference (p = 0.020) was only shown in PMV \u226548 h. The transfusion of rFVIIa, pRBCs, FFP, and PC, respectively, showed a statistical difference on three PMV.In ORPMV\u226524) = 1.045, p = 0.005; ORPMV\u226548 = 1.060, p = 0.002; ORPMV\u226572 = 1.051, p = 0.011]. pRBC  and PC  transfusions were independent risk factors for PMV : 0.999\u20131.000, p = 0.086], on PMV \u226548 h , and PMV \u226572 h . Besides, while considering PMV \u226524 h as the endpoint, AST , ALT , and WBC  were preoperative risk factors. The CPB time  was the predictive factor of PMV \u226548 h. For PMV \u226572 h, WBC  had the predictive effect.Multivariate logistic regression with three different PMV  endpoints showed that age was a risk factor [PMV \u2265 24 h, odds ratio  and coagulation dysfunction , and the respective transfusion volume of pRBCs, FFP, or PC in four different MVT groups had statistical significance. Moreover, after summarizing three multivariate logistics regressions of different PMV endpoints, it was found that intraoperative and postoperative pRBC and PC transfusions are effective predictors for PMV . For lack of targeted research, the mechanism by which pRBC and PC transfusions affect PMV remains unclear. According to the existing studies, we speculated that it might be related to the decreased deformability and oxygen transfer of the allogeneic stored red cells, and the hemolysis of the long-stored red cells will lead to electrolyte disorder  , 28. In On the other hand, transfusion-related immunomodulation is the direct cause of postoperative infections and multiple organ failure. The plasma from pRBCs induced neutrophils to produce superoxide, enhanced cytotoxicity, and stimulated pulmonary endothelial cells , 35. Thep = 0.003) (It had been proven that older age (\u226570 years) means a higher postoperative PMV risk in patients undergoing coronary artery bypass grafting or elective coronary artery surgery, and older age is also an independent risk factor for PMV after total cavopulmanory connection surgery , 40, 41.= 0.003) . When as= 0.003) . After s= 0.003) . Age is = 0.003) . The oppOnly two studies have confirmed that leukocytosis is a risk factor for PMV after cardiovascular surgery, one had PMV \u226524 h after cardiac surgery with CPB, and the other had PMV \u226572 h after acute DeBakey type I aortic dissection surgery , 46. SimMany factors lead to PMV, and our research provided a new idea for ATAAD after TAR to predict PMV. However, for most cardiovascular surgeries requiring blood products transfusion, reducing or restricting the transfusion volume without reference is not a reasonable way to reduce PMV incidence. Restrictive pRBC transfusion failed to show advantages and increased the risk of mortality sometimes \u201349. It mIn patients with ATAAD after TAR, PMV incidence was very high. Blood product transfusion was closely related to postoperative mechanical ventilation time. pRBC and PC transfusions and age increased the incidence of PMV at all three endpoints. FFC had no noticeable impact on PMV.This was a single-center study, which is the main limitation of this research. As a retrospective study, there was a lack of data on the transfusion time (intraoperative or postoperative) and how long the blood products were stored, especially pRBCs.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethical Committee of the Beijing Anzhen Hospital of Capital Medical University (2020100X). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CLi, YG, LS, and JZhu: conception and design. CLi, YG, LS, YL, JZhe, and JZhu: provision of research materials or patients. QX, YZ, CLu, and RG: data collection and analysis. All authors: manuscript writing. All authors contributed to the article and approved the submitted version.This work was supported by the Beijing Major Science and Technology Projects from the Beijing Municipal Science and Technology Commission (No. Z191100006619093) and the Natural Science Foundation of China (No. 81970393).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Helleborus atrorubens is an economically important perennial garden plant with medicinal value. Here, we sequenced the complete chloroplast genome of H. atrorubens. The results revealed the chloroplast genome to be 166,695\u2009bp in length. It possesses a typical quadripartite structure containing one large single copy (LSC) region (84994\u2009bp), one small single copy (SSC) region , and a pair of inverted repeat (IR) regions (31938\u2009bp). This chloroplast genome encoded 130 genes, out of which 85 code for proteins, 37 for transfer RNAs, and 8 for ribosomal RNAs. Simple sequence repeat (SSR) markers and the top variable coding regions were identified.Helleborus genus.Our study lays a foundation for further research, such as species differentiation and phylogenetic reconstruction of the  Helleborus atrorubens is a perennial species native to the Balkan Peninsula with ornamental and medicinal value. While the diversity of phytochemicals produced by H. atrorubens has been well investigated, there is a dearth of genomic resources for this species . The plants were cultivated in Wangcun Village, Linpu Town, Xiaoshan District, Hangzhou, China . A voucher specimen was deposited in the Herbarium of Northwest A&F University (NWFC) under voucher number Shi001 . The genomic DNA was extracted with a plant genomic DNA kit  and sequenced using the Illumina NovaSeq platform according to the manufacturer\u2019s recommendations.H. thibetanus, the closest species with an available plastome, using Bowtie2 v.2.2.6  values were calculated using each gene to determine the hotspots of divergence. A total of 25 genes including psbA, psbD, psbC and others with a high Pi value (>0.01) and suitable length (>500 and <1500\u2009bp) for PCR amplification can be used as candidate molecular markers to study interspecific relationships (Table S1).In this study, 106 chloroplast SSRs (cpSSRs) were identified for H. atrorubens for the first time. The gene order and cp genome arrangement of H. atrorubens were similar to those of related species. The current study provides a valuable contribution that could help species identification, crossbreeding and genetic diversity research.In this study, we elucidated the complete plastome of"}
+{"text": "Brain rhythms emerge from the mean-field activity of networks of neurons. There have been many efforts to build mathematical and computational embodiments in the form of discrete cell-group activities\u2014termed neural masses\u2014to understand in particular the origins of evoked potentials, intrinsic patterns of activities such as theta, regulation of sleep, Parkinson\u2019s disease related dynamics, and mimic seizure dynamics. As originally utilized, standard neural masses convert input through a sigmoidal function to a firing rate, and firing rate through a synaptic alpha function to other masses. Here we define a process to build mechanistic neural masses (mNMs) as mean-field models of microscopic membrane-type (Hodgkin Huxley type) models of different neuron types that duplicate the stability, firing rate, and associated bifurcations as function of relevant slow variables - such as extracellular potassium - and synaptic current; and whose output is both firing rate and impact on the slow variables - such as transmembrane potassium flux. Small networks composed of just excitatory and inhibitory mNMs demonstrate expected dynamical states including firing, runaway excitation and depolarization block, and these transitions change in biologically observed ways with changes in extracellular potassium and excitatory-inhibitory balance. Neural masses are mean field models of neural cell group activities. Neural mass models were first introduced by Walter Freeman in the 1970s  as a modIn the past, networks of NMs  have been constructed and used to understand the origins of evoked potentials , intrinsIt is our objective here to build discrete mean-field model elements - neural masses (NM) - whose dynamics, detailed parameters, and interactions with each other and their environment can be linked quantitatively across scales directly to membrane-level mechanics of single neurons. Such mechanistic neural masses can then be used to build network models that interact with the environment and have measures that can quantitatively be related to physiological measures. Our interest here is especially to embody some of the underlying physiological mechanics that are hypothesized to be involved in epileptic dynamics.Epilepsy - the occurrence of recurrent spontaneous seizures - is a major human health concern, leading to significant deficit in quality of life, higher mortality, and significant economic impact. In developed countries, epilepsy rates are close to 1% of the population. There are pharmacological interventions, but these provide successful abatement of seizures in only as few as 2/3 of patients, and often have significant side effects .At the network level, seizure susceptibility is often described as resulting from an excitatory-inhibitory imbalance. At the cellular level, this can mechanistically result from transient inactivation of inhibitory neurons leading to runaway excitation .Pre- or post-seizure side effects or related neurological phenomena include postictal generalized EEG suppression (PGES) , post-icLe\u00e3o discovered spreading depolarization - which he denoted spreading depression - in 1944 in the context of acute induced seizure models . SD is rAlthough many mechanisms can contribute to SD, the fundamental component is that elevation of the extracelluar potassium concentration leads individual neurons into depolarization block (DB) - a state in which the potassium channels are substantially open, but the potassium current is not enough to repolarize the membrane potential and restore the channels to the normally closed state. In this state, the potassium flux can further increase the extracellular potassium concentration and, through diffusion, induce nearby neurons into DB .These mechanics are well known and captured in membrane and compartment models of single neurons .In this work we define a process to develop mechanistic neural mass (mNM) elements derived from single-cell membrane dynamics. Functionally, these mass elements should serve as plug-in replacements for commonly-used elements in existing neural mass models. These differ in that they parametrize state transitions of the single-cell models to include depolarization block, their sensitivity to slow variables such as extracellular potassium and accommodation state, and their coupling back to those slow variables. We find that small excitatotry-inhibitory networks built from those networks embody dynamical transitions that underlie seizure and spreading depolarization dynamics.It is our expectation that network models constructed with the resulting mechanistic neural masses (mNMs) we develop can reproduce normal dynamics as well as the pathological dynamics of seizures and SD in a way that can be linked to measurable biological features such as extracellular potassium, make quantitative predictions about their effects, and meaningfully give insight as to why they have such effects. As an example, we aim to give insight as to why a modest change in excitatory-inhibitory balance yields a network that is not continually seizing or undergoing SD but is significantly more susceptible to these instabilities.As a primary step, we outline the procedure for deriving mechanistic neural masses and demonstrate it for two canonical neuron types: a non-accommodating mammalian inhibitory neuron, based on the Wang-Buzs\u00e1ki (WB) neuron , and an We then demonstrate that a two-mass inhibitory-excitatory network built from these elements, illustrated in Neural masses  are simpNeural mass dynamics are supposed to represent the activity of groups of hundreds to millions of neurons . While tCritical in the process of creating neural masses is that the dynamics are significantly simplified from the detailed firing/action potential dynamics of Hodgkin-Huxley type membrane dynamics. The simplifications achieved with neural mass dynamics are very low computational load, and loss of action potential timing sensitivity by conversion to firing rates.To achieve this mean field approach from Hodgkin-Huxley type single neuron models - to create mechanistic neural masses - we utilize a time-scale separation approach in which the fast dynamics associated with the action potentials are separated from slower dynamics by treating those other dynamics as quasi-static. We then parameterize the dynamics of that fast model, explicitly fitting as output the firing rate, mean membrane potential, and potassium flux, as a function of those quasi-static inputs. These outputs are then used in the evolution dynamics of the slow input variables, as well as the coupling of the mass to its environment and to other masses.A key idea behind the development of mNMs, is that the time-averages of the fast dynamics of the neuron models can be expressed as simple functions of the total current and quasi-static (slow) state variables. Hence, instead of looking at detailed firing response of neurons in terms of instantaneous changes in membrane potentials, potassium fluxes and firing rates, we look steady-state response of the neuron models and use them to characterize the corresponding neural masses. These steady state responses are then parametrized (fitted) with simple analytic functions of the net input current and the slow state variables. These analytical functions fully characterize our neural mass outputs in terms of mean firing rate, mean membrane potential, and mean potassium flux.\u03bdK) from its nominal value. If the nominal and actual Nernst potentials are \u03bdK, respectively, the change in it is expressed as \u0394\u03bdK.We are interested in dynamics of the masses in the bifurcation space of injected current and changes in the transmembrane potassium Nernst potential  and \u03bdK to obtain membrane potential traces. Following this, we find the corresponding mean firing rate (\u27e8FR\u27e9), and mean membrane potential (\u27e8Vm\u27e9) and mean potassium flux (\u27e8Kflux\u27e9) for each values of Iinj and \u03bdK by averaging the dynamics. As shown in Iinj and \u03bdK values, this subdivides  parameter space into regions of not-firing (NF), firing (F), and depolarization block (DB).Given a Hodgkin-Huxley type single cell neuron model  and Wang- Buzs\u00e1ki (WB) models, described next), the procedure for developing the corresponding mNMs is:Iinj and \u03bdK is characteristically different from not-firing region that occurs at low Iinj values. While the NF region has either negative or zero \u27e8Kflux\u27e9, the DB region has finite and positive \u27e8Kflux\u27e9. Similarly, the \u27e8Vm\u27e9 shows very negative values in the NF region, whereas it shows higher values in the DB region.The depolarization block region that occurs at higher values of Identification of bifurcation boundaries. After identification of the three dynamical regimes of interest, we then analytically identify the bifurcation boundaries.Iinj and \u03bdK state space. For this, we find stability of the fixed points of the models using eigenvalues of the model Jacobian. In practice the fixed points in these models are more easily solved as functions of Vm. The point where the largest eigenvalue crosses zero marks the transitions between stable and unstable regions. The two boundaries of the unstable triangular region are shown as blue (ISSF) and red (ISSDB) lines in top row of \u03bdK  near the thresholds (ISSF and ISSDB). We use these means to parameterize these quantities within the enclosed firing region. Outside these regions the means are analytically defined by the stable fixed point solutions.Note that while the dynamics in the lower triangular region bounded by the DB(\u03bdK) \u2212 \u0394), as shown in green in \u03bdK, for these models are shown in next three rows of FR\u27e9 and other mean dynamical quantities at ISSDB \u2212 \u0394 as their \u201cmaximum\u201d values in the firing region before the model goes into DB.Neural Mass parametrization: Finding functional fits to time-averaged dynamical quantities. After obtaining the fairly smooth boundaries of unstable region and mean dynamical quantities as a function of \u0394\u03bdK, we find and polynomial functions that parameterize the bifurcation boundaries ISSF(\u03bdk) and ISSDB, as well as \u27e8FR\u27e9, \u27e8Vm\u27e9, and \u27e8Kflux (\u03bdk)\u27e9 along these boundaries. So, for any given \u03bdk, these analytical functions would give the F, DB thresholds, and \u201cmaximum\u201d values of the dynamical quantities. These functions are then used to parametrize the dynamical quantities between the boundaries, which appear with nearly square-root behavior. These resulting equations form our mNM behavior.Check Fidelity of neural mass parametrization. Within the firing region, defined by the parametrized boundaries The mean firing rate is a smooth, nearly square-root shaped function of ISS, except near the DB boundary refer , where iIn the next two sections we give the ODE equations and details of the WB and SEAN neuron models that we use to obtain the I and E neural masses.The model equations of the Wang-Buzs\u00e1ki  neuron awhere,\u03c1 = 1.25 is taken from (Ipump), the changes in \u03bdK is fully accounted by [K]o.The expression for the pump current and the value of pump strength ken from . NominalThe activation functions for the ion channels are given by the following equations:KFlux\u27e9 current has contributions from active potassium current (GK(Vm \u2212 \u03bdK)) and the pump current.The values of the model parameters are given in the first column of Vm, n\u221e(Vm), m\u221e(Vm), h\u221e(Vm)), and its stability is computed from the Jacobian of the dynamics at that point. At the fixed point, the input current ISS for a given Vm and \u03bdK is given by:The fixed point of the system of WB model equations for a given membrane potential are given by (F) and Depolarization block boundary (ISSDB) are calculated using the above expression and depicted in the left column of The boundaries of the unstable region in terms of the Firing boundary (ISSIK\u2212AHP) and the pump current (Ipump).We obtain the SEAN model from the Pinksy-Rinzel model  by conveThe equations of the SEAN model are as follows:where,\u03c1 = 1.25 is the pump strength. \u03bdK = \u221275\u00a0mV, \u03bdNa = 60\u00a0mV). Non-hatted versions of these quantities incorporate the changes in Nernst potentials.where Vm are:The activation variables of different ions as a function IK\u2212AHP is the current responsible for accommodation in this model and calcium activation variable q is assumed to be at its steady value:\u03b1q = min  and \u03b2q = 0.001.The potassium after-hyper-polarization current As in , the calIK\u2212AHP into fast dynamical  effects computed at the nominal state and slow modulatory effects that depend on calcium ):We then make the follow approximation to separate the effects of the IK\u2212AHP,fast, where we have chosen Iinj,eff = Iinj + IK\u2212AHP,slow.In practice, the mNM parametrization is computed just using the fast dynamics KFlux\u27e9 current has contributions from the active potassium current (GK(Vm \u2212 \u03bdK)), the pump current, and the accommodation current.The values of the model parameters are given in the second column of Vm and \u03bdK) are determined as:F) and DB (ISSDB) boundaries in terms of steady state current are depicted in The model fixed points are determined similarly as the previous model. The steady state value of the current at given (DB \u2212 \u0394) to parameterize the mNMs. For both the neuron models, we use polynomial functions up to 3rd order to fit these quantities as a function of \u03bdK (x). For instance, for functional fit to ISSF of WB model, we obtain the best linear fit (y = ax + b) with a = 0.014, b = 1.746 as parameters. All the functional best fits for both the models are shown in docs.scipy.org) module was used to obtain the best fit. These functions are used in ascertaining the limits of the three dynamical quantities, for a given \u03bdK.We use the functional fits to the firing and DB thresholds, and the mean of dynamical variables at (adjusted) DB threshold (ISSM) to yield mean dynamical output (y) in terms of mean firing rate, mean membrane potential, or, mean potassium flux.As a next step we separately identify another set of simple functions that would approximate the values of the dynamical variables of the mass, within the predetermined limit, as a function of the injected current. These functions , we first determine the threshold currents, and maximum values of mean dynamical variables from the first set of analytical functions. Next, we use the Iinj, as the independent variable in the next set of functions and find the dynamical variables corresponding to the mass. The choice of these functions was guided by the profiles of average firing rate \u27e8FR\u27e9, membrane potential \u27e8Vm\u27e9, and potassium flux \u27e8Kflux\u27e9 of individual neuron models as function of Iinj. These are shown for three different \u03bdK values for both the neuron models in In summary, for any given combination of  Nernst potentials of the two masses are:The thresholds current values and maximum of the means of FR, V\u03bdK is same for both the masses.The hatted versions of the Nernst potentials , we address them as the neural mass functionals. We present these functionals below for the I and the E masses.The parameters of the above linear and polynomial functions can be used to determine these thresholds for any arbitrary \u0394I), mean membrane potential function KFI) for the I mass are:C1 = \u221260, QI is the normalized total current that the mass receives and is expressed as:The mean firing rate function , mean membrane potential function KFE) for the E mass are:C1 = \u221265 and C2 = 0.1 and normalized total current (QE) is expressed as:The mean firing rate function  from their respective neuron models. Next, we would like to understand how does the coupled E \u2212 I model, in which the two masses interact with each other through synaptic coupling, behaves under the conditions of changing injected currents and changes in the Mass coupling can also be understood from an averaging approach from classic Hodgkin-Huxley (HH) type compartment models. We write this out both for completeness, and to illustrate a slight change from existing NMMs.tap has been characterized by alpha functions \u03b1(t)  .It\u2212tap=\u03c4s is particular to a type of neurotransmitter, and we will in the future denote as a generalization \u03b1x(t) to have time constant \u03c4x. Note that the term Vm \u2212 \u03bds is the difference between membrane potential Vm and the Nernst-potential of carrier ions through the channel of type s. Commonly used time constants include \u03c4p = 10\u00a0ms for pyramidal neurons, \u03c4I,f = 2\u00a0ms for fast (\u03b3-aminobutyric acid GABAA) inhibitory, and \u03c4I,s = 20\u00a0ms for slow (GABAB) inhibitory interneurons  for the ith single cell from population a, assumed to be all of the same cell type:b from action potentials in i will be characterized as,It is now convenient to introduce a pre-synaptic centric conductance function a, composed of neurons i, all with the same neurotrasmitter type, and assumed to have common time delay Da,b. As such \u03b1a,i = \u03b1a. As a result we arrive at an equivalent averaged conductance function ha(t),a as Fa(\u03c4).In our neural mass formalism, we now average over a population ha(t) can be computed without the time convolution by instead integrating the second order ordinary differential equation: It can be shown that Fa(t) = Fa and ha = Fa.Note that under this parametrization, under steady state conditions b, whose presynaptic masses are denoted as members a, will have a total input currentFor computational efficiency, for masses that are coupled into networks with both near and far connections, we only compute the synaptic dynamics once per mass, and accommodate the delays in their connections. Explicitly, for NM for population fb , \u03b8b(t)).Vm in Vm\u27e9. This is commonly further replaced with a nominal value  V^m,bAs described, we have developed mNMs as parameterizations of single-neuron Hodgkin-Huxley style neurons for both inhibitory and excitatory neuron types, based on the WB and SEAN models. As results, we first demonstrate that the individual mNMs parametrizations reproduce the outputs of the demonstrated models.We then investigate the dynamics of the simple 2-element excitatory/inhibitory network illustrated in FR\u27e9ODE, \u03bdK values. We also find out these three quantities  and II(t) are the total contributions to the E and I masses, respectively, expressed as:Ix,inj denote the input currents from outside the network, Ix,pump denotes the pump currents, and the coupling constants gxy and synaptic functions hx(t), and the differential equations that govern them, follow the definitions in Using the methodology as described in Coupled Model section in Methods, we couple the two kinds of masses to each other, so as to study the dynamical behaviour of the E \u2212 I couplet. Here we investigate the system behaviour in terms of the E and I mass firing rates variations in potassium ion concentration, injected current and coupling strength between the masses. Hence, the mean FR of each of the masses is a function of total current expressed as combination of injected current and the mean FR of the other masses that it couples to. The ODEs of the E \u2212 I coupled model are expressed in terms of l (1972) , was ado\u03bbE = \u03bbI = 1, \u03c4E = 10, \u03c4I = 20, which corresponds to slow (GabaB-type) synapses. Nominal coupling constants used here are II,inj = 0.The values of the parameters used for the simulations are, in ms, IE,inj, are shown under four different state conditions in \u03bdK and gIE. At very low pulse height, this small network oscillates. We expect that addition of fast (GABA-A) inhibition could be used to tune that behavior.Some of the fundamental dynamics of this E \u2212 I network, under external injected current input t = 12\u00a0s). At this point the excitatory mass fires at an even higher rate, and is equivalent to the Run-away excitation (RAE). At even higher pulse height, the excitatory mass is driven into depolarization block and both their firing rates go to zero during the pulse. We denote this network state as DB. Note that upon pulse initiation, because the pulses have a sloped front end, both masses initially fire before reaching DB.At moderate pulse height, we observe balanced firing of both the excitatory and inhibitory masses. As the pulse height increases, the inhibitory mass is driven into DB  firing due to failure of the I mass to inhibit its firing. This in turn is because the E mass\u2019s firing rate is high enough to put the I mass into DB. In the BS region, the E mass shows two stable firing patterns for the same injected current depending on whether the I mass is in DB or not - and this depends on history. When the I mass is in the F state and injected current is on, the E mass shows one FR (lower), while when I mass is in the DB state, and the same injected current is still on, the E mass shows another FR (higher). This property marks the bi-stable region in the The analytical boundaries of these regions are shown in 1) The FO boundary is obtained using the parameters to the functional fits as in the equation of I mass goes into depolarization, after reaching its maximum firing rate and as a result the E mass fires in uninhibited manner. Hence, we first find the total current to the I mass (QI) that results in its maximum FR using 2) The RAE boundary is characterized by a point where Steps to obtain the Firing onset (FO), Bi-stability (BS), Run-away excitation (RAE) and Depolarization block (DB) boundaries are calculated as follows, where the Nernst potentials are adjusted using I, and E for the above QI as below. Notice that there is no injected current to the I mass (0 in the equation below).Using the obtained QFRE, we obtain the total current to the E mass, i.e., QE using Using the above obtained IRAE, as follows.E(BS) = FRE(RAE), which implies that QBS = QRAE. Using 3) The bi-stability boundary is obtained by invoking the condition: FRNow since we know the total current, we can again use equation. 18, to find injected current which is our I at BS zero, the second term on the left hand side of the above equation goes to zero.Since FR4) The DB boundary is using the parameters of the fit to the DB threshold as in Hence, we arrive at:\u03bdK vs. These four boundaries separate the \u0394\u03bdK increases all these boundaries shift to lower K]o = 3.5\u00a0mM to [K]o = 6.2\u00a0mM - that the region of bistability is very small.In In \u03bdK = 0, 15\u00a0mV. Here again the boundaries between behaviors are smooth, monotonically increasing functions. Higher \u03bdK leads to smaller areas of the dynamical regions F, BS, and RAE. Beyond the point where the RAE boundary (in green) crosses the DB boundary, the E mass goes into DB right after bistablity. Hence, we shift the DB (in red) boundary to the RAE boundary in the figures.The state space as a function of We note that because of the smooth monotonically increasing shape of these boundaries, that the choice of our nominal value for In the present work our aim was to establish and demonstrate a method to develop computational models of neuronal ensembles - neural masses - that stem from biophysical and coupling characteristics of membrane dynamics resolved cellular models of neurons, whose states and output could quantitatively be mapped across modeling scales, whose bifurcations realistically included depolarization block, and whose dynamics realistically bidirectionally couple to the neurons\u2019 environment.The developed neural mass models are parametrized through simple mathematical functions, show physiologically interpretable behaviors and dynamical transitions from one state to another, as a function of key parameters of neural environment.In this work, we have demonstrated this development process for both an inhibitory neural mass, based on the Wang-Buzs\u00e1ki  (WB), anThese mass descriptions can serve as plug-in replacements for existing NM elements used in other models, in that they produce firing rates as a function of synaptic input from other masses.These masses also receive as input slow variables in the form of extracellular potassium, for both, and accommodation state for SEAN, and as output provide the coupling to those slow variables in the form of average membrane potential and transmembrane in our models, instead of using a sigmoidal function as the base relation between the input and firing rate, we use a fit for actual firing rates measured from ODE implementations of the original compartment models with each\u2019s firing range, and explicitly the thresholds for firing and depolarization block as a function of the slow variables.K]o.Inclusion of depolarization block has not been included in the classic NMMs  before. The explicit parametrization of depolarization block enables these new implementation of neural masses to express the underlying mechanisms of depolarization blocks and runaway excitation  needed fBecause these mass elements are built to parameterize these from the original cellular models, these variables map directly to measurable variables in brain. We investigated the dynamics of a simple two element network formed from coupling an excitatory and an inhibitory mass in which input was delivered only to the excitatory mass. This network itself displays key features that underlie epilepsy. In particular, as a function of input to the network it transitions in a hysteretic manner from firing to runaway excitation to depolarization block. Importantly, as extracellular potassium is increased, the input threshold to unstable dynamics is decreased. More importantly, as the inhibitory coupling to the excitatory network is decreased, this sensitivity to increases in extracellular potassium is greatly enhanced. This latter observation is consistent with the observation that epileptic networks are not seizing all the time, but are more susceptible to seizures and spreading depolarizations.The dynamics of the masses we developed are computationally efficient and can be used as direct plug-ins in existing NMM networks, yet their properties (firing rate vs input and environment) are closer to realistic.This manuscript was built from two well known and established Hodgkin-Huxley style compartment models\u2014that by Wang-Buzs\u00e1ki  and a reWith this choice comes the limitations of these models. For example, we achieve firing rates from our SEAN model that far exceed the maximum firing rate of read pyramical neurons, which is a feature also noted in  for theiWe note that both the WB and SEAN models follow canonical Type 1 transition behavior, with roughly square-root firing rate behavior following. In our modeling, we have only parametrized that behavior and the subsequent transition to depolarization block. We leave for future work parametrization of the host of different transitions that have been articulated for example in . Such efISSF and ISSDB and the maximum firing rate as function of \u03bdK. As illustrated in For the current generated NMMs, we have parametrized the average response of a population of homogeneous neuron types formed with identical detailed parameters. Even when subdivided into common cell types, neurons in real biology will have a distribution of shapes, sizes and even ion channel or pump densities. These different within cell differences will lead to changes in the detailed input/output dynamics that we have parametrized in our models. Given a distribution of such parameters, it is straightforward to build the average mass response based on an average of the parameterized responses given such cell-parameter variations. As long as such cell-parameter variations do not change the firing rate from the square-root shape observed, these will translate simply to shifts in the positions of An interesting future work with these neural masses will include linking them to spatial networks of extracellular space that include tracking and diffusion of potassium as well as glial buffering and cell swelling. These elements should reveal components that spread seizure and SD events as well as express normal physiological rhythms. Through the procedure used to include other slow variables including intracellular sodium concentration, synaptic vesicle reserve, and oxygen dependent ATP production for driving the ionic pumps, it will also be possible to investigate the role of mutated channel dynamics at the network level."}
+{"text": "Yet, they may obtain pro-inflammatory features and thus become proatherogenic. Evidence from animal studies suggests that vaccination against certain major histocompatibility complex (MHC) II-binding ApoB peptides induces an expansion of ApoB+ Tregs and thus confers atheroprotection. To date, in-depth phenotyping of vaccine-expanded ApoB+ T cells has not yet been performed. To this end, we vaccinated C57BL/6J mice with the ApoB-peptide P6 (ApoB978\u2013993 TGAYSNASSTESASY) and performed single-cell RNA sequencing of tetramer-sorted P6+ T cells. P6+ cells were clonally expanded  and formed a transcriptional cluster distinct from clusters mainly containing non-expanded P6+ and P6\u2013 cells. Transcriptomic profiling revealed that most expanded P6+ cells had a strong Treg signature and highly expressed genes mediating suppressive functions. Yet, some expanded P6+ cells only had a residual Treg signature and expressed genes related to T helper 1 (TH1) cells, which are proatherogenic. Modeling the T cell receptor (TCR) and P6:MHC-II interaction showed that only three amino acid residues in the \u03b1 and \u03b2 chain contact the P6 peptide in the MHC-II groove and thus determine the specificity of this TCR to P6. Our data begin to reveal the vaccination-induced response to an ApoB epitope.Atherosclerotic cardiovascular diseases are the major cause of death worldwide. CD4 T cells responding to Apolipoprotein B (ApoB), the core protein of most lipoproteins, have been identified as critical disease modulators. In healthy individuals, ApoB-reactive (ApoB Regulatory T cells (Tregs) are one of the lineages of CD4 T cells that limit inflammatory responses J recombination and template-free filling to generate the CDR3 sequences, which are largely responsible for antigen specificity. Single cell RNA sequencing (scRNA-Seq) can be used to assemble paired TCR\u03b1 and \u03b2 chains \u201318. Singb tetramer sorting with Smart-Seq2 scRNA-Seq (single-cell single-well) (+ CD4 T cells. This method is more expensive and produces fewer (tens to hundreds) but much better transcriptomes than alternative methods   to obtai methods , 21. Our978\u2013993 (P6: TGAYSNASSTESASY) peptide was purchased from A&A Labs . Complete (CFA) and incomplete (IFA) Freund\u2019s adjuvants were purchased from SIGMA . C57BL/6J mice were intramuscularly immunized with 100 \u03bcg P6 peptide in 100 \u03bcl CFA/PBS (50:50%) at week 0 and 100 \u03bcg P6 peptide in 100 \u03bcl IFA/PBS (50:50%) at week 2. For each immunization 50 \u03bcl were injected into the left and right musculus quadriceps femoris. Two weeks after final immunization the inguinal and para-aortic lymph nodes were collected.Seven-week-old C57BL/6J female mice were purchased from The Jackson Laboratory  and maintained under specific pathogen-free conditions. All animal studies were approved by the local Institutional Animal Care and Use Committee. ApoBb) beta chain by a flexible polyglycine linker in the pRMHa-3 expression vector and co-expressed in Drosophila melanogaster S2 cells with the mouse MHC-II (I-Ab) alpha chain and BirA ligase. Biotinylated ApoB:MHC monomers were purified from culture supernatants using nickel affinity chromatography, followed by an additional purification on a Pierce Monomeric Avidin UltraLink Resin column  and coupled to streptavidin-phycoerythrin (PE) or streptavidin-allophycocyanin (APC)  to generate tetramers.ApoB:MHC monomers were expressed as previously described . BrieflyCell suspensions were prepared from draining  lymph nodes. Lymph nodes were passed through a 70 \u03bcm cell strainer and the cell suspension was filtered once more through a 70 \u03bcm strainer before washing . Cells were counted using trypan blue and a Neubauer chamber. Before incubation with tetramers, CD4 + T cells were enriched by a negative magnetic bead separation followed by anti-mouse biotinylated monoclonal antibodies all obtained from Tonbo Biosciences, San Diego, CA, USA:CD8\u03b1, clone 53-6.7, cat.# 30-0081-U500; CD19 clone 1D3, cat.# 30-0193-U500; NK1.1, clone PK136, cat.# 30-5941-U500; CD11c, clone N418, cat.# 30-0114-U500; F4/80, clone BM8.1, cat.# 30-4801-U500; CD11b clone M1/70, cat.# 30-0112-U500; TER-119, clone TER-119, cat.# 30-5921-U500; CD45R, clone RA3-6B2, cat.# 30-0425-M001; and streptavidin-coupled magnetic microbeads . 20 \u03bcl of each antibody and 70 \u03bcl magnetic beads were used for each mouse.2 in 100 \u03bcl of 50 nM Dasatinib (Stem Cell Technologies) in RPMI supplemented with 10% FCS  and 1x Pen/Strep (Thermo Fisher Scientific). Subsequently, 1 \u03bcl  of ApoB:MHC-streptavidin-PE and ApoB:MHC-streptavidin-APC tetramer were added for an additional 45 min incubation period. Cells were washed , the supernatant discarded and resuspended in 100 \u03bcl of Live/Dead Aqua (Thermo Scientific Fisher) diluted 1:1,000 in PBS. After a 30 min incubation on ice in the dark, 100 \u03bcl of staining buffer  were added to the samples and cells were washed . The supernatant was discarded and the cells were resuspended in staining buffer with the following anti-mouse monoclonal antibodies at 1:100 dilution:Enriched CD4 + T cells were > 90% pure, based on surface expression of TCR-\u03b2 and CD4 as measured by flow cytometry. Isolated CD4 T cells were washed  and incubated for 30 min at 37\u00b0C and 5% COCD16/32, clone 2.4G2, Tonbo Biosciences, cat.# 70-0161-M001; CD8a BV421, clone 53-6.7, BioLegend, San Diego, CA, USA, cat.# 100738; CD19 BV421, clone 6D5, BioLegend, cat.# 115538; NK1.1 BV421, clone PK136, BioLegend, cat.# 108732; CD11c BV421, clone N418, BioLegend, cat.# 117343; F4/80 BV421, clone BM8, BioLegend, cat.# 123132; CD11b BV421, clone M1/70, BioLegend, cat.# 101251; Ter119 BV421, clone TER-119, BioLegend, cat.# 116234, CD45R BV421, clone RA3-6B2, BioLegend, cat.# 103251; CD25 BV605, clone PC61, BioLegend, cat.# 102036; CD62L BV785, clone MEL-14, BioLegend, cat.# 104440; TCR-\u03b2 Alexa Fluor 700, clone H57-597, BioLegend, cat.# 109224; CD4 APC-eFluor 780, clone GK1.5, Thermo Fisher Scientific, cat.#47-0041-82; CD44 PE/Cy7, clone IM7, BioLegend, cat.# 103030.After staining for 30 min on ice in the dark, cells were washed , the supernatant was discarded, and cells were resuspended in 150 \u03bcl staining buffer for sorting with a BD Aria Fusion . A 70 \u03bcm nozzle and medium pressure at 800\u20131,200 events/s were used to index sort single cells into single wells of a 384-well plate . For sorting P6:MHC-positive CD4 T cells, 90% of the sample volume were used while P6:MHC-negative CD4 T cells were sorted from the remaining 10%. Each well contained 4 \u03bcl lysis buffer. For 1 reaction of lysis buffer 2 \u03bcl of diluted RNase inhibitor, 1 \u03bcl dNTPs  and 1 \u03bcl of 10 \u03bcM Oligo-dT30VN  were mixed. RNase inhibitor  was diluted 1:20 in 0.2% Triton X-100 , which was diluted in nuclease-free water After sorting, the plate was sealed, gently vortexed, and centrifuged at 2,000 rpm for 30 s ensuring that all cells were collected in the lysis buffer. Subsequently, the plate was incubated for 3 min at 72\u00b0C to hybridize the Oligo-dT primer with the mRNA. The plate was immediately stored at -80\u00b0C until library preparation.Single cell libraries were prepared according to the Smart-Seq2-protocol , 23 with1 (v0.11.7). All cells passed sequence quality score as defined by FASTQC. On average, we captured 2.5 million reads per cell. STAR (v2.6.0a)  for 176 cells were run through FASTQCv2.6.0a)  index wav2.6.0a) . Raw reav2.6.0a) . Post-mav2.6.0a) . Gene boGene counts were obtained from STAR. ENSEMBL gene ids were converted to gene names using the ENSEMBL annotation . Genes wb crystal structures PDB ID 3C60, 2IAD, and 1AIO were superimposed, revealing conserved backbone binding of the three different peptides in the I-Ab binding groove. We used NetMHCIIpan (v3.2) (The three I-An (v3.2)  to predin (v3.2)  to identb peptide binding groove. Of all TCRs, the \u03b2 chain was more conserved in sequence compared to the \u03b1 chain. The TCRs bound to I-Ab with similar footprint. Thus, we assumed that the \u03b2 chain dictated the conserved binding orientation and, therefore, we modeled our \u03b2 chain on top of the TCR\u03b2 chain that has the highest sequence identity. A multiple sequence alignment of these TCRs with the major clone  identified in this study revealed an almost conserved sequence with the TCR\u03b2 chain of YAe62  from one ApoB-P6 immunized mouse and 144 tetramer-negative antigen-experienced CD4 T cells were sorted into single wells. Next, libraries were prepared (NexteraXT) and sequenced. All P6+ and 97 out of 144 P6\u2013 cells passed quality filters (+ cells (71%) and in 53 out of 97 P6\u2013 cells (54%), both TCR\u03b1 and \u03b2 chains were successfully reconstructed. Both productive (expressed) and non-productive rearrangements were used to call clonotypes (+ cells. Although considered a different clonotype (based on productive and non-productive \u03b1 and \u03b2 chains), several other unexpanded cells shared the same \u03b1 or \u03b2 chain with that major clone (We immunized C57BL/6J mice with ApoB P6 (ApoB978-993 TGAYSNASSTESASY) in CFA i.m. followed by P6 in IFA 2 weeks later and harvested the draining  lymph nodes at 4 weeks after the primary immunization . Antigen filters , 3. TraC filters . In 23 oonotypes . Clonal or clone . The mosor clone .+ cells, non-expanded P6+ cells and P6\u2013 cells  was among the P6\u2013 cluster  of Tregs, and several other Treg-related genes such as the co-inhibitory molecule cytotoxic T-lymphocyte associated protein 4 (Ctla4), the TF helios (Ikzf2), the tumor necrosis factor receptor superfamily member 18 , or the capping actin protein (Capg) were upregulated in expanded compared to non-expanded P6+ cells , 2 (TH2), 17 (TH17), and T follicular helper cell (TFH) lineage-defining genes that were highly expressed [imputed counts per million (cpm) =10 in at least three cells] to determine the lineage of each individual P6+ cell. Hierarchical clustering based on these genes showed four clearly distinguishable transcriptomic profiles , or nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). In addition, this cluster showed particularly high expression of Treg marker genes that are also markers of T cell activation  (reg-related cytokines transforming growth factor beta 1 (Tgfb1) (Il10) . These cells showed a weak residual Treg signature and low levels of Rora as well as Ifngr1. The TH2 signature was largely lost. Some of these cells expressed the TFH marker genes Slamf6 and Asap1.We compiled a list of 39 Tprofiles . Most ofor Tregs . The tra, Il2ra)  as well  (Tgfb1)  and inte) (Il10) . The TH1+ CD4 T cells, we threaded the most abundant clone  through the published crystal structure of mouse TCR YAe62 (pdb 3C60). CDR1, 2, and 3 for both \u03b1 (orange) and \u03b2 (green) are shown in b . As expected, residues Y4, A7, S9, and S12 in the 15-mer were anchor residues  vs. adjuvants alone, tetramer+ T cells were not detectable in BALBc mice that express a different MHC-II allele (I-Ae instead of I-Ab in C57BL/6J mice), tetramer binding correlated with a fluorescent marker of antigen-specific TCR signaling (Nur77-GFP) after vaccination with p6, and restimulation with p6 in vitro induced cytokine secretion (IL-17) in tetramer+ T cells  enables detection of these rare cells without the use of tetramers and thereby can help better define the autoimmune response against ApoB in future studies. Every T cell develops a unique TCR through random genetic recombination during maturation in the thymus and, in contrast to the transcriptome of a T cell, the antigen specificity of the TCR is subsequently unaffected by external stimuli , also known as Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), is highly expressed in Tregs and context-dependently modulates their function (Tnfrsf9), also known as 4-1BB/CD137, is directly controlled by Foxp3  is a co-stimulatory molecule which has been implicated in Treg development and functionality. Its presence is necessary to maintain numbers of peripheral Tregs while absence reduces demethylation of the Treg-specific demethylated region (TDSR) at the Foxp3 promotor leading to Treg instability  receptor 1, which is a prototypical TH1 marker  additionally expressed Tbx21, which encodes T-bet, the lineage-defining TF of TH1 cells. Additionally, these cells showed high expression of genes encoding Treg-related cytokines (Tgfb1 and Il10) (Ctla4 and Il2ra)   and T ced Il2ra) . Self-anammation . Hence, or cells , 64. MosTF GATA3 , and the TF Batf . GATA3 ial tract . Batf coesponses . Taken t+ cells exhibited low expression of most lineage-defining genes and might likely represent na\u00efve T cells that have either not encountered P6 or were not sufficiently activated. Some of these cells expressed a TFH profile, led by Slamf6 . This modeling revealed that only three contiguous amino acid residues determine the TCR specificity for P6 . Knowing which features determine the TCR specificity to P6 can be a valuable resource for predicting and evaluating potential cross-reactivities between ApoB and other antigens in future studies.Reconstruction of the TCR \u03b1 and \u03b2 chain allowed us to model and analyze the interaction between IAreg signature and, particularly, showed an upregulation of genes involved in mediating the suppressive function. The successful reconstruction of TCR\u03b1 and \u03b2 in most cells, combined with the known peptide epitope defined by sorting with P6:I-Ab tetramer, provides complete structural information on an ApoB-specific TCR with peptide-loaded MHC-II. Our data is a resource for future studies investigating vaccination strategies with ApoB to modulate proatherogenic autoimmunity.In summary, our study identifies oligoclonal expansion of CD4 T cells in response to vaccination with ApoB-P6. Most of the clonally expanded cells expressed a clear TGSE221281.The data presented in this study are deposited in the GEO repository, accession number KL designed, supervised the study, and provided funding. FN, HW, and YG wrote the manuscript and prepared the figures. FN, HW, KK, CD, and SB performed the experiments. YG, DZ, SA, and PR analyzed the data. TD and MJ contributed to the research materials. All authors contributed to the data research, critically discussed the content, and reviewed the manuscript before submission and have read and agreed to the published version of the manuscript."}
+{"text": "Trace amounts of mycotoxins in food matrices have caused a very serious problem of food safety and have attracted widespread attention. Developing accurate, sensitive, rapid mycotoxin detection and control strategies adapted to the complex matrices of food is crucial for in safeguarding public health. With the continuous development of nanotechnology and materials science, various nanoscale materials have been developed for the purification of complex food matrices or for providing response signals to achieve the accurate and rapid detection of various mycotoxins in food products. This article reviews and summarizes recent research (from 2018 to 2023) on new strategies and methods for the accurate or rapid detection of mold toxins in food samples using nanoscale materials. It places particular emphasis on outlining the characteristics of various nanoscale or nanostructural materials and their roles in the process of detecting mycotoxins. The aim of this paper is to promote the in-depth research and application of various nanoscale or structured materials and to provide guidance and reference for the development of strategies for the detection and control of mycotoxin contamination in complex matrices of food. Aspergillus, Penicillium, and Fusarium at various stages, including production, processing, storage, and transportation  monolithic column for the in-tube SPME of three mycotoxins [1, ZEN, and sterigmatocystin. Therefore, the developed monolithic column based on poly (MAA-co-DVB) had a high recognition ability for three target molecules, effectively overcame the matrix effect of rice food, and realized the highly sensitive determination of three target mycotoxins.Gold nanoparticles (AuNPs) are composed of nanoscale gold atoms (1\u2013100 nm), which not only have unique optical, electrical, and excellent surface enhancement properties but also have a high specific surface area. AuNPs can be used as the carrier of Abs, aptamers, and other specific recognition molecules, making them the most commonly used material in the field of food detection ,83,84. Zcotoxins . The hig\u22121), and recovery (97.6\u2013114.2%) values. Javier and co-workers synthesized core\u2013shell poly (dopamine) magnetic nanoparticles, which were used for the simultaneous extraction of six mycotoxins  from milk products [Dispersive solid-phase microextraction (DSPME) is a non-fiber extraction method, which operates by mixing the solid-phase sorbent directly with the sample medium through vortexing and sonication ,86,87. Aproducts . This DS\u22121) and satisfactory precision (RSD: 1.4\u201315.0%). MOF materials have extremely high porosity, excellent thermal stability, and a large specific surface area. Their tunable pore size, porous channels, and nano-space make them ideal SPME adsorbent materials. Lotfipour and co-workers [3 as a bio-linker and cobalt ions as a metallic center in water, and applied it as a sorbent in a DSPME of patulin and Ochratoxin A (OTA) from fruit juice samples and four aflatoxins  from soy milk  needs to be rapid, high-throughput, and able to meet the requirements of in situ detection and easy popularization. Although highly sophisticated large-scale analytical instruments can provide accurate, sensitive, and reproducible strategies for the detection of mycotoxins in complex food matrices, they often involve complex and cumbersome sample preparation processes and require experienced operators, which present significant drawbacks in terms of cost, convenience, and popularity. In contrast, immune, aptamer, or sensing assays based on the specific recognition or binding of Abs, aptamers, or artificial Abs made up for the above shortcomings and became powerful tools for the rapid screening and detection of mycotoxin contamination ,102. VarCarbon-, metal-, and semiconductor-based nanoscale materials often have large specific surface areas and excellent optical or chemical properties and are inherently rich in binding sites or can be modified to obtain them ,145. The\u22121 (100 pg g\u22121), and the time required for the entire analysis was only 17 min. Li and Zhang\u2019s study introduced a novel time-resolved fluorescence ICA (TRFICA) using two idiotypic nanobodies for the simultaneous detection of AFB1 and ZEN [1 and ZEN  and was 18.3-fold and 20.3-fold more sensitive than mAb-TRFICA. This is the first report about a time-resolved strip method based on AIdnbs for dual mycotoxins. Du et al. [1 by using a signal strategy based on gold growth on the surface of E. coli K12 carrier  are usually a few nanometers to tens of nanometers in size [1, OTA, and ZEN toxins on the surface of QDM and designed an immunochromatographic test strip capable of simultaneously detecting the three toxins . Notably, the proposed multiple toxin detection strategy required only simple manual operation and one portable handheld strip reader, enabling on-site testing to be completed within 45 min. This represents a valuable solution for rapid, sensitive, and on-site detection of multiple mycotoxins. An immunochromatographic assay (ICA) is a rapid, portable, and user-friendly immunodiagnostic tool that has been extensively studied ,148. ICA and ZEN . Enhanceu et al.  proposed carrier b. The la in size ,156. Aft in size ,158. Wan in size  immobilie toxins c. When a1 was designed and developed. Multicolor-emissive NPs doped with lanthanide ions  were functionalized with specific aptamers as bio-probes, and the tungsten disulfide (WS2) nanosheets were applied as the fluorescence quencher. Three toxin targets can be identified in a single solution simultaneously without mutual interference. Under the same excitation wavelength (273 nm), the LODs for three toxins were 0.51 (ZEN), 0.33 (T-2), and 0.40 pg mL\u22121 (AFB1), respectively, demonstrating the remarkable sensitivity. This strategy provided an advanced guidance for the rapid quantitative analysis of multi-toxin targets in a single sample. Through computational simulations, Bagherzadeh and co-workers [1, AFM1, AFG1, AFG2, OTA, and ZEN). After coupling with AuNPs, the developed two aptamer-AuNPs test strips working in competitive mode showed high sensitivity. The F20-T-based strips (LOD: 0.1 ng mL\u22121) were more sensitive than those utilizing F20 (LOD: 0.5 ng mL\u22121). Utilizing a simple strip reader, both of the developed strips were able to analyze AFB1 in maize flour within 30 min.Aptamers are short deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) oligonucleotide sequences with high affinity which show excellent specificity for various target molecules, such as proteins, DNA, small molecules, and metal ions ,161. As -workers  designedSize effect of nanoscale materials endows them with unique optical, electrical, magnetic, and mechanical properties that enable them to generate signals with specific characteristics, providing a signal source for the development of highly sensitive and high-performance mycotoxin detection strategies ,169. The1 [1 molecules, AFB1 directly acted as the quencher of graphene QD fluorescence. Notably, this innovative sensor achieved direct quantification of AFB1 without the use of inhibitors or biometric elements and obtained an acceptable linear response range of 5-800 ng mL\u22121 with an LOD of 0.158 ng mL\u22121. Nanosized MOFs (NH2-MIL-53 (Al)) can undergo hydrolysis in alkaline environments and release a large number of fluorescent ligands (NH2 center dot H2BDC). This property allowed it to be applied as large-capacity nanovesicles for loading signal molecules to investigate various biorecognition processes. Based on this, Fu et al. [1 by using NH2-MIL-53 (Al) as a fluorescent signal probe  modified by thiolated OTA aptamer cDNA as the acceptor. When excited under a 980 nm laser, the emission peak of UCNPs at 657 nm overlapped with the absorption peak of AuNRs at 660 nm. Through the hybridization of nucleic acid sequences modified on the surface of UCNPs and AuNRs, the distance between them is shortened, leading to the quenching of luminescence. Mo et al. reported on a WS2 nanosheet sensing platform based on chemiluminescence RET for OTA detection [2O2-luminol and WS2 nanosheets acted as the energy donors and acceptors, respectively. This chemiluminescence RET sensing system worked based on changes of chemiluminescence intensity caused by the presence or absence of OTA-induced donor affinity or acceptor surface desorption. It is very meaningful to modify the recognition sequence only by using the substrate adapter that can be easily extended to the analysis of other targets.F\u00f6rster Resonance Energy Transfer (FRET) is a non-radiative energy transfer process that is based on a confined radiation-free electric dipole coupling. Energy transfer between nanomaterials can also be achieved through FRET, usually involving two different nanostructures or NPs ,176. Wheetection . The OTA2, OTA, and AFB1 were developed [1, the aptamer formed a complex with AFB1, leading to the denaturation of the stem\u2013loop structure back to the single-strand, thereby recovering the fluorescence signal. This strategy was significantly more selective for AFB1 than other analytes and responded well to AFB1 concentrations in the range of 0.5\u201320 pg mL\u22121 with a low LOD (0.1 pg mL\u22121). A similar principle has been applied in T-2 toxin detection, using a Cu MOF material as a fluorescent quencher with dual-sided FAM labeling [Due to the tunable particle size, optical properties, high stability, and controllability, AuNPs were also extensively employed in signal transduction and enhancement studies ,183. AuNeveloped ,186. Meheveloped . This aplabeling .The unique size and structure of nanoscale materials allowed them to exhibit special electrical and optical properties. These distinct characteristics, different from those of macroscopic materials, have expanded the application of nanoscale materials in various fields and provided new research directions for food safety detection, especially mycotoxin detection ,190.\u22121). This GO-based printed electrode has the advantages of convenient large-scale preparation, a low cost, and broad application prospects. Electrochemical sensors have the advantages of a simple operation, high sensitivity, low cost, and easy miniaturization, making them highly suitable for the rapid screening and on-site detection of food samples on a large scale . Modific\u22121, providing an efficient, simple, and economical solution for DON detection in food and feed samples. Polyaniline (PANI) is a semi-flexible conducting polymer that is environmentally friendly, stable, inexpensive, and biocompatible and has excellent conductivity [1 and FuB1 mycotoxins, with LODs of 0.313 and 0.322 pg mL\u22121, respectively.The electrochemical strategy has also been used for the preparation of novel NPs and the construction of new detection devices during the process of toxin detection. Based on 3D-graphene and iron nanoflorets, Saheed et al. constructed a \u201cDandelion\u201d nanostructure (iron nanoflorets on 3D-graphene\u2013nickel) as the transducer to develop a sensitive biosensing system . The stuuctivity ,201. Duructivity  directly2 functional groups, AuNPs, and FB1-specific aptamers on the fiber end face [1 (R2 = 0.9817), and the LOD was 0.17 ng mL\u22121. Jaroon\u2019s research team designed an unlabeled colorimetric aptasensor, which exploited the selective interaction between the aptamer and AFM1, leading to structural changes while allowing for the aggregation of AuNPs induced by NaCl [\u22121), meeting the maximum residue limit requirement for AFM1.The composition, shape, and size of nanomaterials can regulate their ability to absorb and scatter light, especially noble metals such as Au, Ag, and Pt. When light strikes a noble metal NP, if the frequency of the photon matches the collective oscillation frequency of the electrons in the NP, the NP will strongly absorb the energy of the photon, resulting in a phenomenon known as localized surface plasmon resonance (LSPR) ,204. Altend face . AuNPs n by NaCl . The sol1, OTA, and FB1; high selectivity; and small reagent usage. Li et al. [1, and DON (three toxins) simultaneously. This AuNPs-based SERS assay possessed a wide linear concentration range and a low LOD, which enabled it to rapidly and simultaneously screen multiple mycotoxins in real samples, giving it a great potential for application.Photonic crystal microspheres (PCMs) are microsized particles with an internal photonic crystal structure, possessing diverse optical properties, including abundant optical modes and optical resonances . In a PCi et al.  establisIn recent years, significant advancements have been made in the field of mycotoxin detection in food due to the utilization of various nanoscale materials. These materials have enabled the development of rapid, precise, sensitive, and cost-effective detection methods. Among the diverse array of nanoscale materials, rigid nanomaterials stand out due to their exceptional characteristics, including a high specific surface area, substantial loading capacity, and stability. These attributes make them particularly advantageous in the development of materials for solid-phase extraction (SPE) and solid-phase microextraction (SPME) pretreatment, enhancing the performance of purification processes for precise instrument analysis. Furthermore, flexible nanomaterials, characterized by excellent biocompatibility and specific optoelectronic properties, introduce a new dimension to the rapid mycotoxin detection landscape. They facilitate the preservation of the activity of biorecognition molecules and signal sources, improving the overall efficiency of mycotoxin detection strategies. Despite these valuable advances, several challenges remain. Firstly, nanoscale materials with better properties are still needed, both for efficient and stable pretreatment process and high-throughput rapid detection process. Among them, green synthesis and the high-volume production process of nanoscale materials require more in-depth research. Secondly, there is an urgent need to develop automated and miniaturized detection equipment or devices for mycotoxins in food based on nanomaterials. Such devices would streamline the detection process, minimize operational errors, and promote the widespread adoption of cost-effective detection strategies. Overall, nanoscale materials provide opportunities, as well as challenges, for research on the detection and monitoring of mycotoxins in food."}
+{"text": "Microelectronic components are used in a variety of applications that range from processing units to smart devices. These components are prone to malfunctions at high temperatures exceeding 373 K in the form of heat dissipation. To resolve this issue, in microelectronic components, a cooling system is required. This issue can be better dealt with by using a combination of metal foam, heat sinks, and nanofluids. This study investigates the effect of using a rectangular-finned heat sink integrated with metal foam between the fins, and different water-based nanofluids as the working fluid for cooling purposes. A 3D numerical model of the metal foam with a BCC-unit cell structure is used. Various parameters are analyzed: temperature, pressure drop, overall heat transfer coefficient, Nusselt number, and flow rate. Fluid flows through the metal foam in a turbulent flow with a Reynold\u2019s number ranging from 2100 to 6500. The optimum fin height, thickness, spacing, and base thickness for the heat sink are analyzed, and for the metal foam, the material, porosity, and pore density are investigated. In addition, the volume fraction, nanoparticle material, and flow rate for the nanofluid is obtained. The results showed that the use of metal foam enhanced the thermal performance of the heat sink, and nanofluids provided better thermal management than pure water. For both cases, a higher Nusselt number, overall heat transfer coefficient, and better temperature reduction is achieved. CuO nanofluid and high-porosity low-pore-density metal foam provided the optimum results, namely a base temperature of 314 K, compared to 341 K, with a pressure drop of 130 Pa. A trade-off was achieved between the temperature reduction and pumping power, as higher concentrations of nanofluid provided better thermal management and resulted in a large pressure drop. Recent technological developments for the enhancement of the processing capacity of microelectronics and miniaturization have resulted in increased heat dissipation requirements. Over the period of the last three decades, nanofluids have gained immense popularity in the field of heat transfer. Nanoparticles enhance the thermal properties of the base fluid, thereby increasing heat dissipation. Failing to achieve the required heat dissipation affects the performance and ultimately results in the malfunctioning of the device . ElnaggaBhattacharya and Mahajan  experime2 showed an increase in thermal conductivity. Moreover, an increase in concentration and flow velocity was observed to increase heat dissipation [2O3, SiC, and CuO nanofluids at different concentrations and velocities. SiC and CuO nanofluids gave the highest thermal conductivity and heat flux enhancement, respectively. Moreover, the CuO nanofluid was found most suitable for the cooling of electronic devices [Copper nanofluids and alumina nanofluids with various volume fractions inside silicon microchannel heat sinks were investigated ,13,14,15sipation . A numersipation . The numsipation . Another devices .Bayomy and Saghir  conducte2O3 nanofluid flowed at different volume concentrations. The heat transfer enhancement using the Al2O3 nanofluid in porous media was studied under various flow velocities, heat flux, and particle concentrations. A significant improvement in heat transfer was observed when both metal foam and nanofluids were used. However, chaotic movements, dispersions, fluctuations, and interactions with porous media augmented the heat transfer. Pourfarzad et al. [2O3/water, Al2O3/ethyl glycol, TiO2/water, and TiO2/ethyl glycol nanofluids are passed through porous blocks, porous straight channels, and porous wavy channel heat sinks. In all these cases, water proved to be the better base fluid and the Al2O3 nanofluid showed enhanced heat extraction and temperature uniformity [2O3 nanoparticles suspended in water were used as the nanofluid. The results showed that porously filled channels have increased convective strength; however, they result in a higher pressure drop [Ghaziani and Hassanipour  performed et al.  experimed et al.  experimeiformity . Welsforure drop .Madeeha et al.  studied The literature shows that metal foam and nanofluid have been investigated individually and in combination for electronic cooling in order to tackle the heat dissipation issues in industrial and scientific applications. Most of the studies carried out have been experimental; however, several associated benefits still need to be explored. Therefore, this study uses these combinations and explores the beneficial outcomes of nanoparticles to provide better solutions for the thermal management of devices. The analysis is performed according to the variation in the porosity and pore density for the metal foam and material and concentration of nanoparticle. The base temperature, Nusselt number, heat transfer coefficient, and pressure drop are used to determine the influence of parameters on the performance of the heat sink. It may be predicted that by modifying the fin height, spacing, and fin thickness for a rectangular-finned heat sink, the cooling systems for microelectronics devices can be substantially enhanced. The layout of this paper is as follows: + value for the range of Reynolds numbers used in the calculations, the values fell between 50 and 90. Hence, the k-\u03b5 model was used in all numerical simulations for different combinations of heat sink and metal foam with various concentrations of nanoparticles. The results acquired through numerical simulations were validated using the experimental data available in the literature.The methodology followed in the present research work is depicted in k-\u03b5 turbulence model is used in numerical simulations. The k-\u03b5 model presents two new variables into the system of equations. The continuity equation is written as follows:The MS is the sum of the body forces, and The equation of momentum becomes:k-\u03b5 model is based on the concept of the eddy viscosity, therefore:k-\u03b5 model considers that the turbulence viscosity is related to the turbulence kinetic energy and dissipation and the relation is as follows:The k and \u03b5 derived directly from the differential transport equations for the turbulence kinetic energy and turbulence dissipation rate are as follows:The values of The whole setup was modeled in SOLIDWORKS 19. Different heat sinks were designed based on the variation in fin heights, base thickness, fin thickness, and fin spacing. Aluminum and copper were chosen as the heat sink material. The CAD model for the Heat sink is shown in The CAD model for the metal foam is generated by applying the 3-D scanning modelling approach , Lord KeThe CAD of the heat sink and metal foam are depicted in k-\u03b5 model were used as the governing equations. The equations were solved using the SIMPLE algorithm with a second-order upwind scheme.The assembly was imported to ANSYS FLUENT for further analysis. The continuity, energy, and momentum equation with the standard 2O3, Cu, CuO, SiC, and TiO2 with concentrations ranging between 0.3 and 1.5 % by volume was conducted. The properties of the nanofluid were determined using the following equations [For effective density,Water was chosen as the base fluid and a single-phase model was assumed for the nanofluid. For the nanofluids, a comparative analysis between Alquations :For effeFor effective specific heat capacity,For effective viscosity,For effective thermal conductivity,The boundary conditions applied for the simulations are given below.2;Heat flux on the base = 150,000 W/mVelocity inlet with 0.21 to 0.63 LPM flowrate;Pressure at the outlet = 1 atm;Temperature at the inlet = 300 K.2 was used in this study.The mesh generated for the model after assembly is shown in 2 K was achieved for the selected parameters, respectively.2/K using the selected parameters. Hence, a percentage increase of 19.9% was achieved.After investigating the effect of different fin heights, the influence of various fin spacings on the base temperature and heat transfer coefficient is analyzed. The results of the study are depicted in Furthermore, the effects of the different porosities of the metal foam on the base temperature, Nusselt number, and pressure drop were studied and the results are depicted in Temperature and pressure contours for varying fins spacings are shown in 2 for the 0.01 m/s velocity and at 1200 W/m2 for the 0.016 m/s velocity. The experimental results have been shown with error bars to indicate uncertainty in the results. Similarly, ref. [Firstly, the numerical results are validated with the research work of Ghaziani and Hassanipour . The samly, ref.  investigThe numerical results for the aluminum metal foam of 90% porosity were compared with the research work of . The bas2, the values became relatively constant with a small amount of fluctuation. 2 is reached, followed by a decrease before attaining a relatively constant value. A comparison of the experimental and numerical results from Another comparison was conducted for the Nusselt number with the research work of  at 0.01 3 and removed nearly 170 W of heat. The quarter of the base area of the heat sink used in this investigation removes half the heat, giving a better overall performance than that of a finned heat pipes heat sink. Jajja et al. [Elnaggar  experimea et al.  experimea et al.  numericaThe use of electronic devices in numerous industries has seen a rapid increase in recent times, most notably with smart devices using microprocessors, which are currently used in devices ranging from handheld mobile phones to cars and refrigerators. Due to the working operation requirements of these devices, the heat dissipated leads to overheating and the failure of electronic devices. As a result, thermal management has become an essential step in the field of electronics\u2019 cooling. Conventional cooling systems demand further enhancements to sustain these cooling requirements. Therefore, this research proposes novel methods to find the optimized parameters for a rectangular-finned heat sink. The sink is integrated with metal foam between the adjacent fins, and water-based nanofluids flow through the structure. ANSYS FLUENT is used for all numerical simulations. The following are the key findings of this research work.The current research work revealed the optimum fin height, fin spacing, base and fin thickness, and material for the heat sink. Moreover, the porosity, pore density, and material for the metal foam, and the concentration, velocity, and nanoparticle material are also investigated for the nanofluids.2 and 0.025 m fin height. A base temperature of 314 K is achieved, compared to 341 K without metal foam, with a pressure drop of 130 Pa.The configuration provides optimum results at a base area of 0.062 \u00d7 0.062 mMetal foam has better thermal properties and a much lower relative density compared to its solid metal counterparts.The three-dimensional porous structure provides flow mixing and turbulence that further enhances the performance of the finned heat sink.The addition of nanofluids provides further heat dissipation to the arrangement; however, the pressure drop and, in turn, the pumping power remain relatively unchanged.Experimental validation was carried out to compare the numerical results with the experimental data from the literature. The comparison of the results from both techniques revealed acceptable amounts of error between both methods."}
+{"text": "Scientific Reports 10.1038/s41598-021-02319-7, published online 23 November 2021Correction to: The original version of this Article contained an error in the Results section, where\u201cWe asked each department of the 436 facilities to cooperate in completing the questionnaire, and received responses from 245 facilities (56%), with a total of 941 department heads. Of these, 32 department heads declined to participate in the study and valid responses were obtained from 919 department heads.\u201dnow reads:\u201cWe asked each department of the 436 facilities to cooperate in completing the questionnaire, and received responses from 245 facilities (56%), with a total of 941 department heads. Of these, 22 department heads declined to participate in the study and valid responses were obtained from 919 department heads.\u201dThe original Article has been corrected."}
+{"text": "Chrysomya latifrons) from 15 isolated rainforests and found high levels of gene flow, a lack of genetic structure between rainforests, and low genetic diversity \u2013 suggesting the presence of a single large genetically depauperate population. This highlights that: (1) the blowfly Ch. latifrons inhabits a\u2009~\u20091000\u00a0km stretch of Australian rainforests, where it plays an important role as a nutrient recycler; (2) strongly dispersing flies can migrate between and connect isolated rainforests, likely carrying pollen, parasites, phoronts, and pathogens along with them; and (3) widely dispersing and abundant insects can nevertheless be genetically depauperate. There is an urgent need to better understand the relationships between habitat fragmentation, genetic diversity, and adaptive potential\u2013especially for poorly dispersing rainforest-restricted insects, as many of these may be particularly fragmented and at highest risk of local extinction.Climate change and deforestation are causing rainforests to become increasingly fragmented, placing them at heightened risk of biodiversity loss. Invertebrates constitute the greatest proportion of this biodiversity, yet we lack basic knowledge of their population structure and ecology. There is a compelling need to develop our understanding of the population dynamics of a wide range of rainforest invertebrates so that we can begin to understand how rainforest fragments are connected, and how they will cope with future habitat fragmentation and climate change. Blowflies are an ideal candidate for such research because they are widespread, abundant, and can be easily collected within rainforests. We genotyped 188 blowflies (The online version contains supplementary material available at 10.1007/s00442-023-05333-w. Due to climate change and deforestation, rainforests are becoming increasingly fragmented islands of endemic biodiversity Bowman  that areSpecies that inhabit fragmented rainforests can vary significantly in their extent of population genetic structure and diversity \u2013 spanning a continuum from well-connected to fragmented populations and low to high genetic diversity  are well-suited to this approach because they are widespread throughout rainforests, extremely abundant, and easy to capture in the wild Norris . BlowfliChrysomya latifrons Malloch Ch. latifrons should show high levels of gene flow and limited genetic differentiation between adjacent rainforests. As we expect to observe only broad patterns of isolation by distance, any rainforest fragments inhabited by Ch. latifrons that do not meet these patterns may reflect high levels of habitat isolation.Here, we perform extensive field collections throughout rainforest fragments in southeast Australia and use genotype-by-sequencing (GBS) through the DarTseq\u2122 platform to obtain genetic information , targeting a species that is endemic to the region \u2013 Ch. latifrons has generally been considered a rainforest specialist that is restricted to New South Wales (NSW), Australia  results in the production of specific volatiles that are highly attractive to all Australian Chrysomya species (except Chrysomya flavifrons Aldrich 1925). This bait presumably exploits the preference of Chrysomya species for cues associated with carrion that is in the mid-stage of decomposition, as most Chrysomya are secondary colonisers , which characterises rainforests as \u201cForest dominated by broad-leaved tree species, typically in wet or sheltered environments and with a closed canopy. Can include areas with non-rainforest species as emergents (trees emerging above the canopy), but where rainforest species dominate the character of the site\u201d. We then used QGIS version 3.28.1 (http://www.qgis.org/) to filter this dataset on the rainforest, exported this data from raster to polygons for mapping, clipped to the extents of NSW, and created a 10\u00a0km and 60\u00a0km buffer around each site. We chose both scales as they represent the range of observed dispersal capacities of Chrysomya blowflies  was dissected from the body, placed in a single well of a 96-well plate with 70% ethanol, and supplied to Diversity Arrays Pty Ltd  for a high-density DArTSeq\u2122 assay . The DArTSeq\u2122 extraction and sequencing methods are detailed in Kilian et al.  and GeorCh. latifrons using multiple restriction enzyme combinations and eight specimen replicates. Following sequencing of these test specimens, the optimal restriction enzyme pair was identified as PstI-HpaII, based on the fraction of the genome represented, while controlling for average read depth and the number of polymorphic loci. This restriction enzyme combination was used for all subsequent digestions. Following digestion, all sequence fragment libraries were ligated with Illumina sequencing adaptors and sequenced on an Illumina HiSeq2000 platform.To ensure appropriate DNA fragments were used for subsequent sequencing, restriction enzyme digestion was optimised for Short-read sequences were processed using the DarTseq\u2122 bioinformatic pipeline , as a single specimen from Washpool National Park was removed from the dataset due to low quality reads. The data were then filtered with the \u2018dartR\u2019 package version 1.9.9.1 . Default settings were used in IPYRAD, with the exception of the following: strict filtering of adapters was applied ; and the final minimum length after filtering was set to 60 . The IPYRAD dataset contained a total of 48,912 SNPs across 187 individual flies. The data were then filtered in VCFTOOLS v. 0.1.13 , expected heterozygosity (HE), and inbreeding coefficients (FIS). We also used the \u2018betas\u2019 function from \u2018hierfstat\u2019 to calculate population-specific FST values .To calculate individual blowfly admixture coefficients, the MAF-filtered SNP data were converted into the STRUCTURE format (\u2018.str\u2019) using the \u2018gl2faststructure\u2019 function from the \u2018dartR\u2019 package, then into the \u2018.geno\u2019 format using the \u2018struct2geno\u2019 function of the \u2018LEA\u2019 package version 3.1.4 (Frichot and Francois https://web.stanford.edu/group/pritchardlab/structure.html) across a K value range of 1 to 10 with 10 replications per K. An initial analysis was completed to determine the number of Markov Chain Monte Carlo (MCMC) iterations required to achieve stationarity in FST and log(\u03b1) summary statistics for each K value. The optimal configuration was determined to be 100,000 MCMC iterations, with 5000 discarded as burn in and these parameters were used for all subsequent analyses. Following STRUCTURE analysis, the best K value was selected using the Evanno method . To create plots, results for each K value were permuted across all replicates using CLUMPP v1.1.2 .To verify these results across our different bioinformatic pipelines, we also analysed the IPYRAD-filtered dataset using sNMF (with the same parameters as above) and tested the IPYRAD-filtered dataset with the program STRUCTURE v.2.3.4 (Nm) to infer levels of connectivity between populations.Finally, to calculate relative migration rates among populations, we used the divMigrate implementation . Alternatively, Maxwell\u2019s Rainforest (MA) which appears relatively well connected at a scale of 10\u00a0km is in fact quite isolated at a scale of 60\u00a0km \u2013 and would thus be expected to have the lowest level of broad connectivity to other patches. Nevertheless, at most sites, Ch. latifrons were abundant at the time of collection, ranging from a constant number of five to 20 individuals on the carrion bait. However, at Ulidarra National Park (UL) we only observed three Ch. latifrons as the community was dominated by Ch. semimetallica \u2013 suggesting this may be close to the range edge of Ch. latifrons. At Wyrrabalong National Park (WY) we only observed five flies throughout four hours of collecting  , suggesting minimal genetic differentiation between populations. Likewise, pairwise comparisons of FST values between populations were all\u2009<\u20090.009, and there were no significant differences detectable between geographic populations (Table P\u2009<\u20090.01) (Table ST results.Population-specific F1) Table . Howeverr\u2009=\u2009\u2212\u00a00.053, P\u2009=\u20090.685)  all identified the lowest cross-entropy for a K\u2009=\u20093 .Likewise, the STRUCTURE analysis based on the IPYRAD dataset Fig.\u00a0a was conNm)\u2009>\u20090.5 with the exception of the WY and UL populations (which both had low sample sizes of N\u2009\u2264\u20095), and with no significant asymmetries in migration rates between population pairs. When considering only the three most isolated populations , gene flow was again determined to be high in all cases (Nm\u2009\u2265\u20090.74) with no significant asymmetries in migration rates between population pairs  from Washpool National Park in northern NSW to Maxwells Rainforest on the southern border with Victoria. This is the first comprehensive information on the distribution of Ch. latifrons, which inhabits a large latitudinal transect of\u2009>\u20091000\u00a0km of eastern Australian rainforests and rainforest-adjacent sclerophyll forests. It is almost certain that populations also extend into the rainforests of northern Victoria, where there is suitable habitat available. Remarkably however, Ch. latifrons was completely absent near the Queensland border, where its closest genetic relative, Ch. semimetallica, occurs  suggests they are either generalist consumers of a wide range of animal carrion types, or specialist consumers that utilise types of carrion that are abundant throughout rainforests.Considering the widespread distribution of IS. Rather, they may simply be the result of significant historical bottlenecks , particularly in the context of tolerance to climate change \u2013 as rainforests continue to experience its increasingly severe and frequent effects  can migrate as far as 225\u00a0km  both make long south-easterly migrations in the warmer spring and summer months  and large enough population sizes, there may not have been sufficient time for a resulting lack of gene flow to be reflected in patterns of population structure and diversity  and almost certainly has the capacity to disperse through rainforest-adjacent habitats. Importantly, the lack of population structure we observed does not necessarily imply that there are no signatures of local adaptation in Ch. latifrons. Such questions could be addressed\u00a0by measuring phenotypic differences between populations or with other sequencing approaches  to investigate fine-scale signatures of local adaptation.Finally, the lack of population structure in Our results provide important insights into broad patterns of rainforest connectivity in Australia \u2013 highlighting that rainforest inhabitants with strong dispersal capacities (such as blowflies) can likely\u00a0move between, and connect highly fragmented habitats. Such widespread dispersal between rainforests is unlikely to be representative of all strong dispersers, as it depends largely on species-specific niche breadths and resource distributions that may constrain certain species to specific rainforest patches Supplementary file2 (DOCX 299 KB)Below is the link to the electronic supplementary material."}
+{"text": "Many plants produce chemical defense compounds as protection against antagonistic herbivores. However, how beneficial insects such as pollinators deal with the presence of these potentially toxic chemicals in nectar and pollen is poorly understood. Here, we characterize a conserved mechanism of plant secondary metabolite detoxification in the Hymenoptera, an order that contains numerous highly beneficial insects. Using phylogenetic and functional approaches, we show that the CYP336 family of cytochrome P450 enzymes detoxifies alkaloids, a group of potent natural insecticides, in honeybees and other hymenopteran species that diverged over 281 million years. We linked this function to an aspartic acid residue within the main access channel of CYP336 enzymes that is highly conserved within this P450 family. Together, these results provide detailed insights into the evolution of P450s as a key component of detoxification systems in hymenopteran species and reveal the molecular basis of adaptations arising from interactions between plants and beneficial insects. A specific family of enzymes found in beneficial insects protects them from a plant defense chemical. Plants have evolved sophisticated chemical defense strategies to protect themselves against herbivores and pathogens. These defenses rely on the production of plant secondary metabolites (PSMs), compounds that are not directly involved in primary physiological processes but enhance plant fitness to their environment  and a Michaelis constant (Km) value of 12.77 \u03bcM (~7-fold lower than CYP6AQ1)  .To further explore the role of CYP336A in xenobiotic detoxification in vivo, we analyzed its expression level in different life stages and tissues of the honeybee via quantitative polymerase chain reaction (qPCR). CYP336A1 was found to be highly expressed in late larval instars and adult honeybees. In adult honeybees, its expression was especially high in Malpighian tubules and the brain, key sites of xenobiotic detoxification and the neuronal receptor of nicotine (the nicotinic acetylcholine receptor), respectively (fig. S1). Together, these results provide unequivocal evidence of the key role of CYP336A1 in nicotine metabolism.m/z) 160 (M-2) and 176 (M+14) were identified. By using commercial reference standards, we confirmed that the M+14 metabolite corresponds to cotinine. Subsequent quantification revealed that only approximately 10% of nicotine is transformed to cotinine, while the majority is converted to the M-2 metabolite. On the basis of the literature, there were two strong candidates for the M-2 metabolite: N-methylmyosmine (tautomer of nicotine-\u03941\u2032(2\u2032) iminium ion (1\u2032(5\u2032) iminium ion  resulting from oxidative metabolism of nicotine by CYP336A1. In vivo, several metabolites have been previously identified, with 4-hydroxy-4-(3-pyridyl) butanoic acid, the final product of the C\u20322 oxidation pathway, the major metabolite (fig. S2)  for attack by an electrophile were calculated. Maxima of f\u2212(r) correspond to regions in space, where the electron density of molecules is most likely to be reduced and thus, in many cases, coincide with sites of metabolic attack by oxidative agents, including P450s (Ka (where Ka is the acid dissociation constant) value of 8.1 for the pyrrolidine nitrogen (N-methyl-pyrrolidine ring corresponding to sites of major nicotine metabolites such as cotinine (5\u2032C-oxidation), nicotine N-oxide (N-oxidation), or nornicotine (N-demethylation). In contrast, the Fukui function of protonated nicotine exhibits a high electron density across the pyridine moiety and only minor areas within the pyrrolidine ring surrounding the C\u20322 atom. Together, these results support the hypothesis that nicotine is metabolized by CYP336A1 in its protonated form by oxidative dehydrogenation at the C\u20322 atom of the pyrrolidine moiety (fig. S2).To support this conclusion, Fukui functions nitrogen . The neup-nitroanisole. CYP336A1 exhibited no notable metabolism of any model substrates in this initial panel (table S1). Given its established metabolic activity for nicotine, we widened our substrate panel to explore whether CYP336A1 exhibits specific activity toward additional alkaloids and extended our analysis to include anabasine, atropine, scopolamine, cytisine, and echimidine. From a functional perspective, these substances share the ability to bind to nicotinic or muscarinic acetylcholine receptors, respectively (f\u2212 maxima of the respective Fukui function) (fig. S3). We also included the nonbasic alkaloid caffeine as well as coumarin in our investigation as these two phytochemicals have pharmacologically different actions and do not have a basic nitrogen.Following the identification of CYP336A1 as the major metabolizer of nicotine in honeybees, we investigated the broader substrate profile of this P450. CYP336A1 was initially screened against 13 model substrates routinely used in P450 research, comprising seven model substrates derived from coumarin, five model substrates derived from resorufin, and Incubation of CYP336A1 with the selected set of alkaloids revealed differential metabolic activity for different compounds . The pyrDinoponera quadriceps. Furthermore, interrogation of the CYPome of insects from other orders available on the National Center for Biotechnology Information (NCBI) failed to identify a named CYP336 gene, suggesting that this P450 family is specific to the Hymenoptera.Recent studies have shown functional overlap of evolutionary-related P450s involved in the metabolism of synthetic insecticides across the diversity of bee pollinators . In mostNeodiprion lecontei, Tenthredo koehleri, and Athalia rosae have two, three, and five sequences, respectively, and there is possible evidence of a duplication event before its divergence from other Tenthredinoidea species in A. rosae . With the exception of chromosome 25 in Vespa crabro [~3.6 million base pairs (Mbp)], all chromosomes were\u00a0>15 Mbp long and the pairwise comparisons to N. lecontei reveal a pattern of decreasing numbers of locally collinear blocks (LCBs) with evolutionary distance (table S3). For example, there are 100 LCBs in the comparison of N. lecontei (Symphyta) and Venturia canescens (Ichneumonoidea) chromosomes, but only 59 in the alignment to A. mellifera (Apoidea). The apparent lack of syntenic relationship in V. crabro (17 LCBs) is likely to be explained by the difference in the size of the chromosome containing the CYP336 genes in this species.To further explore the evolution of the CYP336 family within the Hymenoptera, we assessed the conservation of the genomic region harboring these genes. To look for evidence of macrosynteny, chromosomes containing CYP336 genes was interrogated and flanking genes were identified (data S1). When intra-superfamily relationship is considered, the genomic region appears highly uniform with extensive LCBs and conserved gene order within the Symphyta, Vespoidea, Formicoidea, and Apoidea  is found in close association to the CYP336 genes  composed of five to seven genes, found on a separate chromosome in the sawflies, has been inserted into SB1 in the Vespoidea and Apoidea (CYP336 genes (data S1), indicating an interchromosomal rearrangement in this superfamily, which likely occurred after the divergence of the Aculeata, approximately 160 million years (Ma) ago. However, the genes framing the CYP336 sequences are identical to those found in sawflies and the other wasp superfamilies (protein Fe65 homolog and OTU domain-containing protein 7B) .Comparing the genomic region across the Hymenoptera superfamilies, it appears that there is a pattern of intrachromosomal shuffling of syntenic blocks of genes rather than interchromosomal rearrangement (data S1). With the exception of the Formicoidea and parasitic wasps, a syntenic block (SB1) containing five genes . Incubation of the recombinant CYP336A proteins with alkaloids containing a basic nitrogen revealed a conserved capacity to metabolize these phytochemicals  but is less active against the other alkaloids screened (12.94 to 41.05% depletion). In contrast, CYP336A23 was particularly active against the tropane alkaloids atropine and scopolamine , and CYP336A24 metabolized anabasine most efficiently (99.97% depletion) (table S5). Similarly, M. rotundata CYP336A34 exhibited high activity against the pyridine alkaloids nicotine and anabasine (100% depletion) but was unable to metabolize echimidine. CYP336A33 was less active against the pyridine alkaloids (<45% depletion) but showed efficient metabolism of echimidine (82.57% depletion). To gain further insight into the potential divergence of paralogous CYP336A genes, we examined the expression of CYP336A isoforms in the bumblebee and alfalfa leaf-cutting bee. This revealed that paralogous CYP336A genes in these species are divergently expressed in different tissues or body segments (fig. S6). In combination with our functional analyses, this suggests that CYP336A gene duplication events have led to partial subfunctionalization of paralogs coupled with further differentiation in terms of their tissue-specific expression patterns.To investigate whether there is evidence of conserved function within the CYP336 family in terms of activity against alkaloids, seven genes from four representative hymenopteran species that diverged over 281 Ma . Using CAVER Web v1.1 , we hypothesize that D298 functions as a \u201cgatekeeper\u201d by facilitating access and binding of protonated substrates such as alkaloids within the catalytic pocket of CYP336 genes.We extended this analysis by generating a three-dimensional computational model of CYP336A1 using AlphaFold2  or the cabbage looper Trichoplusia ni , a different P450-mediated biotransformation pathway is predominant. Here, N-oxidation and 5\u2032C oxidation have been reported as the major metabolic pathways  . Our analyses also revealed remarkable conservation of the genes framing the CYP336 locus across the superfamilies, from sawflies to bees. Assuming random gene order, the probability of finding two orthologs next to each other in three genomes has been calculated as <4 \u00d7 10\u22126 , leaf-cutting , and parasitism . While the honeybee is an example of a generalist species interacting with many different plants, the sawfly X. alpigena is highly specialized on Pinus cembra  following a single amino acid substitution is remarkable for an insect P450  were obtained from queen-right colonies located in Monheim am Rhein, Germany. Bumblebee colonies were purchased from Agralan Ltd.  and kept in constant darkness at 25\u00b0C, 50% relative humidity. The hives were fed ad libitum with the nectar substitute, Biogluc, and pollen was supplied to colonies every 2 days. Adult alfalfa leaf-cutting bee (M. rotundata) cocoons were obtained from Canada through Bayer AG Crop Science Division  in 2021, brought to emergence as described previously (Honeybees (N-methylmyosmine (CAS 525-74-6) was custom-made and purchased from Carbosynth .All chemicals were of the highest purity available and obtained from Sigma-Aldrich , if not stated otherwise. Investigated phytochemicals included nicotine (CAS 54-11-5) and its metabolite cotinine (CAS 486-56-6), atropine (CAS 51-55-8), anabasine (CAS 13078-04-1), scopolamine (CAS 51-34-3), echimidine (CAS 520-68-3), cytisine (CAS 485-35-8), coumarin (CAS 91-64-5), and caffeine (CAS 58-08-2). p-nitroanisole (CAS 100-17-4).Model substrates included BFC (CAS 220001-53-6), 7-ethoxy-coumarin , 7-methoxy-coumarin , 7-ethoxy-4-(trifluoromethyl)-coumarin , 7-methoxy-4-(trifluoromethyl)-coumarin , 7-hydroxy-4-(trifluoromethyl)-coumarin , 7-benzyloxy-methoxy-resorufin , 7-ethoxy-resorufin , 7-benzyloxy-resorufin , 7-methoxy-resorufin , 7-pentoxy-resorufin , and 7-Benzyloxy-methyloxy-4-(trifluoromethyl)-coumarin  was custom-synthesized by Enamine Ltd. , and 7-pentoxy-coumarin (PC) was synthesized in-house . BOMR was purchased from Invitrogen .A. mellifera NADPH [reduced form of nicotinamide adenine dinucleotide phosphate (NADP+)]\u2013dependent cytochrome P450 reductase using a Bac-to-Bac baculovirus expression system (Invitrogen) in insect High five cells . Site-directed mutagenesis of CYP336A1 was conducted using the Q5 Site-Directed Mutagenesis Kit following the manufacturer\u2019s instructions  and with primers designed for the codon-optimized plasmid sequence .Functional expression of recombinant P450s was performed exactly as described previously  in 15-s intervals for 15 min using the kinetic mode of the microtiter plate reader .The functional activity of recombinantly expressed microsomal P450s was tested against a range of fluorogenic coumarin- and resorufin-based model substrates as recently described ]. Control samples lacked the NADPH regeneration system. Additional controls contained microsomal fractions of insect cells coinfected with an empty baculovirus and A. mellifera cytochrome P450 reductase. The reaction was stopped with the addition of 400 \u03bcl of ice-cold acetonitrile, the samples were stored overnight at 4\u00b0C and centrifuged for 30 min at 3200g, and the supernatant was used for subsequent UPLC-MS/MS analysis.Alkaloid depletion was assessed by incubating 80 \u03bcg of microsomal protein with 10 \u03bcM of the parent compound for 2 hours at 30\u00b0C. The 100-\u03bcl reactions included a NADPH regeneration system [Promega; 1.3 mM NADPMichaelis-Menten kinetic studies were conducted as follows: 20 \u03bcg of CYP336A1 or 160 \u03bcg of CYP6AQ1 was incubated for 30 min with different nicotine concentrations in twofold dilution steps ranging from 100\u00a0to 0.78 \u03bcM. All other factors remained the same.2O) were used in gradient mode. After positive electrospray ionization, ion transitions were recorded on Sciex API6500 Triple Quad . All alkaloids were measured in positive ion mode . Peak integrals were calibrated against a standard calibration curve. Parent depletion was calculated by subtracting the values from +NADPH samples from the average of \u2212NADPH replicates.Alkaloids were quantified by UPLC-MS/MS using Agilent 1290 Infinity II  with a Waters Acquity HSS T3 column  for all alkaloids except scopolamine . Eluents , the supernatant was purified using a SOLA-CX cartridge . Nicotine (15 \u03bcM) was incubated with recombinant CYP336A1 as described above. Sample (360 \u03bcl) was mixed with 2 ml of 5 mM ammonium formate (pH 2.5). The cartridge was conditioned with 500\u00a0\u03bcl of acetonitrile followed by 500\u00a0\u03bcl of 5 mM ammonium formate (pH 2.5) before sample application. The column was washed with 1 ml of 5 mM ammonium formate (pH 2.5) followed by 1 ml of 1% (v/v) formic acid in acetonitrile, evaporated under nitrogen at room temperature before the sample was eluted with 300\u00a0\u03bcl of 5% ammonia (v/v) in acetonitrile.Nicotine metabolite quantification was conducted using purchased reference substances of cotinine and A. mellifera CYP336A1 was generated using the AlphaFold2 structure prediction software (https://saves.mbi.ucla.edu/). The generated model lacked the heme group and so was aligned with CYP3A4 (PDB: 4D6Z) to transfer the heme group using the Schrodinger Maestro suite . The model was refined for further analysis by adding hydrogens, creating zero-order bonds to metals and disulfide bonds. The heme iron charge was set to +3, and the entire enzyme was energy-minimized.A three-dimensional model of Subsequently, the structure was uploaded to the CAVER Web 1.1. tool (www.rdkit.org) for the actual similarity calculations and NetworkX . Key packages for the similarity analysis are RDKit (https://chemcraftprog.com). Molecular geometries were optimized at BP86 level of theory (N(r). The electron densities of the oxidized and reduced species, \u03c1N\u22121(r) and \u03c1N+1(r), respectively, were calculated at the same level of theory. The Fukui functions were obtained numerically by the finite differences . Cytochrome P450s were identified using BLAST2GO (A. mellifera CYP3 clan P450s as query sequences (table S2). Protein sequences were aligned using the MUSCLE algorithm (Genomic and transcriptomic assemblies from hymenopteran species (Hymenoptera: taxid 7399) were retrieved from the NCBI database , Ooceraea biroi , Solenopsis invicta , Vespa crabo , V. canescens , Nasonia vitripennis , and N. lecontei . Syntenic analyses between these chromosomes were performed using Mauve  version 2.4.0 , Cotesia glomerata (MPM_Cglom_v2.3), Ceratosolen solmsi (CerSol_1.0), Polistes dominula (Pdom r1.2), and M. rotundata (MROT_1.0) (table S2). The region ~500 kbp upstream and downstream of the CYP336 locus was examined in more detail for evidence of microsynteny. A series of inter- and intra-superfamily pairwise comparisons were run on these chromosomal extractions, using Mauve  and kept at \u221220\u00b0C for at least 24\u00a0hours, before the dissection of midgut, Malpighian tubules, and brain. Alfalfa leaf-cutting bees were directly divided into three segments  using forceps and scissors on dry ice. Subsequently, tissues were disrupted using 3-mm stainless steel beads in a bead mill at 20 Hz for 2 \u00d7 30 s.\u00a0Total RNA was extracted using TRIzol reagent  following the manufacturer\u2019s instructions. RNA purification was performed using the RNeasy Mini Kit  for M. rotundata samples and the PicoPure RNA isolation kit  for A. mellifera and B. terrestris tissue samples. RNA was eluted in appropriate volumes of nuclease-free water.Expression of bee CYP336A P450s in body parts and dissected tissues was investigated by qPCR. Female bees were snap-frozen in liquid nitrogen and stored at \u221280\u00b0C until further use. Honeybees and bumblebees were transferred to RNAA. mellifera and B. terrestris, PCRs (10 \u03bcl) contained 0.5 ng of cDNA, 5 \u03bcl of SsoAdvanced Universal SYBR Green Supermix , and 0.25 \u03bcM of each primer. Samples were run on the CFX 384 Real-Time System  under the following conditions: 3 min at 95\u00b0C followed by 39 cycles of 95\u00b0C for 15 s, 64\u00b0C for 15 s, and 60\u00b0C for 15 s.\u00a0M. rotundata samples were run under slightly different conditions . A final melt-curve step was included after PCR (ramping from 65\u00b0 to 95\u00b0C by 0.5\u00b0C every 5\u00a0s) to confirm the absence of any nonspecific amplification in all cases. The efficiency of PCR for each primer pair was assessed using a fivefold serial dilution of cDNA. Each reverse transcription\u2013qPCR experiment consisted of at least four independent biological replicates with three technical replicates. Data were analyzed by qbase+ version 3.1 . For honeybees, the reference genes Rpl32 , EF1a (elongation factor 1\u03b1), and RPS5 were used . One microgram was used for complementary DNA (cDNA) synthesis using the iScript cDNA Synthesis Kit  according to the manufacturer. For tissue samples from"
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