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+{"text": "PLoS Biology, Thomas Bartman et al. use the powerful tools afforded by zebrafish genetics to dissect the early steps of heart valve formation. In the process, they provide evidence for a causal relationship between the early function of the heart and its final structure.The heart starts beating and pumps blood through the body long before it has achieved its mature architecture. In theory, this provides a chance for cardiac function to sculpt cardiac structure, an intriguing possibility for developmental biologists, and one of potentially great clinical import for cardiologists seeking to identify the causes of  cardiac anomalies. In this issue of At the time of its first beat, the vertebrate heart is little more than a tube, lined on its outside by a myocardial cell layer whose contractions (the heartbeats) power blood flow, and on its inside by an endocardial cell layer, an extension of the inner wall of the connecting blood vessels. What it lacks still are valves and septae, the fibrous gates that subdivide the mature heart into atrial and ventricular chambers, and control the directionality of blood flow. These structures derive from the endocardium in a process that begins\u2014shortly after the establishment of blood flow\u2014with the local accumulation of endocardial cells into what are known as endocardial cushions (ECs).cardiofunk (cfk), which was devoid of ECs. Genetic mapping of the cfk mutation revealed a single sequence change in a gene encoding a novel actin molecule that is most closely related to the sarcomeric actins found in sarcomeres, the contracting organelles of muscle cells. The result was surprising because contractions are not a property of endocardial cells. Using RNA detection assays, the authors show that the cfk gene is in fact expressed in the myocardium, rather than in the endocardium. It therefore appears that the inability to form ECs in cfk mutants does not reside in the endocardium per se, but is an indirect consequence of a myocardial anomaly.The zebrafish lends itself well to large-scale genetic screens, and powerful genomic tools are now available to efficiently identify the gene affected by any mutation. The authors have used genetic screens to identify several mutations that affect early cardiac function or morphology. Heart anomalies are easy to detect in zebrafish, and can be examined in real time and in live specimens because the embryos develop outside the mother and are fully transparent. Using a fluorescent molecular marker highly expressed in the ECs, the authors narrowed in on mutations that result in valve defects, and identified a mutant they named cfk mutation introduces a single amino acid change in the actin protein, and through detailed biochemical analyses, the authors show that the mutant actin is impaired in its ability to assemble into fibers in vitro. What might be the consequence in vivo? The authors note that cfk mutants display abnormal heart contractions prior to the development of their EC defect. Support for the notion that myocardial contractions are required for EC formation comes from the examination of silent-heart (sih) mutants. sih mutants, which lack a heatbeat, have been shown to harbor a mutation in troponin T, one of the motors of actin contractions; the authors find that sih mutants also fail to develop ECs. The mechanisms linking myocardial contractions and cushion formation remain unclear. Blood flow may be a trigger, though the authors find that ECs can develop even in the presence of pharmacological compounds that abolish it. The characterization of additional mutants should help answer this question.The Valve or septal defects represent 40% of cardiac anomalies in humans. Bartman and colleagues suggest that, by analogy with zebrafish, some may result from congenital defects affecting very early myocardial function. Their work thus opens new avenues for the early detection of human cardiac malfunctions and malformations."}
+{"text": "Likewise, we determined that cushion formation is blocked in \u2212/\u2212)cardiofunk . We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined s cardiofunkCardiac anomolies can result from very early defects in heart development. In zebrafish, such defects have been shown to be caused by a new gene called  The genetic programs and developmental processes that lead to organ formation are still poorly understood. We are currently witnessing an expansion in research that aims to identify the genes responsible for the structural development of organs and their later function. Among the organs of the body, the heart is unique because it begins to function mechanically before structural development is complete, begging the important question of whether myocardial function is required for the morphogenetic events that occur after the heart begins beating. One of the late steps of heart development is the formation of the endocardial cushions (ECs), which are tissue swellings that develop in characteristic locations along the anterior\u2013posterior (AP) extent of the heart tube and contribute to valves and, in four-chambered hearts, to septae. Because of the clinical significance and prevalence of EC defects in humans , an undeDuring the process of EC and valve development, specific endocardial cells undergo multiple poorly understood specification, differentiation, and migration events en route to becoming functional heart valves. The genes involved in one substep of this process, epithelial\u2013mesenchymal transformation (EMT), are gradually being identified. Analysis of EMT during cardiac-cushion development has implicated molecules such as Fibronectin, Transferrin, ES-130, hLAMP-1, TGF-\u03b22, TGF-\u03b23, BMP-2 silent heart , but do undergo looping morphogenesis  boundary. This process is easily visualized at 48 h postfer-tilization (hpf) by examining the endocardial cells expressing green fluorescent protein (GFP) under the control of the mouse promoter  , that fails to accumulate tie2::GFP-positive cells at the AV boundary by 48 hpf  extent of the heart tube before becoming localized to the AV boundary at 42 hpf  zC202M22 , and fv49b10  showed a change of arginine 177 to histidine. The three embryos recombinant at either end of the 52 kb region were homozygous at the site of the R177H lesion, suggesting that we had isolated cfk.To understand better the molecular basis for the d Z10582 . AnalysizC202M22 A. Follow GenScan  and BLAS fv49b10 B. Compar fv49b10 C and hum fv49b10 D and concfk mutation supported our hypothesis that cfk corresponded to an actin gene. Analyses of Cfk indicate that it is a sarcomeric actin by virtue of its homology to zebrafish \u03b1-cardiac and \u03b1-skeletal actins and lack of homology to zebrafish cytoplasmic actin, as well as its conservation of synteny with the human skeletal actin. Further evidence that cfk encodes a sarcomeric actin and not a cytoplasmic actin includes the presence of an extra residue at the N-terminus of Cfk, which is seen in all sarcomeric actins but no cytoplasmic actins, as well as the presence of residues that are stereotypic for sarcomeric actins at all 20 locations where sarcomeric and cytoplasmic actins have unique amino acids , which is distinct from the 3\u2032 UTR sequences of zebrafish \u03b1-cardiac and \u03b1-skeletal actins. These data showed that cfk is expressed in the myocardial but not endocardial cells of zebrafish embryos from 24 to 48 hpf  leads to unstable filaments, occasionally giving rise to a phenotype in heterozygous embryos.During our work with e cfk+/\u2212 A, suggese cfk+/\u2212  demonstr\u2212/\u2212cfk embryos have a dilated heart and lack blood flow and because subsequently approximately 50% of these embryos fail to form ECs, we wondered whether these two phenotypes were causally related or whether lack of EC formation could occur independently of poor early myocardial function. To address this question, 379 embryos from multiple cfk clutches were assayed for cardiac dilation and lack of blood flow at 36 hpf and for lack of endocardial ring formation at 48 hpf. Embryos that were phenotypically wild-type at 36 hpf invariably developed endocardial rings over the next 12 h, whereas all embryos that failed to form endocardial rings had previously shown a functional phenotype at 36 hpf  and fail to generate blood flow, it is impossible to conclusively state whether EC formation is affected in these embryos directly as a result of poor myocardial function or indirectly as a result of the perturbation in blood flow and shear stress caused by poor myocardial function. n = 74) formed an endocardial ring, indicating that blood flow is not required for the initial steps of cushion formation. When myofibril function was further decreased by treatment with 10 mM 2,3-BDM, the percentage of embryos with an endocardial ring decreased to 13% (n = 68). Studies with the anesthetic tricaine confirmed the observation that ECs may form in the absence of blood flow and that the likelihood of forming cushions is inversely proportional to the concentration of tricaine (data not shown). In summary, the results from the 2,3-BDM and tricaine treatments suggest that it is poor myocardial function, and not lack of blood flow, which is primarily responsible for the loss of EC formation in \u2212/\u2212cfk and \u2212/\u2212sih embryos.Because \u2212/\u2212sih embryos fail to beat yet undergo looping morphogenesis and AV boundary specification. The specific absence of EC formation in these embryos clearly demonstrates that myocardial function is required for EC formation. Precisely how myocardial function is required for EC formation is unclear. Prior work has demonstrated a requirement for AV boundary myocardium in the formation of ECs, and several signaling molecules emanating from the myocardium at the AV boundary have been identified , abc-b10 (5\u2032-TCATTCGCAACATTGTCCCATACAT-3\u2032), fv49b10.y1 (5\u2032-CCGACCTAATTCGCTTGGT-CACCAT-3\u2032), and cfk (5\u2032-CATCTTGATGTATTCTTTCTCTGCT-3\u2032). The morpholinos were injected at 4 ng and 8 ng into tie2::GFP embryos at the one-cell stage and examined at 24 to 72 hpf for myocardial function and EC phenotypes. Morpholinos against abc-b10, rab4a, and fv49b10.y1 did not affect myocardial function or EC formation. The cfk morpholino caused no phenotype, an expected result given the ability of actin genes to compensate for each other when down-regulated  to bind to the initiation codon and flanking sequences of the following zebrafish genes: sih , rab4a (egulated  and the s11cfk-AB/SJD hybrid strain using SSLP with various CA repeat markers and SSCP with various ESTs. SSCP was performed by denaturing PCR products at 95\u00b0C for 10 min in 0.7 mM EDTA and 36 mM NaOH and placing on ice before loading on nondenaturing acrylamide gels. BAC library filters for the CHORI-211 (zC) library were obtained from P. de Jong at BACPAC . Linkage of the R177H mutation to the s11cfk allele (through genotyping of mutant embryos) was accomplished by performing PCR across the mutation (forward primer = 5\u2032-ATCGTGCTGGACTCTGGTG-3\u2032 and reverse primer = 5\u2032-GAAAGAATAACCGCGCTCAG-3\u2032) and digesting with NsiI, which cuts only the H177 allele. Synteny analysis was performed through http://genome.jgi-psf.org/fugu3/fugu3.home.html (fugu) and http://genome.ucsc.edu (human).We genotyped diploid mutant embryos from a s11cfk mutant embryos to extract mRNA  and synthesize cDNA . PCR was then performed from the 5\u2032 UTR to 3\u2032 UTR and multiple clones sequenced. To confirm that the amino acid change we saw was a mutation and not a polymorphism in the strain used for mutagenesis, we examined the DNA of four F1 females from the screen .We used a pool of 25 e screen  in whichClutches of dechorionated embryos were placed in 2,3-BDM  at 24 hpf at concentrations between 2 and 20 mM. This concentration range has been shown to affect myofibrillar ATPase in a dose-dependent manner, with 25% of activity remaining at 10 mM . 2,3-BDM2, and 0.1 mM DTT) and used within 2 d. Actin polymerization was induced by the addition of MgCl2 and KCl to final concentrations of 2 mM and 50 mM, respectively, and polymerization was assessed by the increase in light scattering as a function of time at 25\u00b0C in a 120 \u03bcl volume in a thermostatted cuvette using a SPEX Fluorolog 3 fluorimeter with excitation and emission wavelengths set at 360 nm.Site-directed mutagenesis of the yeast-actin coding sequence in a centromeric plasmid and construction of haploid cells producing only the mutant actin were carried out as described previously . Wild-tyVideo S1\u2212/\u2212cfk hearts have complete regurgitation of blood from the ventricle to the atrium and therefore no forward blood flow.Wild-type hearts propel blood through the vasculature, whereas (2.91 MB MOV).Click here for additional data file.Video S2\u2212/\u2212cfk embryos (two different embryos shown) have dilated hearts with weak contractions and are not capable of generating circulation.Wild-type embryos have a narrow, strongly contracting heart and generate effective blood flow. (2.9 MB MOV).Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers discussed in this paper are for cfk (AY222742), zebrafish \u03b1-cardiac actin (AF116824), zebrafish \u03b1-skeletal actin (AF180887), and zebrafish cytoplasmic actin (AF057040).The GenBank ("}
+{"text": "Concerns have recently been raised regarding the safety of potential human exposure to bisphenol A (BPA), an industrial chemical found in some polycarbonate plastics and epoxy resins. Of particular interest is the exposure of young children to BPA via food stored in BPA-containing packaging.In this study we assessed the age dependence of the toxicokinetics of BPA and its glucuronidated metabolite, BPA-Glu, using a coupled BPA\u2013BPA-Glu physiologically based toxicokinetic (PBTK) model.Using information gathered from toxicokinetic studies in adults, we built a PBTK model. We then scaled the model to children < 2 years of age based on the age dependence of physiologic parameters relevant for absorption, distribution, metabolism, and excretion.We estimated the average steady-state BPA plasma concentration in newborns to be 11 times greater than that in adults when given the same weight-normalized dose. Because of the rapid development of the glucuronidation process, this ratio dropped to 2 by 3 months of age. Simulation of typical feeding exposures, as estimated by regulatory authorities, showed a 5-fold greater steady-state BPA plasma concentration in 3- and 6-month-olds compared with adults, reflecting both a reduced capacity for BPA metabolism and a greater weight-normalized BPA exposure. Because of uncertainty in defining the hepatic BPA intrinsic clearance in adults, these values represent preliminary estimates.Simulations of the differential BPA dosimetry between adults and young children point to the need for more sensitive analytical methods for BPA to define, with greater certainty, the adult hepatic BPA intrinsic clearance, as well as a need for external exposure data in young children. Bisphenol A (BPA) is an industrial chemical found in polycarbonate plastics and epoxy resins such as food-can linings, bottles, and dental fillings. The weak estrogenic activity of BPA  has raisIn response to the identification of young children as a higher risk population, we investigated the exposure of children to BPA using physiologically based toxico kinetic (PBTK) modeling. PBTK models have been widely used in human health risk assessment to assess the potential internal human exposure to environmental compounds in the absence of direct human toxicokinetic data . Typicalex vivo method, BPA is completely and rapidly absorbed from the gastrointestinal tract . Using aThe objectives of this modeling study were to estimate the differences in the average steady-state dose-normalized BPA plasma and urinary concentrations between adults and children < 2 years of age after BPA administration and to determine the expected average steady-state plasma concentrations of BPA and BPA-Glu in young children after typical feeding scenarios.http://www.ehponline.org/members/2008/0800073/suppl.pdf)]. The model includes 15 organs as well as arterial, venous, and portal blood compartments. The organs are connected via blood flows, and the circulation system is closed via the lung. We coupled the sub-models on export to MoBi , a software package for the mechanistic and dynamic modeling of biologic processes and drug action. We linked the two submodels such that the hepatic clearance of BPA was the only source function for the BPA-Glu submodel, where the process was defined in the liver.In this study we used a nested coupled PBTK structure consisting of a BPA submodel coupled to a BPA-Glu submodel . We usedWe previously published the body weight, height, blood flows, and organ volumes for children and adults, as used in the PK-Sim software . Table 2fa = 1) of orally administered BPA, these various inputs did not make a life-stage difference. As a result, all simulations used an favalue of 1.The oral absorption model is that of We used the algorithms of V is the volume of the tissue.where O-glucuronide. O-glucuronide. Another study examining the UGT responsible for diclofenac glucuronidation in humans found that rat UGT2B1 and human UGT2B7 both catalyzed this reaction with similar affinities  from adults is converted to an intrinsic clearance  using the well-stirred model (Equation 2) and physiologic information of liver blood flow  and the fraction unbound in plasma (fu):We described the method of scaling clearance from adults to children . In briefu as derived using the method of fu in adults to children based on the age dependence of albumin concentrations in plasma  and multiplied by the percentage of activity relevant to the age of the child to derive a liver weight\u2013normalized intrinsic clearance for the child. Plasma clearances for children are derived by rearrangement of Equation 2 and by using the age-specific liver weight, blood flow, and estimated n plasma .BPA-Glu_kidney during the development of the adult coupled model and compared this with values derived from BPA-Glu_kidney.Urinary excretion is 100% responsible for BPA-Glu clearance in humans . We optiCBPA_urine):We used the following equation to calculate the total BPA (BPA + BPA-Glu) average urinary concentration at steady state , and CLBPA-Glu_kidney. We simulated a BPA oral dose of 5 mg  is calculated as (CLBPA_liver \u00d7 Ccell_liver_BPA \u00d7 fu_BPA/Kliver_BPA)/Vcell_liver, where Ccell_liver_BPA is the concentration of BPA in the liver intra-cellular space, fu_BPA is the unbound fraction of BPA in plasma, Kliver_BPA is the liver:plasma partition coefficient, and Vcell_liver is the intracellular volume of the liver [see Supplemental Material for full differential equations (http://www.ehponline.org/members/2008/0800073/suppl.pdf)].We parameterized the BPA and BPA-Glu models in PK-Sim for an average 30-year-old male of 73 kg and 176 cm . We used of 5 mg . We set BPA-Glu_kidney. We optimized these parameters using the plasma concentration\u2013time data of BPA-Glu for all subjects studied by BPA-Glu_kidney to children as previously described. During the scaling procedure and simulations in children, we allowed no changes to the coupled model structure.The only unknown parameters at this point were the lipophilicity of BPA-Glu and CLBPA_liver, CLBPA-Glu_kidney, and fu.Once we set the adult model of BPA and BPA-Glu, we made changes to scale the model to children. We used the following age-specific parameters: weight, height, organ volumes, blood flows, volume fraction of fat in adipose tissue, tissue:plasma partition coefficients, PS, CLF) asFirst, we completed simulations after a BPA application of 1 \u03bcg/kg once per day to demonstrate the expected differences in the average dose-normalized plasma concentrations at steady state of BPA and BPA-Glu in adults and young children (0\u20132 years of age). Second, we used published environmental exposure data  Table 3Table 3 tAUC is the area under the simulated plasma concentration time curve for an iv or peroral (po) BPA dose.where Cavg_ss). We used its simplest form where an input parameter value (Pin) is changed by 1% and the relative change in outcome is calculated when all other input parameters are fixed:We used local sensitivity analysis here to pri-oritize the impact of the input parameters on the outcome of interest: the average steady-state plasma concentration (fu (1), organ:plasma partition coefficients (15), red blood cell partition coefficient (1), red blood cell permeability (1), organ PS (15), organ-specific PSrbc (18), intrinsic hepatic clearance of BPA (1), and intrinsic kidney clearance of BPA-Glu (1) on Cavg_ss of both BPA and BPA-Glu.We performed all sensitivity analyses based on a BPA dose of 1 \u03bcg/kg/day for an adult. We used sensitivity analyses to assess the variation of both BPA and BPA-Glu organ/tissue volumes (18), organ blood flows (14), hematocrit (1), BPA_liver required to keep the first data point (51 min) below the limit of detection of 10 nM (BPA-Glu_kidney) for the urinary excretion of BPA-Glu and the BPA-Glu lipophilicity were 1.82 mL/min/kg and \u22120.05, respectively. CLBPA-Glu_kidney was equal to that derived by The plasma CLof 10 nM  was 21 mfa = 1), although there was an increase in the efficiency of first-pass metabolism with increasing age. Bioavailability (F) in newborns and 3-month-, 6-month-, 1.5-year-olds, and adults was 88%, 48%, 32%, 23%, and 18%. In all simulations, BPA and BPA-Glu steady state was reached within the first 48 hr. At all ages, 100% of the applied BPA dose was modeled as absorbed to the portal vein  were fu (0.98), hematocrit (0.16), and intrinsic hepatic clearance (0.89). No varied parameter in the BPA model, with respect to BPA-Glu average steady-state concentrations, produced a value greater than 1. No parameter in the BPA-Glu model, with respect to BPA-Glu average steady-state concentrations, produced a value > 1, although a value to note was intrinsic renal clearance (0.80).Values > 1 were of interest in that they identified input parameters that had potential to greatly affect in vivo data from rats after BPA exposure with subsequent scaling to humans. The lumped compartments in the model did not provide an appropriate structure that could account for the relevant physiologic inputs that are required to reliably scale from adult humans to children. This necessitated the use of another model. in vivo data from rats for parameterization. Their model focused on BPA, not BPA-Glu. Further, they measured tissue:plasma partition coefficients 3\u20134 hr after initial exposure, which for a lipophilic compound such as BPA would not be sufficient to reach steady state. The result was a partition coefficient in adipose tissue that was lower than all other organs as well as blood, a result in stark contrast to data from ex vivo experiments that the adipose:plasma partition coefficient was two to three times higher than all other tested tissues. Although in rat this value may not be particularly important for describing the kinetics of a lipophilic compound, because only approximately 4% of body weight is adipose tissue, humans have approximately 15\u201330% of body weight as adipose tissue, so this value is greatly influential. Existing PBTK models of BPA and/or BPA-Glu were not appropriate for use in this scaling study. Optimization of the BPA-Glu lipophilicity parameter as a means to estimate tissue:plasma partition coefficients from the F = 88%) and subsequently a bioavailability decrease with age. Taken with a distribution change over age , the dose-normalized BPA plasma concentrations at steady state were a maximum of 11 times higher than that in adults. As enzyme activity reached that of adults at 1 year of age, the dose-normalized BPA plasma concentrations at this age were lower than in adults because of a higher weight-normalized plasma clearance. This phenomenon has been documented previously  and the hepatic intrinsic clearance of BPA (0.89). For the BPA-Glu concentrations, the parameter renal intrinsic clearance of BPA-Glu (0.80) was of interest. To be confident in our scaled model for children, ensuring the accuracy of these parameters for children is important. We scaled the fraction unbound in plasma from adults based on the method of Because of the relative ease of urine sampling compared with blood sampling, the greatest body of exposure literature is based on urinary concentrations of BPA after sampling of unintentionally exposed individuals. The literature contains little information on children. Based on urinary BPA concentrations, there was a tendency toward higher calculated BPA intakes and urinary concentrations in children and adolescents 6\u201319 years of age  comparedin situ hepatic intrinsic BPA clearance in the adult and urinary concentrations in children < 6 years of age to define daily BPA intake. As a worst-case scenario, these findings suggest that the typical intraspecies uncertainty/safety factor of 10 commonly applied to a threshold dose (In summary, we developed a PBTK coupled model system for BPA and its metabolite, BPA-Glu, in adults and scaled to children based on age-dependent physiologic parameters. Because of low UGT2B7 activity, BPA plasma concentrations could be approximately 11 times greater in newborns than in adults exposed to the same weight-normalized dose. An increase in age to 3 months lowers this ratio to 2, although exposure through food can be greater in this age group than in adults . When coold dose  is just"}
+{"text": "Bisphenol A (BPA), a well-known endocrine disruptor, is highly glucuronidated in the liver, and the resultant BPA-glucuronide (BPA-GA) is excreted primarily into bile. However, in rodents, prenatal exposure to low doses of BPA can adversely affect the fetus, despite the efficient drug-metabolizing systems of the dams. The transport mechanisms of BPA from mother to fetus are unknown.To test our hypothesis that BPA-GA\u2014an inactive metabolite\u2014is passed through the placenta to the fetus, where it affects the fetus after reactivation, we investigated the placental transfer of BPA-GA and reactivation to BPA in the fetus.After performing uterine perfusion with BPA-GA in pregnant rats, we examined the expression and localization of the placental transporters for drug metabolites in the perfusate by reverse-transcriptase polymerase chain reaction and immunohistochemistry. We also investigated the deconjugation of BPA-GA in the fetus and examined uridine 5\u2032-diphospho-glucuronosyltransferase (UGT) activity toward BPA and the expression of UGT isoforms in fetal liver.We detected BPA-GA and deconjugated BPA in the fetus and amniotic fluid after perfusion. In the trophoblast cells, organic anion-transporting polypeptide 4a1 (Oatp4a1) was localized on the apical membrane, and multidrug resistance-associated protein 1 (Mrp1) was localized to the basolateral membrane. We observed deconjugation of BPA-GA in the fetus; furthermore, we found the expression of UGT2B1, which metabolizes BPA, to be quite low in the fetus.These results demonstrate that BPA-GA is transferred into the fetus and deconjugated in the fetus because of its vulnerable drug-metabolizing system. Leaching of BPA out of the products is increased by heating , contactin vitro . Severalin vitro , nervousin vitro , and immin vitro .Generally, adult animals are able to metabolize and eliminate BPA from the body. Previously, we found that BPA is highly glucuronidated by UGT2B1, an isoform of uridine 5\u2032-diphospho-glucuronosyltransferase (UGT) expressed in the rat liver . In addi14C-BPA, 14C-BPA  were purchased from Sankyo Lab Co. . Animals were housed individually under a 12/12-hr light/dark cycle and had  Animals .A schematic illustration of uterine perfusion is shown in 2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 10 mM glucose, 26 mM NaHCO3, 2.5% dextran from Leuconostoc mesenteroides, and 3% Dextran 70) as the perfusate. The perfusate was pumped at a constant rate of 3 mL/min. First, we perfused four animals in a preliminary group with 10 \u03bcM BPA-GA for 20 min to determine whether BPA-GA is transferred into the fetus across the placenta. In this group, we performed a preliminary perfusion with mKRB for 5 min to wash away blood, followed by 20 min inflow of mKRB containing 10 \u03bcM BPA-GA. After the perfusion, the fetus and amniotic fluid were collected.We used modified Krebs-Ringer\u2019s buffer . From the beginning of the BPA-GA perfusion, the perfusate that drained from the caudal vena cava was collected from each animal at 5-min intervals for 90 min. At the end of perfusate collection, the fetus, amnion, amniotic fluid, uterus, and placenta were collected. We also performed a control study using 1-naphthol-glucuronide  as the substrate. Four pregnant rats were used in each perfusion group. Values are expressed as mean \u00b1 SE.Perfusates collected from the caudal vena cava were collected after uterine perfusion and prepared for HPLC and LC/TOF-MS analysis. Each perfusate sample was mixed with a 4-fold volume of acetonitrile and centrifuged at 13,000 rpm for 5 min at 4\u00b0C; each supernatant was then analyzed by HPLC.To prepare tissues for HPLC and LC/TOF-MS analysis, we first added each sample to 0.3 mL methanol; the sample was homogenized and then sonicated for 5 min. The products were centrifuged at 13,000 rpm for 5 min at 4\u00b0C, and the supernatants were concentrated by solid-phase extraction using an Oasis HLB Plus cartridge . The extracts were then analyzed by HPLC.We used an HPLC system  that consisted of a dual pump (DP-8020), a fluorescent photometer (FS-8020), and a column oven (CO-8020). Samples were separated at 40\u00b0C using a reverse-phase column  at a flow rate of 1.0 mL/min under a linear gradient of solution A (methanol/water = 24/76 vol/vol with 10 mM ammonium acetate) and solution B (methanol) for 20 min. BPA-GA was detected at excitation/emission of 275/308 nm and 1-NA-GA was detected at excitation/emission of 283/336 nm. The results were recorded using LC-8020 integration software (Tosoh Corp.); the elution peaks of BPA and BPA-GA were noted and the concentrations compared with the standards.m/z 403\u2013404 by TOF-MS. In the present study, the limits of detection of BPA and BPA-GA in LC/TOF-MS were both 20 pmol/mL (data not shown).LC/TOF-MS was performed using the HPLC system described above and LCT premier XE (Waters Corp.). The flow rate of HPLC was 0.3 mL/min, and BPA-GA was monitored at A23 anti-Mrp1 polyclonal antibody  was purchased from Enzo Life Sciences Inc. . We obtained anti\u2013organic anion-transporting polypeptide (anti-Oatp4a1) polyclonal antibody from Scrum, Inc. ; anti-Oatp4a1 is designed to recognize the amino acid sequence LPSQSSA, which is common between human and rat .Placental tissue samples were analyzed by immunohistochemistry, as described in detail in the Supplemental Material (doi:10.1289/ehp.0901575). Briefly, tissue sections were incubated with anti-Oatp4a1 antibody or anti-Mrp1 antibody and analyzed with a confocal laser scanning microscope (Axionvert 200M) and PASCAL software .Maternal liver, placenta, fetal liver, and fetal intestine were collected on GD18.5. Total RNA was isolated from the tissues using the RNeasy Mini Kit  according to the manufacturer\u2019s instructions. cDNA was synthesized from total RNA using Superscript III  reverse transcriptase according to the manufacturer\u2019s instructions.Sequences of the oligonucleotide primers to amplify gene-specific cDNAs are available in the Supplemental Material (doi:10.1289/ehp.0901575).Mrp1, Mrp2, and Oatp4a1 mRNA in maternal liver and placenta was investigated by real-time RT-PCR using the QuantiTect SYBR Green PCR kit (Qiagen) and analyzed by iQ5/MyiQ Single-Color . Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard. The copy number of each transporter gene was divided by that of GAPDH for normalization. Quantitative values are mean \u00b1 SE of four amplifications for placenta and three for maternal liver for each transporter gene.Quantitative expression of g at 4\u00b0C. After removing the supernatant, the pellets were resuspended in 5 mL William\u2019s medium  containing collagenase  and DNase I  and incubated for 30 min at 37\u00b0C. After the incubation, samples were centrifuged for 5 min at 100 \u00d7 g at 4\u00b0C, and the supernatants were removed. The remaining cells, which had been isolated from two fetuses, were resuspended in 200 \u03bcL William\u2019s medium with or without 25 \u03bcM BPA-GA and incubated at 37\u00b0C for 10, 30, 60, or 120 min. At each time point, cells and medium were pooled and extracts were prepared for HPLC as described above, by adding the same volume of methanol. All experiments were repeated five times. Values express the mean \u00b1 SE.Fetal liver and heart tissues, collected on GD18.5, were washed twice with cold phosphate-buffered saline (PBS). Tissues for two fetuses were combined in one tube and then minced in cold PBS and centrifuged for 5 min at 100 \u00d7 We examined UGT enzymatic activity using hepatic microsomes. We prepared the microsomes from rat liver and performed UGT enzyme analysis as described previously by First, we performed uterine perfusion  using BPNext, to examine the kinetics of BPA-GA in the maternal\u2013placental\u2013fetal unit, we performed perfusion for 20-min inflow with a lower concentration (2 \u03bcM) of BPA-GA and additional inflow without BPA-GA for 70 min , which deconjugates glucuronide conjugates, in both tissues , we did not detect 1-NA-GA or deconjugated 1-NA in the placenta or fetal tissues . These rOatp4a1 and Mrp1 in the placenta by quantitative RT-PCR. High expression levels of Oatp4a1 and Mrp1 in rat placenta have been also reported by Various transporters that either excrete drugs from the fetus to maternal blood or transfer nutrition and physiological compounds from the maternal body to the fetus have been isolated and characterized. Some of these transporters recognize xenobiotics because of their structural resemblance to physiological compounds. Generally, endocrine-disrupting chemicals, including BPA, often mimic endogenous chemical compounds such as estrogen. Moreover, the placenta produces steroid hormones that are used in both the mother and fetus . Therefo42\u2212 in the transport buffer, indicating that SO42\u2212 is not an inhibitor/substrate of the steroid sulfate transporters and that the carbon backbone of the steroid sulfates is a prerequisite for inhibition/transport. The selective transport between BPA-GA and 1-NA-GA, both glucuronide conjugates, could result from the difference in affinity not only for the conjugate residue but also for the parent residue. Further study is required to confirm the affinity of BPA-GA to these transporters. Placenta-specific expression of Oatp4a1 has already been reported . The possibility of transfer of placental deconjugated BPA could not be excluded completely in this experiment. Further work is required to examine the tissue-specific deconjugation of BPA-GA and the possibility of BPA transfer. At any rate, these data demonstrate that once BPA-GA is transferred into the fetus, it is deconjugated (reactivated) to BPA, and that this may be due to uptake of BPA-GA into fetal cells by transporters and subsequent catalysis by \u03b2-Gase.After co-incubation with BPA-GA, we detected deconjugated BPA in fetal liver and to a small degree in fetal heart . We obse tissues . InteresUGT2B1 mRNA in fetal organs was much lower than that in maternal liver. Moreover, in the UGT activity assay using liver microsomes on GD18.5, UGT activities in the fetal liver microsomes toward BPA were quite low. In this experiment, the ratio of fetal UGT activity toward BPA was about 20% of maternal microsomes . There are, however, some important differences between rodent and human placental structure. In rodents, maternal blood and fetal blood vessels are primarily separated by two syncytiotrophoblast layers, but in humans, only a syncytiotrophoblast monolayer separates fetal blood vessels from maternal blood . TherefoUGT2B15 was not expressed as well as other UGT isoforms examined. Both of these studies also suggest that human fetuses also have metabolizing systems vulnerable to BPA.In human liver microsomes, UGT2B15 is a major isoform that glucuronidates BPA . MoreoveUGT2B1 in the fetus. BPA may have been detected in the fetus after the perfusion due to high deconjugation ability of BPA-GA and low UGT activity toward BPA. Thus, we hypothesize that prenatal exposure to BPA may occur at levels sufficient to influence fetal development because of the exceptional drug-metabolizing system of the mother and fetus, which is particular to pregnancy. In 2003, 6 billion pounds of BPA were produced worldwide (Our study had three major findings. First, BPA-GA is transferred into the fetus through the placenta, even if only in small amounts. Second, BPA-GA is deconjugated to BPA in the fetus. Third, fetal liver microsomes have low ability to metabolize BPA to BPA-GA, as supported by the low expression of orldwide . Further"}
+{"text": "Bisphenol A (BPA) is one of the highest-volume chemicals produced worldwide, and human exposure to BPA is thought to be ubiquitous. Thus, there are concerns that the amount of BPA to which humans are exposed may cause adverse health effects. Importantly, results from a large number of biomonitoring studies are at odds with the results from two toxicokinetic studies.We examined several possibilities for why biomonitoring and toxicokinetic studies could come to seemingly conflicting conclusions.We examined > 80 published human biomonitoring studies that measured BPA concentrations in human tissues, urine, blood, and other fluids, along with two toxicokinetic studies of human BPA metabolism.The > 80 biomonitoring studies examined included measurements in thousands of individuals from several different countries, and these studies overwhelmingly detected BPA in individual adults, adolescents, and children. Unconjugated BPA was routinely detected in blood (in the nanograms per milliliter range), and conjugated BPA was routinely detected in the vast majority of urine samples . In stark contrast, toxicokinetic studies proposed that humans are not internally exposed to BPA. Some regulatory agencies have relied solely on these toxicokinetic models in their risk assessments.Available data from biomonitoring studies clearly indicate that the general population is exposed to BPA and is at risk from internal exposure to unconjugated BPA. The two toxicokinetic studies that suggested human BPA exposure is negligible have significant deficiencies, are directly contradicted by hypothesis-driven studies, and are therefore not reliable for risk assessment purposes. Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide, with > 8 billion pounds produced each year and > 100 tons released into the atmosphere by yearly production. Data from multiple sources indicate that the amount of BPA to which humans are exposed may cause adverse health effects; this has raised concerns among regulatory agencies all over the world.As an essential component of polycarbonate plastic, BPA is found in numerous consumer products, including baby bottles, reusable water bottles, reusable food containers, polyvinyl chloride stretch films, papers, and cardboards  is calculated as an acceptable daily human intake, typically 100-fold less than the no observed adverse effect level (NOAEL). However, the RfD for BPA (50 \u03bcg/kg/day) was calculated using the lowest observable adverse effect level (LOAEL) and 1,000-fold safety factors because a NOAEL had not been determined . More thThere is great concern about exposure of human fetuses, infants, and neonates to BPA because of the sensitivity of the developing organs and brain to exogenous hormones . HoweverIn this review, we examined > 80 biomonitoring studies that measured BPA concentrations in human tissues and fluids, specifically focusing on individuals that were exposed to BPA via their environment . These studies, examining thousands of individuals from several different countries, overwhelmingly detected BPA in individual adults, adolescents, and children. Interestingly, results from the large body of research encompassing biomonitoring studies are at odds with the results from two toxicokinetic studies that determined the disposition of BPA in humans after oral administration of BPA , 2005.In this review, we propose several hypotheses to explain why biomonitoring and toxicokinetic studies could come to seemingly conflicting conclusions. Additionally, the reliance of regulatory agencies on the two studies that predict no human exposure, in contrast to the > 80 studies that measure actual internal exposures, is contrary to scientific principles.Biomonitoring studies allow the determination of internal circulating levels and excreted concentrations of a chemical of interest, which account for exposures from all possible sources, rather than suspected exposures from specific sources. In the United States, the Centers for Disease Control and Prevention (CDC) is the major source for information on human exposures to a multitude of environmental contaminants and has developed sensitive assays to reliably measure them . In EuroProper biomonitoring studies take into account the kinetics, bioaccumulative properties, and metabolism of the target chemical to determine which tissue  should be examined. The chemical properties of the substance being examined also have an impact on the matrices that can be examined reliably, because some substances are altered by enzymes in blood and other substances can break down in urine . FurtherMultiple techniques have been used to measure total, unconjugated, and conjugated BPA in human blood, urine, and tissue samples. Gas chromatography (GC) and liquid chromatography (LC) are typically used with various detection methods, including mass spectrometry (MS), tandem MS (MS/MS), and electrochemical or fluorescence derivatization. The CDC has measured BPA using solid-phase extraction coupled with isotope dilution\u2013HPLC (high-performance liquid chromatography)\u2013MS/MS, which is considered the \u201cgold standard\u201d for urine biomonitoring studies because of its high level of accuracy, negligible interference, and ability to identify chemical structures . HoweverThe methods used in biomonitoring studies are crucial for the acceptance of the results . The ELIIn addition to the methods used to measure BPA in human tissues and fluids, the equipment and containers used to collect and store samples are critical for the accurate assessment of BPA concentrations. It has been suggested that the low levels of unconjugated BPA detected in bodily tissues and fluids were due to contamination from collection materials or nonenzymatic deconjugation of BPA during storage . OrganicIn terms of sample collection, studies should control for contamination from syringes used to draw samples, pipettes used to transfer plasma/serum to storage tubes, and the nature of sample storage tubes. If samples are obtained in hospital settings where the patient has an intravenous tube, it is essential to account for BPA leaching from these tubes during saline infusion. Studies need to be performed using appropriate matrix (blood or urine) spiked with known amounts of BPA and subjected to collection procedures similar to that of actual sampling to rule out BPA contribution from these various sources. One study used sheep blood to perform such validations and found very little BPA contamination from these sources . HoweverIn 2001, the CDC conducted the first biomonitoring study of BPA (unconjugated and conjugated) in pooled urine samples . The autBecause most studies of urine used enzymatic treatment with either glucuronidase or sulfatase, or both, many studies reported only the total BPA concentration, that is, unconjugated plus conjugated BPA. Of the seven studies that specifically examined unconjugated BPA, six detected unconjugated BPA in at least one sample ; only onIn contrast to the many small studies detecting BPA and/or its conjugates in urine, one study failed to detect either unconjugated or conjugated BPA in the 19 samples examined . To our The smaller studies described above provide little information about exposure of the general population. In 2005, the first study to measure total BPA concentrations in a reference population was performed by the CDC ; total BUrinary volume is influenced by various factors  that give rise to differing urinary dilutions and, consequently, to changes in the concentrations of excreted substances . Dilute Four additional studies examined fairly large populations of adults. In a study of 172 Korean adults, Levels of BPA exposure and concentrations in bodily fluids and tissues from individuals in less developed countries have been relatively understudied and were previously identified as an important research need . In a ren = 137). Finally, Biomonitoring measurements for adults are generally not informative about internal concentrations or exposure levels in children. The CDC study  examinedFour studies examined total BPA concentrations in urine collected from women during pregnancy, further suggesting exposure of the fetus to BPA during gestation . First, Other studies have estimated associations between BPA concentrations and activities that may increase BPA exposure. For example, Only a few studies have estimated total BPA exposure in the human population. Using exposure estimates from a variety of environmental sources  and from food and beverage contamination , several studies estimated daily human intake of < 1 \u03bcg/kg body weight (BW)/day , 2007. AUsing toxicokinetics, urinary BPA levels have been extrapolated to estimate daily intake levels. For instance, using urine samples collected from 48 women, Like urine, saliva is a preferred bodily fluid for biomonitoring purposes because collection requires relatively noninvasive procedures. To date, studies that have examined saliva for BPA have focused on the effects of dental sealant application to BPA concentrations. Since the 1960s, BPA diglycidyl methacrylate has been used as a component of many dental restorative materials, including those used for sealing molars.Six studies have measured BPA in saliva after dental sealant application, and all were able to detect BPA in the saliva of some of the individuals examined  Table 2Table 2. Seventeen studies have measured BPA in blood and serum samples from healthy male and nonpregnant female adults  Table 3Table 3. r2 = 0.92). Unconjugated BPA was detected in 83% of the samples, and 12% had > 2 ng/mL plasma. Patients undergoing dialysis were also examined in this study, and > 70% of patients had measurable concentrations of unconjugated BPA > 10 ng/mL plasma. In another recent study performed in China, In one of the larger studies of human blood, We are aware of only two studies that were unable to detect BPA in any individual samples of blood from adults . Fung etSeveral studies that examined BPA concentrations in blood or serum from relatively healthy adults also measured BPA in individuals with diseases or health-related conditions. Two studies found women with polycystic ovarian syndrome (PCOS) had higher serum levels of BPA than did healthy control women . These sBecause of concerns that BPA alters the development of rodents exposed during gestation, several human biomonitoring studies have focused on measuring BPA in serum from pregnant women, and in plasma, serum, and tissue from umbilical cords  Tables  and 5. OOf the four studies that measured unconjugated BPA in umbilical cord blood , two detWith the exception of one recent study that measured BPA concentrations in the blood of 300 women and their fetuses , the stuThe overall consensus that can be determined from blood sampling of healthy adults, adults with certain diseases, pregnant women, and fetuses is that internal exposures to unconjugated BPA are in the range of 0.5\u201310 ng/mL, with most studies suggesting an average internal exposure of approximately 1\u20133 ng/mL . These cOnly a few studies have examined additional tissues and fluids associated with pregnancy . In 2002in vitro fertilization (IVF) procedures. Second, With an increase in the number of successful pregnancies that have resulted from assisted reproductive technologies, two studies examining unconjugated BPA levels in follicular fluid raise additional concerns  Table 5Table 5. Kow (octanol\u2013water partitioning coefficient) = 2.2\u20133.4], allowing it to partition into fat and breast milk. Four small studies using analytical chemistry have measured BPA in the breast milk of healthy women   . In anotIn 2008, a large and well-controlled study of the possible health effects of BPA exposure on humans was conducted using samples and information collected for NHANES . By examAnother recent study examined BPA concentrations in urine and blood collected from 516 Korean adults living in urban areas . UrinarySeveral smaller studies have examined the effects of BPA exposure on other health outcomes. For instance, BPA levels in blood have been associated with a variety of conditions in women, including obesity, endometrial hyperplasia, endometriosis, recurrent miscarriages, sterility, and PCOS  were achieved (1.35 hr vs. 4 hr). In addition, the BPA glucuronide levels reported in blood are higher than the total BPA concentrations measured in the same individuals. Finally, the authors indicated that they measured BPA metabolism in three women, a group of three men, and then in a separate group of four men, yet the groups of male volunteers clearly overlap (at least 2 men were subjects in both groups), making the data compiled from combining these two groups questionable.Several inconsistencies in the article by in utero by \u03b2-glucuronidase, an enzyme that is present in high concentrations in placenta and various other tissues  modeling incorporates information on the physiology and anatomy of the experimental animal or human and the biochemistry of the chemical of interest into a conceptual model for computer simulation. Compared with the essentially empiric kinetic models , PBTK modeling has the advantage of being more grounded in physiology. PBTK models are powerful tools for interpolations and extrapolations that are particularly useful in the context of risk assessment, such as dose to dose, route to route, single to multiple exposure, continuous to discontinuous exposure, species to species, \u201cexternal\u201d or administered dose and internal or target tissue concentration, males to females, adults to children, nonpregnant women to pregnant, and so on .A few PBTK models have been developed for describing the toxicokinetics of BPA in rats , pregnanIn a model of oral route exposure to BPA in rats and humans, More recently, a PBTK model was developed to assess the age dependence of the toxicokinetics of both unconjugated BPA and BPA glucuronide in children < 2 years of age . This PBAnother PBTK model\u2014one for BPA in pregnant mice\u2014was developed by in situ; if so, this should be added to future models.Current PBTK models for BPA do not take into consideration the influence of local deconjugation of BPA metabolites at or in the vicinity of target tissues. Finally, a recent study determined that there is a linear relationship between applied doses and circulating unconjugated BPA concentrations . This liThroughout this review we have mentioned research needs; these are summarized below.in utero and neonatal periods) are the most sensitive to BPA; thus, special attention should be given to assessment of total and unconjugated BPA in these vulnerable populations.We suggest that several small biomonitoring studies performed previously should be repeated using large reference populations. First, it is imperative that estimates of urinary BPA levels over 24 hr be undertaken. Second, there is a need for obtaining multiple samples of blood and urine from individuals to assess variability of exposure over time, likely encompassing weeks, months, or years. Finally, large-scale biomonitoring studies across the life span are needed to confirm levels of total and unconjugated BPA in blood. These studies need to compare exposure levels in men and women, adults, neonates, children , pregnant and nonpregnant women, and obese and nonobese individuals, and across disease states. It is clear from animal studies that developmental periods  for assessment of BPA levels and the relationship of those levels and disease outcomes. Several recent studies are excellent examples of using this database to answer important questions relating to BPA exposure and linking BPA exposures to disease outcomes (In addition to these specific data needs, it is important to take advantage of the NHANES data and database (outcomes . The NHAWe believe that human biomonitoring data clearly indicate that the general population is exposed to BPA ubiquitously, including significant internal exposures to unconjugated BPA. More important, animal studies suggest that fetuses and children are particularly vulnerable to BPA exposures and, at the same time, are exposed to higher levels of unconjugated BPA.The two toxicokinetic studies performed to date , 2005, w"}
+{"text": "In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB\u2019s 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection  However, none of the identified antioxidants have proven to be effective for the treatment of any neurodegenerative disease in clinical settings Neurological disorders are estimated to affect as many as 1 billion people globally in vivo models of stroke, Parkinson\u2019s disease, and optic neuropathy 2O2 produced by glucose oxidase Methylene blue (MB), the very first lead chemical structure of phenothiazine and other derivatives, has been used for diagnostic procedures and the treatment of multiple disorders; including methemoglobinemia, malaria, and cyanide and carbon monoxide poisoning in vitro and in vivoMB has long been known as an electron carrier, which is best represented by MB\u2019s action to increase the rate of cytochrome c reduction in isolated mitochondria 2, 95% air). Medium were changed three times weekly and back cultured at confluence (every 3\u20135 days). Cells were observed with a phase-contrast microscope (Zeiss Invertoskop 40\u00b0C). HT-22 cells were used between passages 10\u201330.HT-22 cells, a murine hippocampal cell line derived from the mouse hippocampal HT-4 cell line, were the generous gift of Dr. David Schubert  2. After 12 hours, media was removed and replaced with a 1 \u00b5M solution of Calcein AM in PBS. Cells were incubated for 5 minutes at 37\u00b0C and fluorescence was measured using a Tecan Infinite F200 plate reader (excitation 485 emission 530). For the MTT assay, HT-22 cells were seeded into 96-well, flat-bottomed plates at a density of 3000 cells/well in 100 \u00b5l DMEM  and allowed to attach overnight. Varying concentrations of drug and 20 mM glutamate (or media for control wells) was then added to each well. Plates were incubated for 12 hours at 37\u00b0C with 5% CO2. Plates were removed from the incubator and 20 \u00b5l MTT (5 mg/ml in PBS) was added per well. The plates were agitated gently to mix the MTT into the media and then returned to the incubator for 2 hours. After 2 hours the media was removed and 100 \u00b5l of DMSO was added to each well. The plate was mixed by gentle agitation and the absorbance was measured (560 nm with a reference of 670 nm) with a Tecan Infinite F200 plate reader.Cell viability was determined by Calcein AM and MTT assays. For the Calcein AM assay, HT-22 cells were seeded at a density of 3,000 cells/well and were incubated overnight in 96-well plates in 100 \u00b5l of DMEM (high glucose with 1 mM pyruvate and 10% FBS). Varying concentrations of MB or its derivatives and 20 mM glutamate were added to each well and incubated for 12 hours at 37\u00b0C with 5% CO2. Viability was determined by Calcein AM assay.HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 \u00b5l DMEM  and allowed to attach overnight. Varying concentrations of MB or its derivatives and 5 \u00b5M rotenone (or media for control wells) was then added to each well. Plates were incubated for 24 hours at 37\u00b0C with 5% CO2. Viability was determined by Calcein AM assay.HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 \u00b5l DMEM  and allowed to attach overnight. Varying concentrations of MB or its derivatives and 2 U glucose oxidase (or media for control wells) was then added to each well. Plates were incubated for 3 hours at 37\u00b0C with 5% CO2. After 2 hours, all media was removed and replaced with fresh media containing drugs, but not IAA. The plates were incubated an additional 22 hours at 37\u00b0C with 5% CO2. Viability was determined by Calcein AM assay.HT-22 cells were seeded into 96-well flat-bottomed plates at a density of 3000 cells/well in 100 \u00b5l DMEM  and allowed to attach overnight. Varying concentrations of MB or its derivatives and 20 \u00b5M IAA (or media for control wells) was then added to each well. Plates were incubated for 2 hours at 37\u00b0C with 5% COMitochondrial membrane potential was analyzed by FRET using TMRE/NAO as described previously Mitochondrial membrane potential was also analyzed by flow cytometry and fluorescent microscopy using JC-1 dye. For fluorescent microscopy, HT-22 cells were plated at a density of 10,000 cells/well in a 6-well plate. Cells were incubated for 8 hours in glutamate and indicated drug. After 6 hours, media was replaced with KRH media containing 5 \u00b5g/ml JC-1 dye. Cells were incubated at 37\u00b0C for 15 minutes. After which time, they were washed once with KRH and incubated an additional 10 minutes in KRH at 37\u00b0C. The media was replaced with fresh KRH buffer and the cells imaged.2DCFDA (Anaspec) using a fluorescent microplate reader, flow cytometry, and fluorescent microscopy. For the microplate experiment, HT-22 cells were plated overnight at a density of 3,000 cells/well in a 96-well plate. Cells were incubated with drug and 20 mM glutamate for 12 hours at 37\u00b0C and 5% CO2. The media was then removed and the cells were washed once with PBS then incubated in PBS containing 10 \u00b5M H2DCFDA for 30 minutes at 37\u00b0C. The PBS was removed and cells were incubated for an additional 15 minutes at 37\u00b0C in KRH. Cells were washed twice in PBS and DCF fluorescence was measured using a Tecan Infinite F200 plate reader . Raw data are represented as RFU. The DCF fluorescence was then standardized based on control and Calcein AM cell viability. For fluorescent microscopy, HT-22 cells were plated at a density of 10,000 cells/well in a 6-well plate. Cells were incubated for 8 hours in glutamate and indicated drug. After 8 hours, media was replaced with KRH media containing 10 \u00b5M H2DCFDA. Cells were incubated at 37\u00b0C for 15 minutes, washed once with KRH and incubated an additional 10 minutes in fresh KRH at 37\u00b0C. The media was replaced with fresh KRH buffer and the cells imaged. For flow cytometry, HT-22 cells were seeded at a density of 50,000 cells/well in 6-well dishes (Greiner) and attached overnight. Media was removed and replaced with fresh DMEM  containing vehicle, 10 \u00b5M MB, 20 mM glutamate, or 10 \u00b5M MB and20 mM glutamate. Cells were incubated for 8 hours at 37\u00b0C and 5% CO2. Following the incubation, the media was removed, the cells were washed once with PBS, and incubated in PBS containing 10 \u00b5M H2DCFDA for 15 minutes at 37\u00b0C. The PBS was removed and cells were incubated for an additional 10 minutes at 37\u00b0C in PBS. The PBS was replaced with fresh PBS and the DCF fluorescence was determined with a Beckman Coulter FC-500.Changes in cellular ROS were measured by the ROS reactive fluorescent indicator H5 cells/plate. Cells were allowed to attach overnight. Media was removed and replaced with fresh media containing varying concentrations of drug and incubated at 37\u00b0C and 5% CO2 for 8 hours. Afterwards, the cells were trypsinized for 5 minutes, placed in 1.5 ml Eppendorf tubes, and centrifuged at 1200\u00d7g for 5 minutes at 4\u00b0C. The cells were centrifuged and washed with PBS twice more, and finally lysed with 100 \u00b5l lysis buffer  containing 1% Triton X-100. For protein assays, 40 \u00b5l cell lysate was set aside. The remaining cell lysate (60 \u00b5l), was combined in a 1\u22361 ratio with 2% 5-sufosalicylic acid and centrifuged for 5 minutes at 14,000\u00d7g. 10 \u00b5l of cell lysate was added in triplicate to a 96-well plate along with a GSH standard curve. Prior to reading the plate, 90 \u00b5l reaction buffer containing the following reagents; NADPH, GSH reductase, and diphenyl diselenide was added to each well. Absorbance (415 nm) was measured using a Tecan Infinite F200 plate reader. Protein concentration was measured simultaneously using the Pierce 660 nm Protein Assay (660 nm absorbance).Glutathione was measured using the Anaspec Total GSH Assay Kit (cat#: 72153). HT-22 cells were seeded into 10 cm plates at a density of 2.5\u00d7102, 2 mM KCN, 80 \u00b5M oxidized cytochrome c, and 4 \u00b5M NADH. Changes in absorbance at 550 nm were monitored with a spectrophotometer. The addition of 2 \u00b5g/ml rotenone was used to inhibit complex I activity. For complex II/III assay, mitochondria membrane fractions were added to 50 mM phosphate buffer (pH \u200a=\u200a7.4) containing 20 mM succinate, 500 \u00b5M EDTA, 2 mM KCN, 30 \u00b5M oxidized cytochrome c, and 2 \u00b5g/ml rotenone. Changes in absorbance at 550 nm were monitored with a spectrophotometer. The addition of 2 \u00b5g/ml antimycin was used to inhibit complex III activity.Rat hearts were harvested from 3-month old Sprague Dawley rats after euthanasia following the University of North Texas Health Science Center\u2019s IACUC approved protocol; specifically, rats were anesthetized with ketamine/xylazine and death induced by decapitation (guillotine). Rat hearts were flash frozen in liquid nitrogen and stored at \u221280\u00b0C until use. Hearts were thawed on iced and were then homogenized in 10 mM phosphate buffer (pH \u200a=\u200a7.4) containing 300 mM sucrose, and 2 mM EDTA. The resulting homogenate was centrifuged at 800\u00d7g and the supernatant collected. The supernatant was then centrifuged at 8,000\u00d7g and the resulting pellet containing the mitochondria fraction was re-suspended in 100 mM phosphate buffer (pH \u200a=\u200a7.4). Mitochondria were sonicated 3 times for 30 seconds on low power to break apart mitochondria membranes and expose the individual complexes of the electron transport chain. For complex I/III assay, mitochondria membrane fractions were added to 50 mM phosphate buffer (pH \u200a=\u200a7.4) containing 2 mM MgCl3 reference electrode. Measurements were performed under a blanket of nitrogen in acetonitrile with [Et4N][BF4] (0.1 M) as supporting electrolyte at a scan rate of 100 mV/s. Analyte concentration was kept constant at 10 mg of sample in 10 ml of solution. Ferrocene was used as an internal standard and reported relative to NHE (Fc/Fc+ \u200a=\u200a692 mV vs. NHE).Cyclic voltammograms were acquired at room temperature using a BASiC3 potentiostat equipped with a 3.0 mm glassy carbon working electrode, a platinum wire auxiliary electrode, and Ag/AgNOHT-22 cells were plated at a density of 5000/well in an XF24 plate. Cells attached overnight and the media was exchanged 1 hour prior to the assay for XF24 media. Rotenone (100 nM), carbonyl cyanide-p- FCCP (300 nM), and oligomycin (1 \u00b5g/ml) were diluted into XF24 media and loaded into the accompanying cartridge. Injections of the drugs into the medium occurred at the time points specified. Oxygen consumption was monitored using a Seahorse Bioscience XF24 Extracellular Flux Analyzer.2O2, 10 \u00b5M DCF and in the presence or absence of 165 \u00b5M NADH and mitochondria lysate (19.4 \u00b5g/ml). Assay took place in Greiner 96-well black plates for 30 minutes at 37\u00b0C, at which time the DCF fluorescence was measured with a Tecan Infinite F200 plate reader .Four compounds  were assayed in 10 mM phosphate buffer (pH\u200a=\u200a7.4) with 500 \u00b5M HHT-22 cells were plated at a density of 150000/well in a 6-well plate. Cells attached overnight and either MB or 2-chlorophenothiazine was added to the cells the following day at the indicated concentrations. Cells were grown for 3 days and lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors. Cell lysate was loaded onto a 10% polyacrylamide gel and transferred onto nitrocellulose. Nitrocellulose was incubated with primary antibody overnight at 4\u00b0C at the indicated concentrations . Secondary antibody linked to horseradish peroxidase (Jackson Immunoresearch) was incubated for 2 hours at room temperature (1\u22362000 dilution). Chemiluminescence was detected with a UVP Biospectrum 500.Complex IV activity was analyzed by an in-gel method as previously described All data were presented as mean +/\u2212 S.E.M. The significance of differences among groups with one independent variable was determined by one-way ANOVA with a Tukey\u2019s multiple-comparisons test for planned comparisons between groups when significance was detected. The significance of differences among groups where two independent variables presented were determined by two-way ANOVA with a Bonferronni Post test for planned comparisons between groups when significance was detected. For all tests, p<0.05 was considered significant.The protective action of MB was initially characterized in HT-22 cells using a glutamate neurotoxicity model. In HT-22 cells, glutamate blocks the glutamate/cysteine antiporter with saturating concentrations of extracellular glutamate, resulting in depletion of cellular glutathione 2DCFDA, which is converted into DCF by ROS. In the plate reader assay, glutamate caused a significant increase in DCF fluorescence, which was attenuated by MB with an EC50 of 20.37 nM  IAA is a toxic derivative of acetic acid that inhibits glyceraldehyde 3-phosphate dehydrogenase and disrupts disulfide bonds causing an increase in ROS and lipid peroxidation 2O2 insult, 2 U of glucose oxidase was added to the media of HT-22 cells. The HT-22 glucose oxidase model is defined by generation of extracellular H2O2 from glucose, where antioxidants, such as pyruvate, are highly effective To determine the effect of MB in mitigating extracellular HTo determine the structure-activity relationships of MB\u2019s neuroprotective action, we selected six commercially available compounds and compared their effects to MB in HT-22 cells. These compounds exhibited a wide range of potencies in HT-22 cells against glutamate insult . Three oWe determined the structure-activity relationships of MB and its derivatives on ROS production induced by glutamate in HT-22 cells. To measure intracellular ROS, we employed a 96-well assay format and measured DCF fluorescence in parallel with a cell viability assay after a 12-hour incubation of 20 mM glutamate. Glutamate caused a significant increase in ROS in HT-22 cells after 12 hours, which was significantly reduced by phenothiazine and 2-chlorophenothiazine with EC50s of 57.21 and 84.14 nM, respectively , Table 1We further determined the structure-activity relationships of MB and its derivatives on glutamate induced mitochondria membrane potential depolarization in HT-22 cells. As indicated by the NAO/TMRE FRET assay, a 12-hour incubation with 20 mM glutamate caused mitochondria membrane potential depolarization which was mitigated by TB, MB, phenothiazine, and 2-chlorophenothiazine with the EC50s of 1.54, 17.72, 21.18, and 45.09 nM respectively. Consistent with the cell viability and ROS production assays, promethazine, chlorpromazine, and NR had much lower potencies as compared to TB, MB, phenothiazine, and 2-chlorophenothiazine , Table 1Our further analysis indicated significant correlation between the EC50s for the effect of MB and its derivatives on ROS production and their neuroprotective action . SimilarMB and its derivatives were screened at concentrations between 10 nM to 10 \u00b5M in the rotenone, IAA, and glucose oxidase neurotoxicity models in HT-22 cells. For the purpose of conciseness, the concentration displayed for each compound in the rotenone, IAA, and glucose oxidase assays is based on the EC50 value calculated from the glutamate assay. All the tested compounds except for chlorpromazine were protective in the rotenone model of cellular toxicity . For theWe determined the effect of MB and its derivatives on the activity of mitochondria complexes I-III  and complexes II-III . Both assays rely on measuring the rate of cytochrome c reduction in terms of changes in cytochrome c absorbance, which is typically reduced by complex III. In the complex I-III assay, with NADH as the electron donor, MB and TB significantly increased the rate of cytochrome c reduction, while no effect was found in the other tested compounds . To veriWe determined the effect of MB and its derivatives on cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse XF24 Flux Analyzer. OCR and ECAR were measured under five conditions. The first set of measurements established a baseline for 35 minutes followed by injection of media containing MB or each of its derivatives. Upon injection, NR, MB and TB increased OCR, while 2-chlorophenothiazine and chlorpromazine had no effect on the OCR . Cells rChanges in cellular oxygen consumption are often mirrored by opposing changes in lactate production We determined the redox potential of MB and its derivatives and standardized them to a normal hydrogen electrode (NHE). Our analysis indicated that MB, NR and TB have distinct redox potential from the other derivatives . InteresWe compared the effect of MB and 2-chlorophenothiazine on the expression of mitochondria complex IV subunit I (Cox1). Previous studies have reported that MB increases the activity of complex IV as well as the expression of subunit II of complex IV, coded by mitochondrial DNA 2O2 and 10 \u00b5M H2DCFDA in phosphate buffer with the addition of each MB related compound ranging in concentration between 10 nM and 10 \u00b5M. Each compound was tested in both the presence and absence of 165 \u00b5M NADH and fractionated heart mitochondria for 30 minutes. NADH served as an electron donor and was necessary for MB to reduce DCF fluorescence. MB reduced DCF fluorescence at concentrations of 100 nM, 1 \u00b5M, and 10 \u00b5M in the presence of mitochondria and NADH. However, without NADH and mitochondria present, MB increased DCF fluorescence at 100 nM, 1 \u00b5M and 10 \u00b5M . These compounds were highly efficacious and potent in the IAA, glutamate, and rotenone neurotoxicity assays, but had no effect on anaerobic glycolysis, cellular oxygen consumption or the complex I-III shunt. The second group of compounds are those with amine side chains attached to the 3, 7 carbons of the phenothiazine nucleus (MB and TB). Both MB and TB had high potencies and efficacies in the neurotoxicity assays, coupled with their ability to enhance cellular oxygen consumption and decrease anaerobic glycolysis. Both compounds were also unique in their ability to act as an intermediate between complex I and cytochrome c. The third group was made up of the compounds with a side chain attached to the 10 nitrogen of phenothiazine (chlorpromazine and promethazine). Promethazine and chlorpromazine were less potent in the glutamate, IAA, and rotenone neurotoxicity assays. In addition, promethazine and chlorpromazine are less efficacious in the IAA assay as compared to the two previous groups with the exposed nitrogen motif in the phenothiazine nucleus (MB and phenothiazine). Besides being less potent, neither promethazine nor chlorpromazine had any effect on cellular oxygen consumption, anaerobic glycolysis, or the complex I-III shunt. The fourth group contained only one compound, NR. NR has a substitution of a nitrogen in place of the 5 sulfur yielding a phenazine nucleus with side chains on the 3, 7 carbons. NR had decreased neuroprotective potency relative to MB in the glutamate, rotenone and IAA assays as well as a decreased efficacy in the IAA assay. However, NR was capable of enhancing cellular oxygen consumption, but did not aid in electron transfer between mitochondria complexes I and III.in vivo in a C elegans model of Parkinson\u2019s disease, with phenothiazine again being highly efficacious The addition of a side chain to the 10 nitrogen caused a significant loss of potency and efficacy as demonstrated by the differences between phenothiazine and chlorpromazine in the glutamate, IAA, and rotenone assays. Chlorpromazine and promethazine have previously been reported to have minor protective actions with micromolar potency, which corresponds to our results from the neurotoxicity assays The position 5 sulfur is as equally important as the availability of the free 10 nitrogen motif evidenced by the differences between TB and NR in the glutamate, IAA, and rotenone assays. The substitution of a nitrogen in place of the sulfur in the heterocyclic nucleus of the molecule (phenothiazine backbone replaced with phenazine backbone) significantly decreased both the potency and efficacy of NR as compared with MB.+MB\u2019s neuroprotective effects have been demonstrated in models of Alzheimer\u2019s disease, Parkinson\u2019s disease, stroke, optic neuropathy, and hypoxia The distinct neuroprotective action of MB was suggested by our mitochondrial lysate oxidation assay, where MB requires mitochondria and NADH to reduce oxidative stress. We predict that MB accepts electron(s) from NADH via mitochondria complex I and is reduced to leuco-MB, which can act as a direct free radical scavenger and recycle back to the oxidized form of MB. This unique action of MB makes it a mitochondria specific regenerative anti-oxidant. On the other hand, phenothiazine and 2-chlorophenothiazine can function as direct free radical scavengers independent of the presence of mitochondria and NADH. In addition, the enhancement of complex IV expression and activity associated with MB was not observed with 2-chlorophenothiazine indicating a distinct mechanism between these two compounds. With the addition of a side chain to the 10-nitrogen, chlorpromazine enhanced the oxidative reaction independent of the presence of mitochondria and NADH explaining its low neuroprotective potency.2MB has previously been shown to directly accept electrons from NADH, NADPH, and FADHIn conclusion, our structure-activity relationship study of MB has demonstrated the distinct anti-oxidant properties of MB. MB acts on superoxide generated due to the blockage of the mitochondria electron transport chain by providing an alternative mitochondrial electron transfer carrier to bypass complexes I-III. In addition, reduced leuco-MB can directly scavenge superoxide and recycle back to the oxidized form MB . As a moFigure S1Effect of MB and its derivatives on rotenone neurotoxicity in HT-22 cells. Calcein AM cell viability assay after 24 hour exposure of 5 \u00b5M rotenone with co-treatment of (A) MB, (B) 2-chlorophenothiazine, (C) NR, or (D) chlorpromazine. * p<0.05 compared to 5 \u00b5M rotenone in media.(TIF)Click here for additional data file.Figure S2Effect of MB and its derivatives on IAA neurotoxicity in HT-22 cells. (A) Calcein AM cell viability assay after 24 hour exposure of 20 \u00b5M IAA with co-treatment of (A) MB, (B) 2-chlorophenothiazine (C) NR, or (D) chlorpromazine. * p<0.05 compared to 20 \u00b5M IAA in media.(TIF)Click here for additional data file.Figure S3No protective action of MB on direct oxidative insult induced by 3 hours exposure of 2 U glucose oxidase. MB enhances direct oxidative insult induced cell death at 1 and 10 \u00b5M. Pyruvate significantly attenuates the direct oxidative damage acting as an ROS scavenger. * p<0.05 compared to 2 U glucose oxidase in media.(TIF)Click here for additional data file.Figure S4Effects of phenothiazine and promethazine on OCR and ECAR. (A) OCR and (B) ECAR recording at baseline and cumulative treatment of each drug , oligomycin, FCCP, and rotenone. Promethazine and phenothiazine had no effect on OCR and ECAR. (C) OCR and (D) ECAR recordings at baseline and cumulative treatment of MB, oligomycin, FCCP, and rotenone. Wells containing media only were used as blank controls. MB dramatically enhances OCR and inhibits ECAR, but exhibited no effect on OCR and ECAR in blank controls.(TIF)Click here for additional data file."}
+{"text": "The aim of this study was to estimate the prevalence of selected atherothrombotic risk factors and several clinical manifestations of atherothrombosis, as well as the utilization rates of selected vascular interventions in Greece.During December 2009, 3,007 adults  recruited in a random-digit dialed telephone survey (response rate: 16%). The sample size was selected following a multistage and stratified by gender, age group, and Greek region procedure in order to be more representative. Data regarding medical history and socio-demographic characteristics of the participants were collected.Overall, 6.5%, 17.7% and 14.0% of participants reported that they had been diagnosed with diabetes mellitus, hypertension and hypercholesterolemia, respectively. In the overall sample, 2.5% of participants reported that they had been diagnosed with angina, 2.0% with myocardial infarction, 1.6% with stroke and 2.5% with peripheral artery disease. Overall, 1.5% of participants reported that they had undergone percutaneous coronary intervention, 1.4% coronary artery bypass grafting, 0.6% angioplasty of a peripheral vessel, and 0.7% surgery of a peripheral vessel.Despite the limitations may occur due to the sampling procedure, the findings of the present study indicate that atherothrombosis affects a large portion of the population in Greece and it is expected to impose a significant economic burden. The data of the current study could contribute in obtaining an accurate estimation of the economic burden of atherothrombosis in Greece because people who are aware of their condition/disease are those who use health care resources. Atherothrombosis is defined as an unpredictable, sudden disruption of an atherosclerotic plaque, which leads to platelet activation and thrombus formation. Atherothrombosis is the underlying condition which causes coronary artery disease (CAD), including myocardial infarction (MI) and angina pectoris (stable or unstable), stroke, and peripheral artery disease (PAD). CAD andIn Greece, an accurate estimation of the total economic cost of atherothrombosis has never been provided due to lack of data. This highlights the need to collect data in order to obtain an accurate estimation of the total economic burden of atherothrombotic disease. The data required for this purpose is the prevalence of several clinical manifestations of the disease and several atherothrombotic risk factors as well as the utilization rate and cost of various health services related to the atherothrombosis management.Age, smoking, hypertension, diabetes mellitus and hypercholesterolemia are major atherosclerotic risk factors. As concerns the prevalence of the last three risk factors, few data are available in Greece -11. HoweIn Greece, there are only few data regarding the awareness and treatment of the aforementioned risk factors. Moreover, there are limited data regarding the prevalence of the clinical manifestations of atherothrombosis -15, whilDuring December 2009, 89,526 phone numbers were randomly selected in order to recruit men and women from all Greek regions to participate in a random-digit dialled (RDD) telephone nationwide survey. From the study design, it had been decided that the sample would be stratified by gender, age group and Greek region  in order to be more representative. Moreover, it had been decided that 0.03% of the total population of each Greek region, with only one person per household, would be interviewed in order to obtain 3,000 completed questionnaires , \"overweight\" (25 < BMI < 29.9 kg/m2) and \"obese\" (BMI > 29.9 kg/m2). Participants' smoking habits were also recorded .A structured questionnaire  that was developed specifically for the purposes of the current study was used in order to retrieve various data from participants. In particular, participants were asked to report their age , marital status , educational status (years of education), nationality , region of residence , and occupational status . In addition, participants were asked to report their weight (kg) and height (m) so that their body mass index (BMI) could be determined. Then, participants were categorised as \"normal-weight\" , hypercholesterolemia (defined as diagnosed by doctor or use of lipid lowering agents) and diabetes mellitus (defined as diagnosed by doctor or use of hypoglycemic treatment), as well as the time of the first diagnosis, were recorded. Any history of angina, MI, stroke, or PAD diagnosed by a doctor, as well as the time of first diagnosis were also recorded. Finally, participants were asked to report whether and when they had undergone any of the following interventions: PCI, CABG, angioplasty of a peripheral vessel or surgery of a peripheral vessel.Categorical variables are presented as absolute (n) and relative frequencies (%). The chi-square test without the correction of continuity was used in order to evaluate the associations between categorical variables. The prevalence of one, two, and three or more atherothrombotic risk factors was determined for the overall study population and men and women, separately. Then, the multiple multinomial logistic regression analysis was conducted in order to evaluate the association of several socio-demographic factors (independent variables) with the probability of having 0, 1 or \u22652 atherothrombotic risk factors (dependent variables). The results are presented as odds ratios (OR) and 95% confidence interval (CI).A probability value of 5% was considered as statistically significant. All statistical calculations were performed using SPSS version 17.0 software .The socio-demographic characteristics of the participants are presented in Table The prevalence of the clinical manifestations of atherothrombosis in relation to participants' socio-demographic characteristics is presented in Table The prevalence of several atherothrombosis-related interventions in relation to participants' socio-demographic characteristics is presented in Table The distribution of selected traditional CVD risk factors in relation to participants' socio-demographic characteristics is presented in Table Considering clusters of smoking, diabetes mellitus, hypertension and hypercholesterolemia, it was found that 48% of participants had none of these risk factors, 40% had one, 9.5% had two and 2.5% had three or more risk factors. Including obesity in the aforementioned risk factors, 41.4% of participants were found to have none, 40.7% to have one, 13.4% to have two and 4.7% to have three or more risk factors. A statistically significantly difference was detected in the distribution of number of atherothrombotic risk factors between genders. In particular, women were more likely to have three risk factors or more (6%) compared to men .Then, multinomial logistic regression analysis was carried out to evaluate the association of selected socio-demographic risk factors with the probability of having one or two and more risk factors among the five risk factors . This analysis revealed that participants' age, educational status and gender were significantly associated with the probability of having one risk factor versus none, while only age and educational status were associated with the probability of having two or more risk factors versus none Table .The present study provided data on the self-reported prevalence of CVD risk factors, main clinical manifestations of atherothrombosis and utilization of vascular interventions at a nationwide sample of 3,007 adults from Greece, in 2009. Although a considerable number of previous studies have presented similar data, they have been conducted earlier than 2005 ,10,16-18Although the results of the present study are not comparable with those reported in the majority of the previous studies, their findings as well as their sample size, age of population, period of study, etc are presented in Table The differences observed between the findings of our study and those of the previous large-scale epidemiological studies could be attributed to the fact that in the latter, clinical examination has been used to identify subjects suffering from several conditions or diseases. Moreover, comparison of the present results with those of epidemiological studies indicates that the present study seems to underestimate the actual prevalence of the aforementioned risk factors in Greece. However, the current data provide an accurate estimation of the prevalence of people who are aware or/and treated of their condition, since data of epidemiological studies indicate that less than half of sufferers tend to be aware of their condition. For instance, although the prevalence of hypercholesterolemia was estimated to be 37.7% in the ATTICA study, less than 50% of them were aware of their conditions corresponding to about 17% of the total sample [As concerns the prevalence of the several clinical manifestations of atherothrombosis, previous data for Greece are limited. In particular, only one study was found to provide data regarding the prevalence of MI . The resFinally, as concerns the prevalence of the use of several atherothrombosis-related interventions, only limited data are available for Greece. For example, it is known that almost 19,000 PCIs/year are performed, that is 0.2% of the total adult population in Greece undergo a PCI each year [Despite the relatively large size of the sample, the results of the present study may be affected by several methodological factors related to the study design. Above all, in conducting a survey by telephone instead of face-to-face at the homes of respondents leads to automatically exclusion of individuals without telephone service ,21. MoreApart from the low response rate, telephone surveys may suffer from poor response quality compared to face-to-face surveys . First oIn the present study, the prevalence of atherothrombotic disease, as well as the prevalence of selected traditional atherothrombotic risk factors, was estimated in Greece. These data indicate that atherothrombosis is a prevalent disease in Greece. Therefore, specific programmes aiming to primary prevention of atherothrombosis should be developed and implemented in Greece. Moreover, the data of the current study could contribute in obtaining an accurate estimation of the economic burden of atherothrombosis in Greece because people who are aware of their condition/disease are those who use health care resources. Therefore, this portion is useful to estimate the total direct health care cost related to atherothrombosis at a national level.The authors declare that they have no competing interests.All authors contributed in writing of the manuscript and interpretation of the results, reviewed its content and approved the final version submitted for publication. Furthermore, NM designed the study and supervised data collection; GK contributed to data management and carried out the statistical analysis; VF contributed to data management and preparation of database.The study was sponsored by a grant from Sanofi-Aventis. The study sponsor had no role in the design and conduct of the study; the collection, management, analysis and interpretation of the data; the preparation, approval and submission of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/11/16/prepubQuestionnaire. The file contains the questionnaire used to collect data.Click here for file"}
+{"text": "Platanus are widely planted in the Shanghai urban area. A great deal of allergenic Platanus pollen is released from the trees and suspended in the atmosphere during its flowering season, ultimately causing allergic respiratory diseases. Few papers have focused on the distribution of this type of pollen and its expression of allergenic proteins. In order to investigate any differences in protein expression in Platanus pollen following exposure to gaseous and particulate pollutants, a special apparatus was designed. Exposure condition  of Platanus pollen and gaseous pollutants can be simulated using of this apparatus. Fresh Platanus orientalis pollen, pollutant gases , and typical urban ambient particles  were mixed in this device to examine possible changes that might occur in ambient airborne urban pollen following exposure to such pollutants. Our results showed that the fresh P. orientalis pollen became swollen, and new kinds of particles could be found on the surface of the pollen grains after exposure to the pollutants. The results of SDS-PAGE showed that five protein bands with molecular weights of 17\u201319, 34, 61, 82, and 144\u00a0kDa, respectively, were detected and gray scale of these brands increased after the pollen exposure to gaseous pollutants. The two-dimensional gel electrophoresis analysis demonstrated that a Platanus pollen allergenic protein  increased in abundance following exposure to pollutant gases and VEPs, implying that air pollutants may exacerbate the allergenicity of pollen.Being major ornamental street trees, species of  Okuyama et al. . After branches with\u00a0inflorescences were cut off the tree, the inflorescences were air-dried at room temperature and kept from any pollutants off. Pollen grains were collected from the dried inflorescences. The collected pollen was kept at \u22124\u00a0\u00b0C until subsequent use.Fresh Platanus pollen induced by air pollutants, an apparatus was designed to simulate the exposure of pollen to ambient urban gaseous air pollutants, including SO2, NO2, and NH3  as well as vehicle exhaust particles . The exposure devices included an airflow meter, a humidification device, a mixer for gases, a temperature and humidity sensor, a pollen container, an exposure chamber, an electronic fan, a sampler inlet head with filter, and a low capacity pump  took place. The particle capture device was used to capture the exposure pollens with a polycarbonate membrane filter.In order to investigate allergenic protein changes in ump Fig.\u00a0. The exp2 (0.1\u00a0L/min), NO2 (0.54\u00a0L/min), NH3 (0.03\u00a0L/min) were transferred into the chamber. The mole ratio of SO2, NO2, and NH3 was kept 30:200:30 in the chamber   with the following parameters: voltage, 30\u00a0kV; resolution, 3\u00a0nm; and scanning distance, 15.0\u00a0mm. The chemical composition on the pollen was investigated by X-ray energy dispersive spectroscopy (EDX) .The EDX spectrometer was Link ISIS spectrometer with a Si(Li) detector, which allows X-ray detection from elements higher than carbonate (Platanus pollen was defatted with acetone (3.44\u00a0mol/L), dried, and extracted in 40\u00a0ml of 0.01\u00a0M phosphate and 0.15\u00a0M NaCl phosphate-buffered saline ; the mixture (1:20 wt/vol) was stirred for 12\u00a0h at 4\u00a0\u00b0C. The suspension was filtered through a cellulose filter paper , dialyzed against PBS, and sterilized by 0.22\u00a0\u03bcm filtration. The total protein content of the pollen extracts was determined by the Bradford protein assay using bovine serum albumin (BSA) as the standard. Extracted proteins were separated by 10\u00a0% SDS-polyacrylamide gel electrophoresis (90\u00a0\u03bcl of each pollen extract per well) and visualized by Coomassie blue staining.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for protein analysis. A modified version of the pollen protein extraction protocol described by Varela et al.  was usedThe two-dimensional gel electrophoresis (2-DE) protocol was as described by Sheoran et al. . An equaPlatanus protein identification by mass spectrometry (MS) was carried out using the protocol described by Sheoran et al.  plus mass spectrometer. Data were acquired in a positive MS reflector using a CalMix5 standard to calibrate the instrument. Both the MS and MS/MS data were integrated and processed with the use of the GPS Explorer v3.6 software with default parameters. Employing the combined MS and MS/MS spectra, proteins were successfully identified based on 95\u00a0% or higher confidence interval of their scores in the MASCOT v2.1 search engine .n et al. . BrieflyP. orientalis pollen grains were suboblate, with a diameter 20\u201330\u00a0\u03bcm, and with trenches on its surface. The surface of fresh pollen grains became swollen after exposure to the mixture of gases. Further, EDX spectral analysis demonstrated that C, N, and O were the main elements on the surface of pollen before exposure to the air pollutants . After pollen exposure to gaseous pollutants for 1, and 8\u00a0h, the main protein bands became dark, suggesting more protein was expressed by the pollen grains. In this study, a new protein band was found after the exposure of pollen to gaseous pollutants and VEPs for 8\u00a0h. The presence of the new protein band, with an approximate molecular weight of 26.5\u00a0kDa, implies that VEPs stimulate fresh Platanus pollen to express new proteins.The results of SDS-PAGE analysis showed that protein bands with molecular weights of 17\u201319, 34, 61, 82, and 144\u00a0kDa could be found in samples of fresh pure P. orientalis pollen to air pollutants. Grayscale values , size (M), and hydrophobicity, proteins can separated from one another with this technique. Figure\u00a0Two protein spots (p1007 and p8000) in the 2-DE gel were selected to identify their protein constitution using of mass spectrometry Fig.\u00a0.Fig.\u00a05TZea mays]); and gi|116782336 (unknown [Picea sitchensis]). None of these proteins are a documented allergenic protein. Only one protein was found in protein spot p8000: gi|29839547 . The amino acid sequence of the Pla a 1 is: MKLSFSLCIF FFNLLLLLQA VISADIVQGT CKKVAQRSPN VNYDFCVKSL GADPKSHTAD LQGLGVISAN LAIQHGSKIQ TFIGRILKSK VDPALKKYLN 101DCVGLYADAK SSVQEAIADF KSKDYASANV KMSAALDDSV TCEDGFKEKK GIVSPVTKEN KDYVQLTAIS LAITKLLGA. This amino acid sequence further demonstrated the identity of the allergenic protein expressed by the Platanus pollen.According to Mascot Score Histogram, four proteins were found in spot p1007 Table\u00a0: gi|1222Platanus trees are widely grown as street and shade trees around cities in the world. This kind of tree includes Platanus occidentalis, Platanus orientalis, and Platanus acerifolia. Our field survey results showed that most of Platanus tree is widely planted in parks and avenues in Shanghai is P. orientalis. And this fielding survey results agreed with our microscopic characterization results, which the Platanus pollen collected in Shanghai was the same as P. orientalis pollen provided by GREER  (data not shown).Platanus pollen is a major contributor to pollinosis symptoms during March and April. Extensive studies on P. acerifolia allergy have been reported, however, there are few reports on allergenic protein released by P. orientalis trees  have been tested to contribute to the symptoms of patients with pollinosis. Platanus pollen in Shanghai urban atmosphere is obviously greater abundance compared with that in the suburban and rural area during its flowering season (data not shown). Importantly, main air pollutants in urban atmosphere , could be absorbed on the surface of Platanus pollen   and two major allergens (Pla a 1 and Pla a 2), with molecular weights of ~18 and ~44\u00a0kDa, respectively  pollen allergy  pollen, and suggested that the 17\u00a0kDa band was the allergenic protein. Asturias et al. (Platanus (acerifolia) extracts, but only two of them, Pla a 1(18\u00a0kDa) and a 43 kDa were allergenic protein. While in China, several studies focused on the identification of proteins from Platanus pollen, for example, Li et al.  and claimed that those 22\u201371\u00a0kDa in size were the major allergenic proteins based on Western blot analysis. It must be pointed out that Platanus pollen was defatted with acetone (3.44\u00a0mol/L) in the procedure of protein extracts in this study, and this chemical regent might affect the quantity and quality of allergenic content which accumulated in the intine  and mass spectrometry were employed. The 2-DE result clearly demonstrated grayscale values of 14 protein spots . A new protein band, with an approximate molecular weight of 26.5\u00a0kDa, also was found after Platanus pollen exposure to VEPs in this study. Therefore, our results might demonstrate that air pollutants could affect allergen liberation from Platanus pollen. Further experiments dissecting this phenomenon will be needed.Behrendt et al.  reportedPlatanus pollen, Pla a1 (18\u00a0kDa protein) was responsible for 79\u00a0% of the Ig-E binding capacity and could be as a reliable diagnosis of Platanus (acerifolia) pollen in the allergenic reaction  after P.orientalis pollen exposure to the pollutant gases and particles, the pollen became swollen, and new kinds of particles could be found on the surface of the grains; (2) the results of two-dimensional gel electrophoresis and mass spectrometry analysis demonstrated that the expression of an allergenic protein (Pl a a1) of P.orientalis pollen is increased after exposure to pollutant gases and VEPs, implying that air pollutants can exacerbate the allergenicity of pollen of this genus.A special apparatus was designed to investigate any differences in protein expression in"}
+{"text": "Introduction: Quantitative electroencephalography (QEEG) brain frequency and network analyses are known to differentiate between disease stages in Parkinson's disease (PD) and are possible biomarkers. They correlate with cognitive decline. Little is known about changes in brain networks in relation to apathy.Objective/Aims: To analyze changes in brain network connectivities related to apathy.Methods: 40 PD patients  were included. All patients had extensive neuropsychological testing; apathy was evaluated using the apathy evaluation score . Resting state EEG was recorded with 256 electrodes and analyzed using fully automated Matlab\u00ae code (TAPEEG). For estimation of the connectivities between brain regions, PLI (phase lag index) was used, enhanced by a microstates segmentation.Results: After correction for multiple comparisons, significant correlations were found for single alpha2-band connectivities with the AES . Lower connectivities, mainly involving the left fronto-polar region, were related to higher apathy scores.Conclusions: In our sample of patients with PD, apathy correlates with a network alteration mainly involving the left fronto-polar region. This might be due to dysfunction of the cortico-basal loop, modulating motivation. Apathy is beside depression one of the most common non-motor, neuropsychiatric symptoms in patients with Parkinson's disease (PD) , including frequency, connectivity and brain network analysis, are correlating to the disease stage and the cognitive decline, related to PD dementia  was used, a connectivity measure little influenced by effects of volume conduction  has been analyzed. All patients were on dopaminergic medication and were tested while they were in the ON state.Fifty-eight patients with PD were recruited between October 2011 and April 2013 from the movement disorders clinic of Hospital of the University of Basel or through advertisements. The patients were participants of a Cognitive training-study  . Subjects were instructed to relax, but to stay awake and to minimize eye and body movements. A continuous EEG with closed eyes was recorded for 12 min. During data acquisition, a subset of electrodes was monitored online by a technician to check for vigilance and artifacts.All EEG data were automatically preprocessed using TAPEEG, as described in a previous publication  therefore, this variable was square root transformed, leading to a non-significant p-value (p = 0.34) of the KSLT. After z-score transformation, the executive function variables were averaged; variables indicating good performance in smaller values were reversed  were used . Lower connectivities were related to higher apathy scores .FH helped conducting the study, processed the EEG data and drafted the manuscript. AM and ZR helped conducting the study. PF and UG conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.UG: Support of research from Mach-Gaensslen-Foundation, Gossweiler Foundation, Parkinson Schweiz, Synapsis Foundation, Botnar Foundation, PF: Support of research from Swiss National Science Foundation, Mach-Gaensslen-Foundation, Gossweiler Foundation, Parkinson Schweiz, Synapsis Foundation, Botnar Foundation, Freiwillige Akademische Gesellschaft Basel, Novartis Research Foundation, Novartis, Roche, AbbVie, Hedwig-Widmer-Stiftung. The other 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": "Several clinical studies have reported the application of dipeptidyl peptidase-4 (DPP-4) inhibitors as treatments for type 1 diabetes mellitus (T1DM). This study aims to review the outcomes of these existing studies and to discuss the therapeutic effects of DPP-4 inhibitors on T1DM.  We thoroughly searched the Medline, Embase, PubMed, and Cochrane Library databases and ClinicalTrials.gov for studies concerning the use of DPP-4 inhibitors in patients with T1DM.  In preclinical trials, DPP-4 inhibitors improved the pathogenesis of T1DM. However, only a portion of the studies showed potential efficacy regarding clinical glycemic control and other clinical parameters. From this meta-analysis, pooled data from 5 randomized controlled trials revealed that the additional use of DPP-4 inhibitors resulted in a greater decrease in glycated hemoglobin A1c (HbA1c) levels ) than insulin monotherapy, although the decrease was not significant. A small decrease in postprandial glucose or insulin consumption was confirmed.  Although DPP-4 inhibitors may be beneficial for T1DM, existing studies do not strongly support these positive effects in clinical practice. Further optimized clinical trials are needed. The proportion of diabetes mellitus patients diagnosed with T1DM is estimated to be 5%\u201310% [Type 1 diabetes mellitus (T1DM) is considered a chronic immune-mediated disease that is characterized by the selective destruction of e 5%\u201310% \u20134, but te 5%\u201310% . New thee 5%\u201310% . Only a e 5%\u201310% .\u03b2-cells in a glucose-dependent manner, suppresses glucagon secretion from \u03b1-cells, and inhibits hepatic glucose production, eventually contributing to the antihyperglycemic effect. In addition, DPP-4 inhibitors preserve the \u03b2-cell mass [Dipeptidyl peptidase-4 (DPP-4) inhibitors, which have been widely used as outstanding blood glucose-dependent antidiabetic agents for patients with type 2 diabetes mellitus (T2DM), show promise. These inhibitors prevent the degradation of incretin (glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)) by dipeptidyl peptidase-4 enzymes and therefore elevate endogenous GLP-1 levels. GLP-1 stimulates insulin secretion from ell mass .+CD25+FoxP3+cells, which were reduced in patients with T1DM [Recently, an increasing number of studies on the topic of DPP-4 inhibitors and T1DM have discovered their mutual characteristics of immune destruction. Insulitis was alleviated in a T1DM animal model treated with DPP-4 inhibitors , and theith T1DM , increasith T1DM . Glucagoith T1DM \u201315 in seOur research was a study-level systematic review and meta-analysis of clinical trials. Therefore, ethical approval was not necessary for this study.http://www.ncbi.nlm.nih.gov/pubmed/) and the Cochrane Library (http://www.cochranelibrary.com/) was  AND (\u201ctype 1 diabetes\u201d OR t1dm). Publications were searched in Medline and Embase using the same strategy on one website (http://www.embase.com/). Additionally, all references of the articles downloaded from these databases were rapidly checked, and we manually searched 360 relevant articles. We did not employ any restrictions on publication year, specimen, or language.This systematic review was performed according to PRISMA guidelines . Two invWe used the following selection criteria to ensure the inclusion of all relevant studies: (1) controlled clinical studies on the topic of DPP-4 inhibitors; (2) studies in which subjects were restricted to a diagnosis of type 1 diabetes, including patients with latent autoimmune diabetes in adults (LADA); and (3) studies reporting changes in control and experimental groups after treatment with respect to at least one indicator .The exclusion criteria were as follows: (1) studies that did not meet any of the above inclusion criteria; (2) comments, reviews, abstracts, and articles other than original papers; and (3) duplicated data from the same laboratory.Two investigators (QX Wang and M Long) independently searched the titles and abstracts as well as the full text, if necessary. All ambiguities were clarified by discussion with a third investigator (HT Zheng).\u03b2-cell function (detectable C-peptide levels) and glycemic control  for the blood glucose levels during a specific period). Insulin dosage, cholesterol, triglycerides, and adverse events were classified as secondary outcomes. We extracted the following data: treatment option, length of study, sample size, average age, duration of T1DM, body mass index (BMI), study design, and number of research centers. Briefly, all relevant data available that might impact the results were extracted and input into a predesigned table.The outcomes were recorded in detail. The primary outcome parameters studied here were classified as variations in We used the Cochrane Risk of Bias Tool for Randomized Controlled Trials to assess the quality of the literature and the risk of bias.N), means (M), and standard deviations (SD) for females and males, we adopted the formula from chapter 7.7.3 of the Cochrane Handbook to synthesize M1+2 and SD1+2 data for systematic review. The formulas wereWe used the change in the value of the outcome to evaluate the therapeutic effect and emphasize the benefits of the combination of DPP-4 inhibitors with insulin versus insulin monotherapy. Because some articles reported the sample sizes  was used to synthesize the data, and 95% confidence intervals (95% CI) were calculated. We reported the mean difference (MD) for indicators of efficacy, including the HbA1c, fasting blood glucose, and 2-hour postprandial blood glucose levels and other continuous variables. The relative risk ratio (RR) was used for dichotomous variables, such as indicators evaluating safety. If the indicators were reported in fewer than 3 studies, we summarized the result of each study individually. Statistical significance was set to an tatistic . If signThe literature retrieval process is shown in \u03b2-cells in patients with LADA. Regarding indicators, the earliest initiated study was an 8-week crossover study from Sweden [Two hundred fifty-three participants, including 120 C-peptide-positive patients, from 5 RCTs were included in this meta-analysis and systematic review. Regarding medicine options, four studies , 18\u201320 em Sweden  that focm Sweden  focused We evaluated the risk of bias using Review Manager version 5.2. (1) \u03b2-Cell Function: C-Peptide. The additional effects of DPP-4 inhibitors on the C-peptide level remain controversial. One study [P > 0.05). In the study by Griffin et al. [\u03b2-cell function, could not be confirmed in the group treated with DPP-4 inhibitors. Therefore, whether patients with a higher baseline C-peptide level, a shorter duration, and differential insulin usage will benefit more from combination therapy with any DPP-4 inhibitor remains unknown.ne study  reportedne study , 19, 20 ne study  did not ne study  selectedn et al. , the AUCn et al.  were ini(2) Glycemic Control: HbA1c. As a main outcome indicator, specific data on the HbA1c levels were available in four RCTs. Individually, three RCTs did not observe a significant improvement in the HbA1c levels when the patients were treated with sitagliptin [P = 0.23, >0.05).agliptin , 20 or vagliptin  in additagliptin  did not agliptin  was remoagliptin . However(3) Glycemic Control: Glycemic Fluctuation. Amazingly, after 16 weeks of treatment, the group receiving DPP-4 inhibitors had lower postprandial blood glucose levels and lower glucagon levels in the study by Garg et al., although the 4-hour AUCs for glycemia were not different between groups [n groups . Howevern groups . Zhao etn groups . The remn groups , 19 did (1) Other Relevant Indicators. Regarding the insulin dosage, two studies [ studies , 20 prov studies  revealed studies  reported studies  showed t studies , the ins2, P < 0.05) in the insulin monotherapy group, and the body weight was also increased but did not significantly differ between groups. Garg et al. [According to Garg et al. , the GLPg et al.  consiste(2) Overall Adverse Events and Side Effects. We believe that hypoglycemia is one vital adverse event that deserves more attention when antidiabetic agents are added to insulin therapy. The study by Hari et al. [i et al.  observedi et al.  reportedi et al. . In the i et al. , a low ii et al. , hypoglyi et al. , 19, 20 i et al. . Records\u03b2-cell proliferation [\u03b2-cell mass and number of proliferating \u03b2-cells [Due to their recommended use as first-line antidiabetic agents by the American Association of Clinical Endocrinologists (AACE), DPP-4 inhibitors are currently widely used as outstanding monotherapeutic or combination therapeutic agents for T2DM. However, no DPP-4 inhibitor for T1DM has received authorization for application. No currently available treatment has shown lasting disease remission, although insulin is life-saving. Meanwhile, the life expectancy of patients with T1DM has substantially improved in recent decades. Therefore, additional management strategies are needed. Any novel treatment option (newer) in addition to insulin may produce a qualitative leap. Emerging studies have reported evidence that an elevation of serum DPP-4 activity is related to the pancreatic autoimmune process, intestinal proinflammatory alterations, and hepatobiliary injury \u201323 , which was originally intended to investigate the efficacy of linagliptin compared to glimepiride administration for 104 weeks as an add-on therapy to preferably >1500\u2009mg of metformin in patients with T2DM with insufficient glycemic control. Some of the participants were reclassified as LADA depending on the tested autoantibodies during the trial. Further investigation found that these LADA patients demonstrated progressively increasing C-peptide levels and slightly decreasing HbA1c levels after linagliptin-metformin treatment compared to the baseline levels [Here, we systematically reviewed all relevant publications and performed a meta-analysis of five randomized controlled clinical trials on the effects of DPP-4 inhibitors on T1DM. We were also interested in another clinical trial , and a report of full-scale adverse effects should be the focus of future studies.As discussed above, more studies should be recruited to elucidate the roles of DPP-4 inhibitors in appropriately selected patients with T1DM. A larger sample size, a longer follow-up, unified characteristics of participants (such as diabetes duration and C-peptide levels), the comprehensive monitoring of glycemia and relevant hormone levels, the use of immunological indicators (such as CD4"}
+{"text": "The \u03b12Na+/K+-ATPase subunit isoform is predominantly expressed in astrocytes, which us the sharp Na+-gradient maintained by the sodium pump necessary for astroglial metabolism. Prolonged ischemia induces an elevation of [Na+]i, decreased ATP levels and intracellular pH owing to anaerobic metabolism and lactate accumulation. During ischemia, Na+/K+-ATPase-related functions will naturally increase the energy demand of the Na+/K+-ATPase ion pump. However, the role of the \u03b12Na+/K+-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice for the loss-of-function disease-mutation G301R in the Atp1a2 gene (\u03b12+/G301R) to study the effect of reduced \u03b12Na+/K+-ATPase expression in a moderate contusion spinal cord injury (SCI) model.The Na2+/G301R mice display significantly improved functional recovery and decreased lesion volume compared to littermate controls (\u03b12+/+) 7\u00a0days after SCI. The protein level of the \u03b11 isoform was significantly increased, in contrast to the \u03b13 isoform that significantly decreased 3\u00a0days after SCI in both \u03b12+/G301R and \u03b12+/+ mice. The level of the \u03b12 isoform was significantly decreased in \u03b12+/G301R mice both under na\u00efve conditions and 3\u00a0days after SCI compared to \u03b12+/+ mice. We found no differences in astroglial aquaporin 4 levels and no changes in the expression of chemokines  and cytokines  between genotypes, just as no apparent differences were observed in location and activation of CD45 and F4/80 positive microglia and infiltrating leukocytes.We found that \u03b12 isoform in the spinal cord is protective following SCI. Importantly, the BMS and lesion volume were assessed at 7\u00a0days after SCI, and longer time points after SCI were not evaluated. However, the \u03b12 isoform is a potential possible target of therapeutic strategies for the treatment of SCI.Our proof of concept study demonstrates that reduced expression of the \u03b1 Spinal cord injury (SCI) results in massive cell loss at the site of the lesion. Injury to the central nervous system (CNS) causes a range of cellular and molecular changes that make changes to the local environment and impede regeneration. Cytokines and chemokines are released at the lesion site serving to recruit peripheral leukocytes to the injury site . AstrocyIn the intact spinal cord there is limited proliferation of astrocytes. However, in response to injury, inflammatory cytokines cause adult astrocytes to proliferate and give rise to reactive astrocytes , 3.+ and regulating synaptic concentrations of the neurotransmitter glutamate. This is accomplished through the Na+-dependent glutamate transporters EAAT1 and EAAT2 i, increasing the energy demand of Na+/K+ ATPase, reversal of the Na+/glutamate co-transporter, cell swelling and activates volume regulatory processes i oscillations [Egr-1, Fos, June, Nr4a2, Hes1 and Gabre) [Increasing evidence points towards the Nallations \u201329, and d Gabre) .2+ concentration can modulate transcription in two ways; (1) by promoting translocation of nuclear factor-kappa B (NF-\u03baB) from the cytosol to the nucleus, and (2) by phosphorylating the cAMP response element binding protein (CREB) [Interestingly, increasing the Can (CREB) .2 isoform following contusion injury to the spinal cord, we took advantage of a newly generated knock-in mouse model harboring the Familial Hemiplegic migraine type 2 (FHM2) disease-related loss of function mutation G301R in the Atp1a2 gene [2+/G301R) display pathological relevant symptoms related to Familial Hemiplegic migraine type 2 (FHM2), and showed impaired glutamate uptake in in vitro-matured hippocampal mixed astrocyte-neuron cultures from \u03b12G301R/G301R E17 embryonic mice [2(+/G301R) behavioral phenotypes [2G301R/G301R) die immediately after birth [2+/G301R mice to elaborate on the role of the \u03b12Na+/K+-ATPase after SCI. We demonstrate that \u03b12+/G301R mice display significantly improved functional recovery and decreased lesion volume compared to littermate controls (\u03b12+/+) already 7\u00a0days after SCI. Although long-term evaluations after SCI were not assessed, however, this study, suggests that decreasing the level of the \u03b12 isoform might serve as a new potential therapeutic target in SCI treatment.To explore the role of the \u03b11a2 gene . Heteroznic mice . Moreoveenotypes . Mice hoer birth . In this2+/G301R mice with reduced levels of the \u03b12 isoform in various brain structures [2 isoform levels in the spinal cord under na\u00efve conditions, Western blotting was performed. As expected, \u03b11 and \u03b13 isoform levels were comparable between \u03b12+/G301R and \u03b12+/+ mice, whereas \u03b12 isoform levels were reduced approximately 40% in \u03b12+/G301R mice compared to littermates  and counterstained with Hoechst, and revealed no gross morphological differences between the \u03b12+/G301R and \u03b12+/+ mice under na\u00efve conditions . In both \u03b12+/G301R and \u03b12+/+ mice, the \u03b12 isoform was preferentially detected in the white matter, both the posterior, lateral and anterior funiculi, with little astrocytic-localized \u03b12 isoform in the grey matter.Previously, it was shown that the G301 mutation confers haploinsuffiency in heterozygous \u03b1ructures . To inve+/K+-ATPase in SCI has not been addressed despite its essential role in astroglial functions. To examine the role of the astrocytic \u03b12 isoform, we performed SCI on the \u03b12+/G301R mice and compared the effect to \u03b12+/+ mice after 7\u00a0days in order to assess any correlations. The \u03b12+/G301R and \u03b12+/+ mice were subjected to SCI and subsequently allowed 7\u00a0days post-surgical survival. The \u03b12+/G301R mice significantly improved their BMS score compared to \u03b12+/+ mice . To evaluate the injury site, luxol fast blue (LFB) and glial fibrillary acidic protein (GFAP)/Nissl/DAPI staining was performed. Both analysis of LFB and GFAP/Nissl/DAPI stained sections revealed significantly reduced lesion volumes in \u03b12+/G301R mice compared to \u03b12+/+ mice 7\u00a0days after SCI  \u03b12 isoform levels in their spinal cords under na\u00efve conditions. Interestingly, while both \u03b11 and \u03b13 isoform levels were significantly altered in both genotypes following moderate SCI, the \u03b12 isoform was only significantly decreased in \u03b12+/+ mice following SCI injury, whereas the \u03b12 isoform level remained comparable between na\u00efve and SCI-injured tissues in the \u03b12+/G301R mice. This might reflect the fact that there is a lower threshold for \u03b12 isoform expression and cell survival in the \u03b12+/G301R mice.The \u03b12+/G301R mice was superior to \u03b12+/+ mice and correlated with a significantly reduced lesion size in the \u03b12+/G301R compared to \u03b12+/+ mice. In support of these findings, it was recently found that knock-down of the \u03b12Na+/K+-ATPase in astrocytes deficient of the mutant superoxide dismutase 1 (SOD1) was able to protect motor neurons from degeneration in co-cultured primary motor neurons [Atp1a2 gene by lentiviral-mediated RNAi in the spinal cord of SOD1 mutant mice suppressed motor neuron degeneration and subsequently increased the life span of mutant SOD1 mice [2Na+/K+-ATPase upregulated mRNAs encoding mitochondrial respiration and expression of secreted inflammatory factors  in SOD1 mutant astrocytes.After moderate SCI injury, the recovery was evaluated by functional outcome, estimation of lesion volumes and changes in pro- and anti-inflammatory cytokines and potential changes in microglial/leukocyte activation. Interestingly, the functional recovery of the \u03b1 neurons . HeterozOD1 mice . Moreove2+/G301R compared to \u03b12+/+ mice. Overall, there was no difference in AQP4 level between the genotypes nor was there any significant difference between na\u00efve and SCI-treated mice. Previous studies on cerebral edema found that reduced levels of AQP4 significantly improved the outcome [2Na+/K+-ATPase works independently of AQP4 in the area tested, or maybe the effect would be more evident in later stages after SCI, which awaits future testing.We interrogated the possibility that AQP4 might be regulated differentially in \u03b1 outcome , 26, whi2+/G301R and \u03b12+/+ mice compared to na\u00efve conditions, however, we observed no statistical differences between the two genotypes. CCL2 [We found that chemokines CXCL1, CCL2 and CCL5 and cytokines TNF, IL-6 and IL-10 were significantly upregulated in the lesioned spinal cord of \u03b1es. CCL2 , IL-6 [3es. CCL2 , 39 and es. CCL2  have pre+, and excitotoxic cell death. A reduction or defect to extrude Na+ by the Na+/K+-ATPase pump as a consequence of the mutation introduced will result in increased intracellular Na+ levels, which might increase even further following SCI. In fact, increased intracellular Na+ concentration has been suggested to exacerbate the effects of compression trauma to the spinal cord in rats [+ and K+ gradients following SCI is expected to induce a reversed operation of the Na+-dependent glutamate transporters, leading to glutamate efflux and subsequent activation of glutamate receptors causing substantial Ca2+-dependent injury [2G301R/G301R mice display significantly reduced uptake of glutamate, and subsequently, increased glutamate levels [2+/G301R mice will be altered.The pathophysiology of SCI involves a primary mechanical injury and a delayed secondary injury due to a number of proposed mechanisms including ischemia, abnormal intracellular shifts of ions including Na in rats . Furthert injury . It has e levels . It rema+/K+-ATPase can also act as a signal transducer and a NF-\u03baB activator by interacting with neighboring membrane proteins and organized cytosolic cascades of signaling proteins [+/K+-ATPase is modulated by glutamate by NMDA receptor-nitric oxide production, leading to activation of cyclic GMP-PKG [Tnf-\u03b1, Il-1\u03b2, and Bdnf mRNA levels [The Naproteins , 43. The GMP-PKG , 45. IntA levels .+/K+-ATPase activity to SCI. A rat model of SCI induced by extradural compression of the cord resulted in a 50% decrease in the activity of synaptosomal Na+/K+-ATPase 30\u00a0min after the compression injury [Several other studies have correlated reduced Nan injury .2+/G301R mice recover highly significantly after SCI compared to littermate \u03b12+/+ control mice tested 7\u00a0days after SCI. The molecular mechanisms regarding this rapid recovery as a consequence of less Na+/K+-ATPase activity in astrocytes in relation to inflammatory responses remain to be elucidated, and these results serves as a proof of concept study and open promising potential towards therapeutic applications towards SCI.In conclusion, the \u03b1Animals were housed in ventilated cages at a 12-h light/dark cycle, under controlled temperature and humidity, and free access to food and water. Mice were cared for in accordance with the protocols and guidelines approved by The Danish Animal Inspectorate under the Ministry of Food and Agriculture, Denmark (J. No. 2013-15-2934-00924 to KLL and J. No. 2013-15-2934-00815 to KLH); experiments performed in accordance with the ARRIVE guidelines, and all efforts were made to minimize pain and distress. All animal procedures were approved by Institutional Animal Care and Use Committee at the University of Aarhus and Southern Danish University, Denmark. All experiments were performed blinded.2+/G801R\u00a0mice [\u00ae 96 Real-Time PCR System) using primers F-5\u2032-ggatgagggacagaacgaag and R-5\u2032-catggagatcgagcatttca (Sigma-Aldrich).Heterozygous \u03b101R\u00a0mice  were genA ketamine /xylazine  cocktail was used to anaesthetize mice, and mice were laminectomized between vertebrae T8 and T10, and the impactor lowered at a pre-determined impact force resulting in an approximate displacement of 500\u00a0\u03bcm (moderate injury) . ContusiFunctional post-SCI recovery of hind limb function was determined by scoring of the locomotor hindlimb performance in the open field using the Basso Mouse Scale (BMS) system, a 0 to 9 rating system designed specifically for the mouse . Under oMice were deeply anaesthetized using an overdose of pentobarbital (200\u00a0mg/ml) containing lidocaine (20\u00a0mg/ml) and perfused through the left ventricle with cold 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Spinal cords were quickly removed and tissue segments containing the lesion area (1\u00a0cm centered on the lesion) were paraffin-embedded and cut into 10 parallel series of 15\u00a0\u03bcm thick microtome sections. Sections were stored at room temperature.2O, immersed briefly in lithium carbonate (0.05% Li2CO3 in distilled water) and differentiated in 70% EtOH, before rinsed thoroughly in distilled H2O and immersed in 0.05% lithium carbonate to stop further differentiation. Sections were submitted to hematoxylin, rinsed in running tap water and immersed briefly in eosin solution. Finally, sections were rinsed in 70% EtOH, followed by 3\u00d7\u00a099% EtOH, placed in 3\u00d7\u00a0xylene prior to mounting with Depex. Paraffin embedded sections were deparaffinized 3\u00d7 3\u00a0min in xylene, 3\u00d7 2\u00a0min in 99% EtOH and 2\u00d7 2\u00a0min in 96% EtOH, before staining.For evaluation of lesion pathology, one series of sections from each animal was stained in Luxol Fast Blue (LFB) (0.1% LFB in 95% ethanol (EtOH) and 0.05% acetic acid) at 60\u00a0\u00b0C for 12\u00a0h. Sections were rinsed in 96% EtOH and distilled H+) as chromogen (DAKO). Nuclei were counterstained using Mayer\u2019s haemalum w/4.5% chloralhydrate. As negative control, the primary antibody was omitted to check for any unspecific reaction from the detection system. As positive control for antibody-specificity, the staining was tested using a mouse multi block containing several different tissues including lymphatic organs. The activation state of CD45+, and F4/80+ microglia and leukocytes and AQP4 expression were investigated in 5 sections  from each animal centered on the lesion epicenter.Heat-induced antigen retrieval was done on the paraffin embedded sections by boiling the sections in Tris-EGTA buffer, pH 9.0 (CD45), TRS buffer  (F4/80), or TEG buffer, pH 9.0 (AQP4), first 15\u00a0min at 900W, then 9\u00a0min at 440W. The sections were allowed to cool in the buffer before blocked for endogenous peroxidase and biotin activity. Sections were then incubated with anti-CD45 ; BD Pharmingen), anti-F4/80 , or anti-AQP4  antibodies and detected using biotinylated rabbit anti-rat IgG (DAKO) (CD45 and F4/80) or biotinylated donkey anti-rabbit IgG diluted 1:200 followed by ready-to-use anti-rabbit horse-radish perioxidase (HRP)-labelled polymer  with diaminobenzidin  diluted 1:400 for 1\u00a0h at room temperature and hereafter over night at 4\u00a0\u00b0C. Next day, sections were placed at room temperature for 30\u00a0min before they were rinsed in TBS for 10\u00a0min and then in TBS with 0.1% Triton X-100 for 10\u00a0min. The sections were then stained with NeuroTrace\u00ae 530/615 Red Fluorescent Nissl Stain (ThermoFischer Scientific) for 20\u00a0min, and further rinsed 2\u00d7 10\u00a0min in TBS before the sections were immersed in a TBS solution containing 10\u00a0\u03bcM diamidino-2-phenylindole (DAPI) for 10\u00a0min. The sections were shortly rinsed in distilled water before they were mounted with ProLong Diamond. Control reactions were performed by omitting the primary antibody or by substituting the primary antibody with Alexa Fluor\u00ae 488 conjugated mouse IgG1\u03ba (ThermoFischer Scientific). Sections were devoid of staining in the FITC imaging filter.One series of sections from each animal was deparaffinized and rehydrated by placing the sections\u00a03\u00d7 3\u00a0min in xylene, 3\u00d7 2\u00a0min in 99% EtOH, 2\u00d7 2\u00a0min in 96% EtOH, 2\u00a0min in 70% EtOH and finally 5\u00a0min in running tap water. The sections were demasked using TEG-buffer by placing the sections in warm TEG-buffer in a steamer for 15\u00a0min, then letting them cool for 15\u00a0min at room temperature before rinsing them for 15\u00a0min in running tap water. Sections were then rinsed 3\u00d7 15\u00a0min in tris-buffered saline (TBS) before they were pre-incubated with 10% fetal bovine serum (FBS) in TBS with 0.5% Triton X-100 for 30\u00a0min. Sections were incubated with Alexa Fluor\u00ae 488-conjugated donkey anti-rabbit antibody and Alexa Fluor\u00ae 568-conjugated streptavidin (Life Technologies) diluted 1:350 in 1% donkey serum PBS with 0.025% Triton X-100 for 1\u00a0h at room temperature. Hoechst  (Life technologies) was used to counterstain the nuclei. Sections were then mounted with fluorescence mounting medium (Dako) and subsequently analysed on a LSM510 laser-scanning confocal microscope using a 40\u00d7 C-Apochromat water immersion objective NA 1.2 (Carl Zeiss). Zen 2011 software (Carl Zeiss) was used for image capturing and analysis.Sections were prepared as desribed above for GFAP staining. Primary antibodies \u03b12 (Merck Millipore) diluted 1:300 and NeuN  diluted 1:300  were appThe volume of the injury was determined from the area of every tenth section sampled by systematic uniform random sampling. Area of the lesion site was estimated as previously described . Digital2+/G301R and \u03b12+/+ littermate mice exposed to SCI and allowed 3\u00a0days survival in addition to na\u00efve \u03b12+/G301R and \u03b12+/+ littermate mice were prepared as described [1, \u03b12, \u03b13 and AQP4) gels and electro-blotted onto nitrocellulose membranes (Pharmacia-Amersham). Membranes were blocked in PBS with 5% skimmed milk and 0.5% Tween-20 and incubated with the following primary antibodies: anti-\u03b11 diluted 1:2000 , anti-\u03b12 diluted 1:1000 (Merck Millipore), anti-\u03b13 diluted 1:1000 (Merck Millipore), anti-AQP4 diluted 1:1000  anti-GAPDH diluted 1:1000 (Abcam), or anti-\u03b2-Actin diluted 1:2000 (Sigma-Aldrich) overnight at 4\u00a0\u00b0C.Whole spinal cord protein samples from \u03b1escribed . Equal aNext, membranes were incubated with HRP-conjugated secondary antibodies (swine anti-rabbit HRP diluted 1:2000 (Dako) or rabbit anti-mouse HRP diluted 1:2000 (Dako)) for 1\u00a0h at room temperature. Visualization of blots was done in a LAS 3000 imager (Fujifilm) with Amersham ECL Western Blotting Detection Kit . Post densitometric analysis and image processing of blots were performed in Image J.To measure cytokine protein levels by the MSD Mouse Proinflammatory V-Plex Plus Kit  under na\u00efve conditions and 3\u00a0days after SCI, we used a SECTOR Imager 6000  Plate Reader according to the manufacturer\u2019s instructions. The same samples as those used for Western blotting were diluted two-fold in Diluent 41 prior to measurement and 50\u00a0\u00b5l dilution was loaded in each well. Data was analyzed using MSD Discovery Workbench software .t test analysis or Bonferroni post hoc, or by Student\u2019s t test. Analyses were performed using Prism 4.0b software for Macintosh, (GraphPad Software). Statistical significance was established for p\u00a0<\u00a00.05.Comparisons were performed using repeated measures (RM) or regular two-way ANOVA followed by multiple"}
+{"text": "Connexin-36 (Cx36) electrical synapses strengthen transmission in a calcium/calmodulin (CaM)/calmodulin-dependent kinase II (CaMKII)-dependent manner similar to a mechanism whereby the N-methyl-D-aspartate (NMDA) receptor subunit NR2B facilitates chemical transmission. Since NR2B\u2013microtubule interactions recruit receptors to the cell membrane during plasticity, we hypothesized an analogous modality for Cx36. We determined that Cx36 binding to tubulin at the carboxy-terminal domain was distinct from Cx43 and NR2B by binding a motif overlapping with the CaM and CaMKII binding motifs. Dual patch-clamp recordings demonstrated that pharmacological interference of the cytoskeleton and deleting the binding motif at the Cx36 carboxyl-terminal (CT) reversibly abolished Cx36 plasticity. Mechanistic details of trafficking to the gap-junction plaque (GJP) were probed pharmacologically and through mutational analysis, all of which affected GJP size and formation between cell pairs. Lys279, Ile280, and Lys281 positions were particularly critical. This study demonstrates that tubulin-dependent transport of Cx36 potentiates synaptic strength by delivering channels to GJPs, reinforcing the role of protein transport at chemical and electrical synapses to fine-tune communication between neurons. Synaptic plasticity at glutamatergic chemical synapses involves highly orchestrated molecular and morphological processes that fine-tune communication between neurons. Short- to long-term functional and morphological adaptations require dynamic trafficking and turnover of channel and receptor proteins. While the molecular machinery involved in chemical synaptic plasticity and the respective signaling network has been extensively investigated, fewer details are known about the plasticity of electrical synapses. In vivo, modifiable responses of electrical synapses have been found in both lower and higher vertebrates ,2. In vit manner ,4,5. TheThe regulation of synaptic strength through ion channels, NMDA and \u03b1-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor recruitment, and turnover is potentiated by calcium entry through NMDA receptors ,10,11,12Using the mouse Neuro-2a neuroblastoma cell line as a platform for exogenous expression of wild-type and mutant Cx36 proteins, we demonstrate that Cx36 binds tubulin through a conserved binding motif, which is distinct from the confirmed motif of Cx43 ,21. The Rattus norvegicus Cx36 (Accession number: NP_062154.1) was used in this study. Wild-type Cx36 tagged with enhanced green fluorescent protein (EGFP) and the carboxyl-terminal mutant Cx36\u0394279\u2013292-EGFP (denoting the deletion of amino acids 279\u2013292) expression plasmids were generated previously [\u00ae, CCL-131, Manassas, VA, USA) were cultured in high-glucose Dulbecco\u2019s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% nonessential amino acids (NEAA), and 1% penicillin/streptomycin as described [6) overnight in 19 mm (24 well), 100 mm, or 35 mm MatTek culture dishes. Cells were transfected using the Effectene Transfection Reagent Kit  with 200 ng DNA, as per the manufacturer\u2019s protocol, and all analyses were performed 48 h post-transfection.Mouse neuroblastoma 2a (Neuro-2a) cells . Membranes were blocked with 1\u00d7 iBind\u2122 Solution (horseradish peroxidase (HRP) or alkaline phosphatase (AP) detection), and immunoblotting was carried out using the iBind Western System  as per the manufacturer\u2019s protocols. Primary antibodies and enzyme conjugates were diluted 1:10,000 , 1:1000 , and 1:2500 . Secondary antibodies were diluted 1:10,000  and 1:15,000 . Fluorescent signals were detected using the Odyssey\u00ae CLx Infrared Imaging System  as per the manufacturer\u2019s protocol using default settings. Transiently transfected Neuro-2a cells were washed once with divalent free (DF) phosphate-buffered saline (PBS) prior to lysis. Whole-cell protein lysates were prepared directly in 1\u00d7 Laemmli sample buffer , 3% \u03b2-mercaptoethanol) and heated for 3 min before gel separation. Twenty micrograms of proteins were fractionated by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE) on 5% stacking and 10% running gels and transferred onto a 0.2-\u00b5m nitrocellulose membrane using the Trans-Blot6) transfected Neuro-2a cells expressing Cx36 wild-type or Cx36\u0394279\u2013292-BirA* fusion proteins were incubated with 50 \u00b5M biotin for 24 h as previously described [g and incubated at 4 \u00b0C overnight with 180 \u00b5L of Dynabeads . Collection of the beads with protein candidates attached, washes, elution, and preparation for western blot or mass spectrometry analysis were performed as described [On a 100-mm dish,  or Total Internal Reflection Fluorescence (TIRF) studies. Tubulin-dependent trafficking of Cx36 to the gap-junction plaque was analyzed using Neuro-2a cells expressing EGFP-tagged wild-type Cx36 protein treated with either Colchicine  or Paclitaxel  to disrupt or stabilize microtubules, respectively. Neuro-2a cells were incubated with a drug at 37 \u00b0C, 5% CONeuro-2a cell pairs expressing EGFP-tagged wild-type Cx36 or Cx36\u0394279\u2013292 were used for dual whole-cell patch clamp recording. In brief, Neuro-2a cells were perfused in a bathing solution supplemented with 100 \u00b5M Colchicine for 1, 3, or 24 h and the run-up was induced as previously described . Whole-c2) was measured using the ImageJ  free-hand tool. The percentage of Cx36 gap-junction-paired cells versus the total paired Cx36-expressing cells was measured as previously described [Transfected Neuro-2a cells were fixed with 4% formalin for 15 min at room temperature. Cells were subsequently rinsed with PBS and distilled water prior to mounting with Fluoroshield\u2122 with DAPI  mounting medium. Mounted slides were visualized using a Zeiss LSM 700 confocal microscope controlled by the ZEN 2010 software program  at room temperature. Cells were imaged using the Plan-Apochromat 63x/1.40 Oil DIC M27 or the EC Plan-Neofluar 40x/1.3 Oil M27 oil immersion lenses. Images were generated at a high resolution of up to 2048 \u00d7 2048 pixels and an average of 4 scanning repetitions in the single plane or each plane of a Z-stack. The single or multi-track mode was selected accordingly for the compilation of localization studies. The gap-junction plaque area (\u00b5mf = (Fmax \u2212 F0)(1 \u2212 ekt\u2212)) and half-time of recovery (T1/2 = ln 2/k) were calculated by the mono-exponential association equation in GraphPad: Ft = F0 + (Fmax \u2212 F0)(1 \u2212 ekt\u2212), where Ft is the fluorescence after background subtraction at time t; F0 and Fmax are the fluorescence values immediately after and at the end of recovery following the bleaching event, respectively; and k is the first-order rate constant for recovery. Repopulation figures and X-Y scatterplots were done using the R software. Sample sizes ranged based on the efficiency of gap-junction plaque formation.For the quantitative analysis of connexon trafficking and gap-junction regeneration, FRAP was performed 48 h post-transfection using the Zeiss LSM 700 confocal microscopy in combination with Zeiss 63X  oil immersion lens and the Zen 2010 software. The microscope was equipped with an incubation chamber to maintain cells at 37 \u00b0C in DMEM without phenol red. Since previous reports  have des2 levels at 5%. Cells were imagined in DMEM without phenol red. Images were acquired at a resolution of 512 \u00d7 512 pixels in 20-s intervals for a total duration of 400 s. Images were processed in Imaris  by tracking single particles expressing EGFP within the TIRF field. For all experiments, Neuro-2a cells were plated onto 35-mm MatTek dishes and co-transfected with pShuttle-mCherry-Tubulin and the pEGFP-Cx36 wild-type or mutants as previously outlined. Time-lapsed TIRF microscopy was performed using the Zeiss Observer.Z1 spinning-disk microscope in combination with the Zeiss 100X  oil immersion lens, Photometrics Evolve\u2122512 camera, and the Zen 2 (2014) software. The microscope was equipped with an incubation chamber to maintain the temperature at 37 \u00b0C and COA helical segment spanning amino acids 47\u201362 of the RB3 protein stathmin-like-domain X-ray structure  was subsp < 0.05 was considered significant. XY plots of the mobile fraction and half-time of recovery represented the mean \u00b1 CI. All comparisons were made to the wild-type (WT) control unless otherwise stated. Sample sizes and p values are provided throughout the manuscript and figure captions. Beeswarm boxplots were generated in GraphPad Prism 6 . Plaque repopulation graphs and mobile fraction vs. half-time of recovery XY scatterplots were created in R-statistical program . All data are represented as the mean \u00b1 SEM. Unless otherwise stated, statistical analysis was carried out using SPSS  and significance was determined by the Kruskal\u2013Wallis test. For the FRAP data, statistical analysis was carried out using GraphPad and statistical significance was determined by the Kruskal\u2013Wallis test followed by a Dunn\u2019s multiple comparison test. The interaction between tubulin at the carboxy-terminal (CT) of Cx36 was initially identified by mass spectrometry using both affinity-purified samples and immunoprecipitated protein complexes from murine brain lysates . This reIn previous studies ,4,5, we n = 6; 1 h Colc: 0.2 \u00b1 0.03, n = 5, p < 0.01 \u00d7 10\u22122; O/N Colc: 0.3 \u00b1 0.02, n = 6, p < 0.01 \u00d7 10\u22122)   or 80% (24 h) compared to untreated controls  C,D. An i \u00d7 10\u22122) . In a re \u00d7 10\u22122) E,F. Thes2, n = 28; \u0394279\u2013292: 1.6 \u00b1 0.1 \u00b5m2, n = 26, p = 0.17 \u00d7 10\u22121)   D and les \u00d7 10\u22121) E. We conn = 30; \u0394279\u2013292: 0.57 \u00b1 0.01, n = 25, p < 0.01 \u00d7 10\u22121) and Golgi organelles , indicating that Cx36 vesicles were not primarily associated with the ER\u2013Golgi complex. A marked reduction in co-localization to the tubulin cytoskeleton was observed for the Cx36 mutant expression in comparison to the wild-type protein . Co-localization of Cx36\u0394279\u2013292 to caveolin-1 was indistinguishable from wild-type    the ER retention signal KDEL, (ii) amino acids 1\u201360 of human galactosyltransferase, or (iii) caveolin-1. This approach permitted the visualization of the microtubule network, endoplasmic reticulum (ER), Golgi, and transport vesicles. Mander\u2019s overlap coefficient (MOC) was used to quantify co-localization as the overlap between corresponding fluorescent signals contributed by Cx36 and cell organelles. Cx36\u0394279\u2013292 displayed less co-localization to the ER  ; however \u00d7 10\u22121) G. Our ren = 475; \u0394279\u2013292: 6.4 \u00b1 0.5 \u00b5m, n = 304, p = 0.26 \u00d7 10\u22121), and mean speed   B. We conHow were Cx36 connexons delivered to GJPs in the absence of tubulin binding? In general, connexons can replenish GJPs by two distinct pathways. Targeted direct delivery of connexons to the GJP relies on a complex of cytoskeletal and adherence-junction proteins . Alternaf) in %, GJP: 25.6 \u00b1 0.6, n = 28; PM: 69.6 \u00b1 3.6 n = 12, p < 0.01 \u00d7 10\u22122; half time of recovery (T1/2), GJP: 11.5s; PM: 23.5s) , suggesting that no changes were made to the route of delivery. Cx36\u0394279\u2013292 expression also led to a T1/2 that was faster at the PM and GJP  than the WT  was used to determine recovery kinetics of EGFP-tagged Cx36 wild-type or \u0394279\u2013292 proteins imaged at 1.0-s intervals pre- and post-bleach iterations. For visualization and quantification of connexon recovery, the fluorescence intensity of each region of interest (ROI) during the post-bleach recovery phase was corrected for background noise and normalized against the pre-bleached fluorescence intensity values. After bleaching, we observed that the plasma membrane (PM) of wild-type Cx36 recovered more efficiently than at the GJP (mobile fraction (M: 23.5s) A,D. The escribed ,35. In Cn the WT B,D. We cp < 0.01 \u00d7 10\u22122). This loss of stability was also accompanied by a large reduction in the T1/2, reflecting dynamics that were consistent with diffusion outcomes    was included in this investigation since both tubulin and actin-dependent transport has been found at chemical synapses . Immunob = 0.01) C. GJP fo \u00d7 10\u22121) D. This on = 40; Colchicine: 0.12 \u00b1 0.07, n = 21, p < 0.01 \u00d7 10\u22121) and Paclitaxel treatment . Considering that Paclitaxel stabilizes microtubules yet less Cx36 interacted with the tubulin cytoskeleton, we propose that binding of paclitaxel creates a steric hinderance for Cx36 binding. In this way, paclitaxel treatment would act as a competitive inhibitor, controlling the quantity of Cx36 able to bind to tubulin. Our results reaffirmed that Cx36 preferentially interacts with an intact and dynamic tubulin cytoskeleton, promoting transport to the plasma membrane and subsequent coalescence at the GJP. Treatment with Cytochalasin D had no effect on Cx36 co-localization to the tubulin cytoskeleton   to the tubulin cytoskeleton was significantly impaired with both Colchicine  . This con = 475; Colchicine: 6.5 \u00b1 0.4 \u00b5m, n = 337, p = 3.01 \u00d7 10\u22121) or mean speed  with Colchicine treatment in comparison to the untreated wild-type. To consider the effectiveness of drug treatment in an overexpression model and the sensitivity of TIRF detection under these circumstances, the experiments were repeated in single transfected cells for Cx36-EGFP. Here, colchicine application significantly impaired the displacement length  and mean speed   emphasizing the importance of a dynamic tubulin cytoskeleton on trafficking vehicles in vivo. Since paclitaxel treatment was sufficient to establish a response in the TIRF field, a single transfected analysis was not further explored. To account for the actin contribution, Cytochalasin D treatment was also included here. Inhibition of vesicular transport was also apparent with Cytochalasin D treatment , suggesting that actin can serve as a compensatory modality for Cx36 transport and confirming that Cx36 delivery to the PM is both actin and tubulin dependent  . In doubependent . 1/2 that was uniformly faster across all treatment groups . Here, the incorporation of Cx36 into the GJP after actin- or tubulin-cytoskeletal disruption was consistent with diffusion dynamics . No significant difference in recovery was found with Paclitaxel treatment , likely attributable to the direct stabilization of the GJP via tubulin stabilization. Although the GJP remained stable under paclitaxel treatment, it is important to highlight that transport into the GJP was impaired as shown previously (p < 0.01 \u00d7 10\u22122)  , confirmA previous report demonstrated that a 26 amino-acid peptide of Cx43 mediates binding to \u03b1- and \u03b2-tubulin isoforms. This region adopts a helical conformation upon binding to tubulin and is regulated by phosphorylation . Here, w2, n = 7, p < 0.01 \u00d7 10\u22121; I280A: 1.7 \u00b1 0.3 \u00b5m2, n = 19, p = 1.60 \u00d7 10\u22122; K281A: 1.1 \u00b1 0.1 \u00b5m2, n = 9, p = 1.00 \u00d7 10\u22123; L282A: 1.3 \u00b1 0.1 \u00b5m2, n = 16, p = 3.00 \u00d7 10\u22123; V284A: 1.1 \u00b1 0.2 \u00b5m2, n = 26, p < 0.01 \u00d7 10\u22121; R285A: 1.6 \u00b1 0.3 \u00b5m2, n = 12, p = 2.90 \u00d7 10\u22122; G286A: 1.1 \u00b1 0.2 \u00b5m2, n = 10, p = 1.00 \u00d7 10\u22123)  . Cx36-L282A was indistinguishable from the wild-type control .Sequential alanine-scanning mutagenesis from K279 to G286 was performed to investigate positional roles of individual amino acids in supporting the binding and physiological mechanisms between Cx36 and tubulin. In contrast to the similar expression observed between Cx36\u0394279\u2013292-EGFP and WT Cx36 A, all po \u00d7 10\u22123) C. Specif \u00d7 10\u22121) D as did n = 37, p < 0.01 \u00d7 10\u22121). Amino acids K279A and K281A also had a marked reduction in co-localization to the tubulin cytoskeleton , suggesting that lysine residues have a significant role in direct binding or anchoring to tubulin. The remaining mutants were indistinguishable from the wild-type   . Based on = 273, p < 0.01 \u00d7 10\u22121; R285A: 9.8 \u00b1 0.6 \u00b5m, n = 302, p < 0.01 \u00d7 10\u22121; G286A: 9.6 \u00b1 0.6 \u00b5m, n = 286, p = 0.02 \u00d7 10\u22121). All else were indistinguishable from the WT  , and all else were indistinguishable from the WT   A. Simila \u00d7 10\u22121) B. Togeth1/2 was recorded for several mutants in comparison to the wild-type control in FRAP investigations . Furthermore, these select mutants displayed a significantly higher Mf . Our results confirmed that single amino-acid changes affected the mobility of Cx36, again, highlighting the sensitivity of this motif. Amongst these mutants, a less controlled mechanism of GJP incorporation, through the loss of protein-binding partnerships, was being promoted. Consistent with our previous model , paired with an excessive increase in T1/2 (32.18 s)   . Please  \u00d7 10\u22122) .This research uses a neuroblastoma model of synaptic plasticity to explore the interactions between Cx36 and tubulin. By sequence similarity to Cx43 , a tubul6-E-X4-L core consensus sequence, surrounded by several conserved polar, hydrophobic, and charged amino acids [The Cx36\u2013tubulin-binding motif differs from other microtubule-binding motifs found in proline-rich regions of several microtubule-associated proteins (MAPs) or the motor proteins kinesin and dynein. For example, the microtubule-binding motif in both motor proteins has a P-Xno acids ,47. SimiThree-dimensional structural modeling of the Cx36\u2013tubulin-binding site to the engineered stathmin-like domain (SLD) predicted similarities between Cx36 and SLD. The Cx36 carboxyl terminus likely adopts a helical structure upon binding with tubulin similar to previous reports on Cx43\u2013tubulin interactions . The preThe Cx36\u0394279\u2013292 mutant exhibited less co-localization to the ER\u2013Golgi complex but more co-localization with caveolin-1 vesicles. Although the ER stress response was not explored in this study, equal protein expression of wild-type and mutant proteins as well as lack of signs of protein degradation suggest that involvement of the endoplasmic reticulum-associated degradation (ERAD) pathway is minor. We propose a mechanism in which the mutant protein is prematurely released from the ER\u2013Golgi complex prior to its packaging into transport vesicles. Since we have demonstrated that Cx36\u0394279\u2013292 is still transport competent, a different mechanism of transport may be favored. Interaction with actin is a possibility. The increase in the association between Cx36\u0394279\u2013292 and caveolin-1-positive vesicles could be an indication of alternative transport pathways.The pharmacological manipulation of the microtubule network and genetic manipulation of the tubulin-binding motif confirmed that the trafficking of Cx36-carrying vesicles and the formation of GJPs were critically dependent on an intact and functionally dynamic cytoskeleton. The processes observed appear to follow the generally accepted chain of events in which connexons packaged in vesicles emerge from the Golgi reach the cell membrane via microtubules at multiple nonrandom insertion sites ,33,34. Af was reported to be 56% and subsequently decreased to 41% after sequential bleaching. Wang et al. also found half-time of recovery to be 1.55 \u00b1 0.22 s [1/2 is typically reflective of weaker binding mechanisms, incorporation of Cx36 at the GJP under these circumstances was likely mediated by diffusion rather than a controlled transport mechanism.Lauf et al. previously described the directed transport of Cx43 to the plasma membrane, demonstrating mobility of approximately 70%, before incorporation into GJPs. At GJPs, Cx43 has been described as exhibiting either low or high mobility states at the lateral ends. Categorization of the mobility state was found to be independent of GJP size but, rather, to be determined by the C-terminal domain influencing channel density, protein interaction candidates, or both . Our stu\u00b1 0.22 s . In thisTheoretical and practical considerations suggest that other transport mechanisms must exist alongside tubulin-dependent transport. In neurons, microtubules participate in axonal vesicular transport and tubulin entry into dendritic spines is activity-dependent or associated with development . LateralWayakanon et al. investigated the consequences of GJ transport and internalization of various CT truncated Cx43 mutants. Deletion of amino acids 235\u2013242, which corresponds to the tubulin-binding motif, resulted in the absence of GJPs . In contWhat is the physiological relevance of Cx36 interaction with microtubules? Cx36 has one of the lowest voltage sensitivities  and single-channel conductance (10\u201315 pS) among other connexin isoforms . The detOur investigation provides insight on a critical step towards molecular and functional asymmetry at a vertebrate electrical synapse. Tubulin-dependent transport connects two major bookends of the life cycle of Cx36. Before interaction with tubulin, direct interaction with CaM occurs primarily at the ER/Golgi complex . Vesicul"}
+{"text": "A novel method for numerical modelling of noncollinear and nonlinear interaction of femtosecond laser pulses is presented. The method relies on a separate treatment of each of the interacting pulses by it\u2019s own rotated unidirectional pulse propagation equation (UPPE). We show that our method enables accurate simulations of the interaction of pulses travelling at a mutual angle of up to 140\u00b0. The limit is imposed by the unidirectionality principal. Additionally, a novel tool facilitating the preparation of noncollinear propagation initial conditions - a 3D Fourier transform based rotation technique - is presented. The method is tested with several linear and nonlinear cases and, finally, four original results are presented: (i) interference of highly chirped pulses colliding at mutual angle of 120\u00b0, (ii) optical switching through cross-focusing of perpendicular beams (iii) a comparison between two fluorescence up-conversion processes in BBO with large angles between the input beams and (iv) a degenerate four-wave mixing experiment in a boxcar configuration. Even more fundamental approach, which drops the paraxial approximation and treats nonlinear polarization (and possibly free currents) in a general way can be derived from the vectorial Unidirectional Pulse Propagation Equation (UPPE)3. These models enable numerical simulation of nonlinear processes in the presence of frequency dependent diffraction, dispersion and spatial walk-off. They do not, however, immediately enable noncollinear beam propagation. Still, as they were the most accurate models avaiable they have been used for noncollinear simulations of Optical Parametric Chirped Pulse Amplifiers (OPCPA)7. Only a small range of noncollinearity angles has, however, been considered  to preserve the accuracy of calculations.One of the major achievements in modeling of ultrafast nonlinear processes is a framework for numerical simulation of second-order nonlinear processes based on only two assumptions: unidirectionality of propagation and paraxial approximation10, therefore, their representation requires huge numbers of points in the simulation grid. Consequently, the full 3D models are used only when the signal pulses are moderately chirped11. In other cases the numerical reality forces simplifications and, therefore, additional assumptions. For example, three dimensional simulations with neglected higher order dispersion and diffraction are often in use14. Another common practice applied to lower the memory requirements is to reduce the dimensionality. Thus two dimensional simulations with one of the Cartesian dimensions dropped17, sometimes with no diffraction and no walk-off included19, are used. Noncollinear OPA geometry is known to provide very large bandwidths22, the simpler\u2013collinear propagation model is, however, often used to model its operation. A good example of this approach are the two dimensional models with cylindrical coordinates23. Beams in such simulations are intrinsically collinear. To account for noncollinearity ad hoc spatial overlap parameters are sometimes included25. Dispersionless models with spatial effects26 and finally, one dimensional simulations with dispersion treated up to the second order27, or even with no dispersion30 can also, without question, be of value in some applications.Accurate as they are these models are also resource consuming. In the case of OPCPA the challenge comes from the fact that the interacting pulses are not only extremely broadband but can also be stretched up to nanosecond durations31. Therefore, a search for more accurate simulation methods and mathematical tools which could reduce the computational requirements for these methods is justified.The extra assumptions pointed out above are well justified when general qualitative results are of interest. However, a more accurate modeling is always tempting as it may reveal some subtle effects obscured by too crude approximations. Recently, some of us have shown that the use of a correct ab initio approach to nonlinear pulse interaction simulations applied to a design of a nonlinear optical device can result in the threefold efficiency increase with respect to the efficiency of previous solutions and an unprecedented reduction in the size of the device34 can serve as a good example. Apparently, to this day no propagation simulation approach to these problems have been attempted.The need for a robust noncollinear simulation is even more pronounced in the case of broadband frequency mixing at large angles. The fluorescence up and down conversion processes with 10\u00b0\u201330\u00b0 angles37, Raman based40 and (with a proper model for the medium nonlinear polarization response) 2D-IR41 experiments can be considered. While these experiments are often performed in a noncollinear configuration the literature on appropriate propagation models is minimal or nonexistent.Yet another area of interest for noncollinear propagation is a variety of four-wave mixing (FWM) processes. Here degenerate and two-color resonant FWM42. First, it requires resoling the whole simulation domain of interest (not only the vicinity of the pulse) with a resolution high enough for the features of the pulse itself to be resolved. Additionally, the time step is limited so to fit the spatial grid resolution (Courant condition). Therefore, a supercomputer cluster instead of a laptop and a time of several days instead of minutes is required per single simulation performed with FDTD. Also, FDTD presents complications when nonlinear effects  have to be implemented43. Finally, the accuracy of FDTD is much smaller than that of the spectral  methods44.Although, the direct solution of Maxwell equations with finite difference time domain (FDTD) methods can, in principle, be considered as an alternative to unidirectional methods, it suffers from signifcant flawsIn the present paper we describe a novel method for numerical simulations of noncollinear pulse propagation and nonlinear interaction using just the unidirectionality approximation. In this method the angle between the interacting beams is limited only by the unidirectionality principle and, therefore can exceed 140\u00b0 (depending on the beam size). The method relies on expressing the separate UPPE describing propagation of each of the beams in a single common spatial coordinates system. In this new system the interaction of pulses through nonlinear terms can easily be calculated while the rotated linear terms assure accurate propagation. Our approach is valid as long as the volumes of the spectral space used for representation of the particular interacting pulses with the same polarization do not overlap.45. This technique can be easily applied to rotations of a complex number based electric field. It is more than three orders of magnitude faster than the linear 3D interpolation even for relatively small grids. For large grids the Fourier rotation becomes an enabling tool as the rotation time can be reduced from days (as in case of interpolation) to minutes. It is also expected to be more accurate than the common interpolation techniques45.Our method enables simulation of noncollinearly propagating pulses. Some preparation of the input pulses  is, however, required. We, therefore, present a Fourier transform based 3D rotation procedure inspired by the image processing techniqueslinear interference of ultrashort pulses.optical switching through cross-focusing of beams crossing each other at a 90\u00b0 angle.a fluorescence up-conversion in a BBO crystal where we compare type II, and type I configurations .a degenerate four-wave mixing experiment in the boxcar configuration for which we observe spectral narrowing as the angle between the beams is increased.To demonstrate the capabilities of our method, we present a number of linear and nonlinear propagation examples:3:p represented in Fourier space with \u1ff6\u2013the optical frequency and kx, ky\u2013the spatial frequencies or wavevector z or more specifically only the part of electric field propagating towards positive values of z as this is a unidirectional equation. The ep\u2009=\u2009Ep/|Ep| is a unit polarization vector and |.| describes length of vector. The wavevector component along the propagation axis kx, ky and refractive index n via the dispersion relation:The most accurate unidirectional model of propagation in nonlinear media, derived from Maxwell equations with a single assumption of unidirectionality is the one based on the vectorial UPPEp\u2009=\u2009o) and extraordinary (e) modes in uniaxial or slow (s) and fast (f\u2009) modes in biaxial materials. In homogeneous medium representation of two modes is also required and one can select p\u2009=\u20091, 2 in this case. The dependence of kx, ky) is responsible for dispersion and diffraction, respectively. The additional dependence of refractive index on kx, ky present in biaxial materials is responsible for spatial walk-off (double refraction). All the above mentioned linear effects are treated exactly in UPPE. The PNL\u2009=\u2009PNL and j\u2009=\u2009j are the nonlinear part of the polarization vector and the free current vector47. UPPE is solved for electric field components perpendicular to z axis. The missing Ez component of electric field vector required for calculation of PNL and j can, however, be obtained from Ex and Ey48.For the case of birefringent materials Eq. . can be Ep:\u03c9R is a certain reference frequency . This substitution is equivalent to variable change (shift operation) in the Fourier space, the Fourier transform of Eq.  in a xl\u2009\u2212\u2009yl\u2009\u2212\u2009zl system, will require solution of its own UPPE represented in a new coordinate system x\u2032\u2009\u2212\u2009y\u2032\u2009\u2212\u2009z\u2032:The central idea of our work is to represent each of the interacting pulses in a separate discrete cuboid grid and write a rotated UPPE for each of the pulses separately see Fig.\u00a0.. The grAi, i\u2009=\u20091, 2, 3) in the t\u2032\u2009\u2212\u2009x\u2032\u2009\u2212\u2009y\u2032 space is required. Often it is interesting to simulate propagation of complicated shape of electric field. It can involve Hermite and Laguerre spatial modes as well as secant, sinc, super-Gaussian or just arbitrary temporal shape known from SPIDER or FROG measurement of an experimental pulse. While, discretization of such shapes in arbitrarily rotated coordinates (t\u2032\u2009\u2212\u2009x\u2032\u2009\u2212\u2009y\u2032) is difficult it is easy in non rotated grids. It is, therefore, of essence to find a procedure for arbitrary rotation of electric field within discretized grids. Moreover, this procedure is required at the end of simulation for back rotation of the fields and retrieval of output temporal and spatial profiles. In the Methods\u2019 \u201cArbitrary Fourier rotation\u201d section we present such a procedure based on Fourier transform and shear operations. This method can be faster than interpolation by as much as 3 orders of magnitude /c\u2009=\u2009\u03c9 nR/c is the normalized detuning from the reference frequency. Here 4 crystal for the angle between z and zC axes of 48\u00b0 . The dispersion comes from slight deviation from linear growth of the phase along the vertical axis in Fig.\u00a0kz along the horizontal axis. And finally the presence of walk-off reveals itself in slight asymmetry of the colour pattern around the vertical axis. There are no propagating wave solutions for In the process of simulation the linear phase term (from Eq. . or K\u2032p from Eq. .) has toentclass1pt{minimax\u2032\u2009\u2212\u2009y\u2032\u2009\u2212\u2009t\u2032 space\u2013this requires inverse 3D Fourier transformation and back transformation of the mixing result to the \u03ba\u2032 space. Finally, Eq.\u00a0In each step of the simulation the nonlinear polarization is calculated in the kx\u2009\u2212\u2009ky\u2009\u2212\u2009\u03c9) used for representation of particular interacting pulses with the same polarization do not overlap . We have verified that this condition is easily fulfilled for a standard NOPA setup.Our approach is valid as long as the volumes of the spectral space  for beam sizes above 10\u2009\u03bcm, for which the paraxial approximation is valid, have been compared with with SNLO50 and Hussar software31. The results were found to be in perfect agreement. Then the results of propagation for various values of \u03b8 have been compared to results of the forward propagation. Both linear and nonlinear (SHG) propagation through 5\u2009mm BBO crystal have been tested for various beam sizes (3\u2013300\u2009\u03bcm) and pulse durations (3\u2013300\u2009fs). The linear propagation tests have also been performed for constant width of 10\u2009\u03bcm and pulse durations down to 1.15\u2009fs (1.4 cycle) and for various width (w0\u2009\u2264\u20091\u2009\u03bcm) and constant duration of 10\u2009fs in 1\u2009mm of fused silica. The nonlinear propagation requires multistep solver, thus, the Integrating Factor Runge-Kutta 4, 5 Dormand-Prince method (IFRK45)51 with adaptive step-size control52 have been used. The linear problem can be solved in a single propagation step as in this case: 53, which does not subdivide the steps into smaller parts was used. Both: Integrating Factor and Exponential methods are specially designed for problem with large linear therm.First the results of forward propagation  the error in beam waist, pulse duration and energy was bellow 3\u00b710\u22123 for the single step linear cases (for pulses longer then 1.15\u2009fs the errors are bellow 10\u22125). For the nonlinear case the error depends on the step size. We have found, however, that, for a given distance, it can be easily reduced down to 10\u22124 for reasonable step sizes\u2013a multiple of the light wavelength (grid size 1024(t)\u2009\u00d7\u20091024(x)). The detail description of the tests of the method is present in the supplementary material.In case of rotated UPPE method the ultimate limitation appears when pulse contains components that would propagate in the negative z-marching schemes were used\u2013IFRK45 and an EE equipped with Richardson extrapolation52 for automatic step size selection. The step sizes were selected automatically so that the relative errors bellow 10\u22126 in each step were assured. The sizes and densities of the grids were increased to a point at which relative differences in parameters resulting from consecutive simulations: energies, beam waists and pulse durations were below 10\u22123 . Additionally, the spectral, spatial and temporal profiles were also compared visually. This convergence has occurred for grid sizes that enabled simulation on a 16\u2009GB RAM laptop computer. The results have been, however, confirmed with simulations using larger grids run on a 64\u2009GB RAM PC.For each of the examples, two \u03bcm waist and central wavelength of 800\u2009nm through 20\u2009\u03bcm of ZnSe. A \u201ctop view\u201d of the initial pulse and the result of propagation are presented in Fig.\u00a0The noncollinear but linear propagation has a potential application for solving interference problems. Here an example interference pattern for two pulses crossing each other at a mutual angle of 120\u00b0 is visualized. The pulses are created by propagation of two 3\u2009fs FWHM Gaussian pulses with 3\u200958. It has been obtained eperimentally in gases59 where the band of a supercontinuum of a probe beam was controlled through interaction with the pump. In this work apart of phase-modulation the Raman effect played an important role. Beam direction switching through attraction of beams traveling in the same direction has been studied for coaxial54, and displaced beams55. It has been studied extensively in fibers56 also for supercontinuum generation purposes57 and in fiber gratings58. Cross-focusing of coaxial beams have also been extensively studied in the context of plasma physics62.The cross-phase modulation and resulting cross-focusing has been considered as a candidate mechanism for optical switching in dielectric mediay axis  in 50\u2009\u03bcm-thick fused silica plate  and II (eoe) up-conversion processes in BBO crystal are compared. The fluorescence in range from 500\u2009nm to 900\u2009nm and the gate beam at 1020\u2009nm were selected. For type I interaction the following geometry is assumed: \u03b8G\u2009=\u200931.9\u00b0, \u03b8F\u2009=\u200958\u00b0 and \u03c6\u2009=\u200990\u00b0 (deff\u2009=\u20091.4 pm/V) with indexes G and F corresponding to the gate and fluorescence beams, respectively. For type II: \u03b8G\u2009=\u200934.3\u00b0, \u03b8F\u2009=\u200953.4\u00b0 and \u03c6\u2009=\u20090\u00b0 (deff\u2009=\u20091.18 pm/V). The mutual beam angles are 26\u00b0 and 19\u00b0, respectively. The gate pulse with a Gaussian temporal profile (100\u2009fs FWHM), energy of 11\u2009\u03bcJ in a Gaussian beam was selected to simulate conditions of the experimental setup at IPC PAS63. The beam waist was assumed to be w0\u2009=\u20090.5\u2009mm. The fluorescence was modelled by a pulse with a super-Gaussian (flat top) spectrum ranging from 510\u2009nm to 865\u2009nm. The fluorescence pulse was chirped with 50\u2009fs2 to the duration of 100\u2009fs (FWHM). Figure\u00a0In this section we will present the first ever 3D numerical simulation of the fluorescence up-conversion process under high mutual angles of the fluorescence and gate beams. The search for optimal conditions for efficient and broadband up-conversion in spectroscopy is a subject of a live debate lasting at least since the begining of the centuryISum) normalized to the fluorescence spectral power density (IF) for three thicknesses of the BBO crystal and for the two phase-matching types are presented in Fig.\u00a034 for retrieving fluorescence from the up-converted light. The type II phase-matching delivers both: more efficiency and a broad spectrum up-conversion. This confirms its superiority over type I phase-matching for 1020\u2009nm gate and is consistent with results obtained experimentally for 1340\u2009nm gate34. Note, that for the 150\u2009\u03bcm crystal the quantum up-conversion efficiency exceeds 0.1.The spectral power density of the up-converted signal (n2\u2009\u2248\u200910\u221218\u2009m2/W64) together with a broadband  signal pulse stretched to around 1.1\u2009ps through introduction of 16 000\u2009fs2 of dispersion.Degenerate four-wave mixing between pulses at 1550\u2009nm in a box car configuration is considered in the present section. Two narrowband pump pulses  are sent into a highly nonlinear 5\u2009mm ZnSe crystal  pulse is created. The idler pulse is broadband and its phase is similar to that of the signal pulse with, however, an opposite sign  first. Then the temporal centers and finally tails of the pulses have the chance to interact.The situation suggests that, as well as in the collinear situation, all the frequencies will be amplified in a similar way. This is, however, not the case because the idler pulse is generated mostly in the area where the other three beams overlap. Figure\u00a0We estimate that performing an analogous simulation with FDTD would require ~3500 times more RAM (above 1\u2009TB) and ~85 times more computational steps, which can translate into days of simulation instead of 1\u2009minute with the current approach.We have presented a novel method for numerical simulation of noncollinear pulse propagation and nonlinear interaction. We have found that the method works properly even for the mutual angles between the propagation directions of the interacting pulses as high as 140\u00b0, it is limited by unidirectional approximation. The techniques have been tested on linear and nonlinear propagation examples.We have also presented a novel method for arbitrary 3D roation of the complex electric field. Our method, in comparison to interpolation presents a speed-up on a level of three orders of magnitude.Our simulations shown that optical switching is possible through cross-focusing even for very short interaction lengths i.e. even in the case of perpendicular pulse routes. We have shown that the type II phase-matching delivers both: more efficiency and a broad spectrum up-conversion and, thus, it is a preferable method for fluorescence up-conversion in BBO. Finally, we have shown that increase of noncolinearity angle in degenerate four-wave mixing experiment can lead to spectral narrowing of the generated signal pulse.z\u2032 axis by \u03b4 (Rz\u2032(\u03b4)), around y\u2032 axis by \u03b8 (Ry\u2032(\u03b8)) and again around z\u2032 by \u03d5 (Rz\u2032(\u03d5)). The \u201cnormalized frequency\u201d (\u03ba) and time  becomes:It is convenient to use three Euler angles and to represent the actual pulse direction as a result of three consecutive rotations: around PNL,p\u2009=\u2009epPNL where the envelope defined by Eq. (To simplify the notation in this section UPPE (Eq. ) is writd by Eq. . was useWith the above described variable change rotated UPPE yields:xB, yB, zB), ,  represents the system of the beam, crystal and simulation, respectively.In multi-pulse propagation simulations it is convenient to set the direction of one of the beams with respect to the simulation coordinates system and to define the direction of all the pulses with respect to the (often birefringent) crystal. We therefore give expressions for transitions between the three coordinate systems:\u03d5B, \u03b8B and \u03b4B, then:Ra in which the very matrices are represented , thus, they have a simple classical form, similar to that of the upper left part of second transformation matrix in Eq.  and the beam orientation with respect to the crystal orientation  is known, then:If, on the other hand, the crystal orientation with respect to the simulation frame  are not in general parallel to the unit vectors defining the simulation coordinate systems. These vectors are, however, available as the columns of RCS matrix and the form of the matrix defining a rotation about an arbitrary vector can be found in reference69.Note that the matrix \u03d5B, \u03b8B, \u03b4B) and the material orientation with respect to the beam  are known:xB, yB, zB) are the columns of RBS matrix. The matrix RBS and angles \u03d5, \u03b8, \u03b4 are required for calculation of the linear propagation phase term K'p as will be shown in the next section. The angles \u03d5, \u03b8, \u03b4 can be obtained from RBC70.Finally, if beam propagation direction  a corresponding set of coordinates from the beam reference frame  is calculated through rotation Eq.  and \u03d5\u2013angles defining the direction of propagation of each of the plane waves  with respect to the crystal orientation are initialized to given values defining the general beam direction (\u03b80 and \u03d50). Again \u03b8 and \u03d5 are matrices numbered by \u03ba\u2032, kx\u2032 and ky\u2032, the corresponding values of \u03ba, kx and ky are, however, known from the previous step.Then, matrices of \u03b8 and \u03d5 the values of refractive index np\u2009=\u2009np are calculated .The iteration can be stopped when the change of Q\u2032p can already be calculated from Kp and Eq.  is uniquely defined by \u1ff6, kx and ky. The procedure for finding epC\u2013the electric field vector direction in the crystal coordinate system\u2013is known73 and implemented in Hussar software31. We will assume that this vector does not change due to nonlinearity. We have verified that, for a \u201cworst case crystal\u201d with linear properties of highly birefringent YVO 4, and Kerr constant of 10\u221218\u2009m2/W characteristic for highly nonlinear ZnSe64 illuminated with intensity of 3400\u2009GW/cm2\u2013the damage threshold intensity of highly resistant BBO crystal for 25\u2009fs pulses at wavelength of 800\u2009nm73, the actual change of ee components is around 1%. For more common conditions this error will not exceed the one coming from currently obtainable accuracy of the refractive index measurements\u201310\u22124 (see refractive index measuremnt references in73). Total electric field can, therefore, be decomposed into:p\u2260q. Moreover, r, s\u2009=\u2009x, y. Then, for a medium with second order nonlinearity characterized by nonlinear suscetibility \u03c7(2):Note first that in absence on nonlinearity birefringent media electric field vector of a particular mode (q\u2009+\u2009q\u2009\u2192\u2009p) and SFG (p\u2009+\u2009q\u2009\u2192\u2009p) becomes:\u03c7eff represents the effective nonlinear coefficients. We have verified that for a set of 18 nonlinear crystals  if the x axis is selected along the polarization vector for the wave propagating exactly along the z axis (\u22122 for Gaussian beams with waist above 1.4\u2009\u03bcm at 532\u2009nm (divergence of ~7\u00b0) and less than 10\u22123 for beam widths above 4.5\u2009\u03bcm (divergence of ~2\u00b0). Note, therefore, that in practice it is safe to assume \u03c7(2)  give results with accuracy of 5\u201310% at best73.Which for SHG  to a mixed space, multiplication by a phase factor and back transformation to . The phase factor has to depend linearly on the Fourier space variable as well as on one of the remaining real variables. Two shear operations are required for single rotations. The definitions of the operators and corresponding Fourier transform operations are listed in the Table\u00a0Here, we describe a convenient way of rotating an arbitrarily shaped pulse without the use of interpolation which is erroneous and time consuming when applied to a 3D case. The inspiration for the method comes from the raster image rotation well known in computer graphicsRz(\u03c6)Ry(\u03b8)Rz(\u03b4). The three Euler angles corresponding to consecutive rotations around z, y and z axes  are used to achieve complete freedom of pulse manipulation. Figure\u00a0Rz(\u03c6\u2032)Rx(\u03b8\u2032)Rz(\u03b4\u2032) with second rotation performed around x axis. Therefore, the above described rotation can be constructed in the following way:A traditional 3D rotation can be constructed from 3 rotations: It is worth to note that the order of the shear operations for each rotation can be reversed (eg.: ters Eqs  and 31)\\document45. Any rotation by angle \u03b1\u2009>\u200945\u00b0 can be, however, decomposed into a trivial 90\u00b0 rotation and rotation by 90\u00b0\u2009\u2212\u2009\u03b1. Thus, to perform rotations by higher angle values we propose following procedure:\u03c6\u2009\u2208\u2009\u2009\u2229\u2009 set \u03c6\u2009\u2192\u2009\u03c6\u2009\u2212\u200990\u00b0, \u03b4\u2009\u2192\u2009\u03b4\u2009+\u200990\u00b0, \u03b8\u2009\u2192\u2009\u2212\u03b8, perform second rotation around x axis.if \u03c6\u2009\u2208\u2009 set \u03c6\u2009\u2192\u2009\u03c6\u2009\u2212\u2009180\u00b0, \u03b8\u2009\u2192\u2009\u2212\u03b8, use standard rotation.if \u03c6\u2009\u2208\u2009 set \u03c6\u2009\u2192\u2009\u03c6\u2009\u2212\u2009270\u00b0, \u03b4\u2009\u2192\u2009\u03b4\u2009+\u2009270\u00b0, perform second rotation around x axis.if \u03b4 back into a \u2009\u2229\u200945\u00b0, 135\u00b0] replace E with Eif \u03b4\u2009\u2208\u2009]135\u00b0, 225\u00b0] replace E with Eif \u03b4\u2009\u2208\u2009]225\u00b0, 315\u00b0] replace E with Eif The use of Fourier transform for rotations is limited to around 60\u00b0\u2009\u03b8\u2009\u2208\u2009. For \u03b8\u2009>\u200960\u00b0 a scheme involving 90\u00b0 rotations in the x\u2009\u2212\u2009\u03b6 and y\u2009\u2212\u2009\u03b6 plane would be required. Small corner regions of the rotated surface are affected by artifacts arising from the periodic nature of the Fourier transform algorithm45. This, however, is a minor concern when the electric field is concentrated in the center of the x\u2009\u2212\u2009y\u2009\u2212\u2009\u03b6 plane, which is the usual case with an optical pulse.The scheme presented above can be used for The speed advantage of the Fourier transform base rotation with respect to the 3D interpolation (MATLAB\u2019s griddata function) for different grid sizes is presented in Fig.\u00a0Chirp pulse interference.Up conversion process.Four-wave mixing in boxcar configuration.Supplementary information"}
+{"text": "Scientific Reports 10.1038/s41598-018-35086-z, published online 09 November 2018Retraction of: Authors retract this Article.In the study the Authors used an RNA-seq dataset that was not generated in their laboratory and was not publicly available for re-use.All Authors agree to the retraction."}
+{"text": "In this study, phosphorylated birchwood xylan was produced under alkali conditions using trisodium trimetaphosphate. Three single-factor experiments were used to explore the influences of time, temperature, and the molar ratio of trisodium trimetaphosphate to xylan on the degree of substitution (DS) and charge density of xylan. The response surface methodology was used to explore the interaction of these three factors. Phosphorylated xylan with a maximum DS of 0.79 and a charge density of \u22123.40 mmol/g was produced under the optimal conditions of 80 \u00b0C, 4 h, and a molar ratio of xylan/sodium trimetaphosphate (STMP) of 1/3. Fourier transform infrared (FTIR), ascorbic acid method analyses, and inductively coupled plasma\u2013atomic emission spectrometer (ICP-AES) analyses confirmed that the phosphate groups were successfully attached to xylan. Thermogravimetric analysis confirmed that phosphorylated xylan was less stable than birchwood xylan. Furthermore, the phosphorylated xylan was applied as a flocculant for removing ethyl violet dye from a simulated dye solution. The results indicated that more than 95% of the dye was removed from the solution. The theoretical and experimental values of charge neutralization for the dye removal were close to one another, confirming that charge neutralization was the main mechanism for the interaction of dye and phosphorylated xylan. The impacts of salts on the flocculation efficiency of phosphorylated xylan were also analyzed. Xylan is an abundant natural polymer with a structure similar to that of cellulose; it accounts for 15\u201330% of the biomass . The polEthyl violet (EV) is a triphenylmethane dye with extensive industrial applications . It has Codonopsis pilosula polysaccharide was synthesized and characterized, and its ability to inhibit the virulence of duck hepatitis A virus was compared with that of an unmodified Codonopsis pilosula polysaccharide [Chrysanthemum indicum polysaccharide with a degree of substitution (DS) of 3.17 using the STMP-STPP  method [Among the modification methods, phosphorylation modification methods can be used to significantly enhance bioactivities , which cccharide . In anotccharide . Song anccharide . Ghimiciccharide . Ming an) method . HoweverThe current study investigated the production of phosphorylated xylan with STMP and the interaction of reaction parameters to obtain phosphorylated xylan with a high DS and charge density under alkali conditions. Furthermore, the characteristics of the prepared phosphorylated xylan and the application of phosphorylated xylan as a flocculant for the removal of ethyl violet dye from a simulated dye solution were analyzed. The main novelties of this work are the following: (1) the production of phosphorylated xylan and the optimization of the phosphorylation reaction of xylan by grafting STMP onto a xylan backbone; and (2) the assessment of the performance of phosphorylated xylan as a flocculant in isolating cationic dye segments from a simulated ethyl violet dye solution.Xylan from birchwood, STMP used as received without further purification, and polydiallyldimethylammonium chloride (PDADMAC) diluted to 0.001 M prior to use were purchased from Sigma-Aldrich, Shanghai, China. EV dye was also obtained from Sigma Aldrich and diluted to 100 mg/L for use. All other chemicals used herein were of analytical grade and without further purification. The chemical structure of ethyl violet is presented in The phosphorylation procedure of xylan was carried out using STMP as a crosslinker, according to the method stated earlier ,21. BircThe reaction mechanism of xylan with STMP (as a crosslinking agent) involved in this study was a reaction between the hydroxyl groups in the xylan and metaphosphate groups in STMP, leading to phosphate ester (O\u2013P\u2013O) linkages between two polysaccharide moieties to produce phosphorylated xylan ,22,23, aResponse surface methodology (RSM) is a powerful technique that can be applied to explore the interactions between factors in experiments and to optimize the operating conditions to obtain the best results . To expl\u22121 within the range 400 to 4000 cm\u22121.FTIR analysis was conducted on the xylan and phosphorylated xylan samples using a FTIR spectrophotometer . In this measurement, unmodified and phosphorylated xylan samples of 0.01 g each were used. Each spectrum was recorded with 32 scans in transmittance mode with a resolution of 0.5 cmApproximately unmodified and phosphorylated xylan samples of 0.02 g each were dissolved in 100 mL of deionized water and ultrasounded at 30 \u00b0C for 1 h. The solution was used to measure charge density using a particle-charge detector  against a 0.001 M PDADMAC standard solution.P% represents the phosphorus content; a is the molecular weights of the unmodified xylan\u2019s monomeric unit, 132 g/mol; b is the mass of phosphorous atom, 31 g/mol; and c is the added molecular weight when \u2013OH in xylan was substituted by \u2013OPO3Na2 [The phosphate radical content of the phosphorylated xylan was determined using the ascorbic-acid method . Accordi\u2013OPO3Na2 , 124 g/m3 by stirring at 500 rpm for 36 h at 35 \u00b0C, and the solutions were then filtered using a 0.2 \u00b5m nylon filter. The filtered solutions were used for molecular weight analysis. The molecular weight analysis of the samples was carried out using gel permeation chromatography, Heleos-II GPC  with a multi-angle laser light scattering detector. The columns of PolyAnalytic PAA 206 and PAA 203 were set up at 35 \u00b0C, and a 0.1 mol/L NaNO3 solution was used as a solvent and an eluent. The flow rate was set at 0.50 mL/min, while poly(ethylene oxide)s were used as standard samples for the calibration of this aqueous system. The degree of polymerization of unmodified and modified xylan was calculated based on Equation (3).nM is the number averaged molecular weight of xylan, g/mol. The molecular weight of xylan\u2019s monomeric unit is 132 g/mol. DS is the degree of substitution of modified xylan. The added molecular weight when \u2013OH in xylan was substituted by \u2013OPO3Na2 is 124 g/mol.Dried unmodified and phosphorylated xylan samples of approximately 5 mg each were dissolved in 0.1 mol/L NaNOElemental analysis of phosphorus in xylan and phosphorylated xylan was carried out using an inductively coupled plasma\u2013atomic emission spectrometer . The sample was dissolved in 0.1 mol/L HCl and further diluted with water. The measured concentration of phosphorus in aqueous solution was converted into actual phosphorus content, taking into consideration the dilution factor . The conThe thermal analysis of unmodified and phosphorylated xylan samples was performed on a thermogravimetric analyzer . Samples of 3\u201310 mg were heated from room temperature to 600 \u00b0C at the rate of 10 \u00b0C/min under a nitrogen environment to examine the thermal stability properties.The viscosities of xylan and phosphorylated xylan were measured at different concentrations at 25 \u00b0C using a Brookfield DV-II + Pro viscometer . The viscosities were measured in aqueous solutions (pH 7) at room temperature. In this set of experiments, different concentrations of xylan or phosphorylated xylan solutions were placed in the spindle No. S61 of the viscometer, and the spindle rotation was adjusted from 1 up to 100 rpm to measure the viscosity of the samples.C0 refers to the concentration of the ethyl violet dye in the control sample (mg/L); and C refers to the concentration of the dye after treatment with phosphorylated xylan (mg/L). To investigate the impact of salts on the efficiency of phosphorylated xylan in removing the dye from the solution, dye solutions (100 mg/L) containing 0.01 mol/L NaCl, NaNO3, and FeSO4 were prepared. Phosphorylated xylan was added to 30 mL of a dye solution containing salts at pH 9. After that, the dye solution was treated as stated above , and the removal of the dye was determined according to Equation (4). The experiments were repeated three times, and the average values reported in this work.In this set of experiments, ethyl violet dye solutions were prepared at different concentrations by dissolving the dye in deionized water, and the pH of the dye solutions was adjusted to the desired value by adding a HCl or NaOH solution to it. Then, different amounts of phosphorylated xylan solution were added to the dye solutions, and the mixtures were continuously stirred at 30 \u00b0C for 30 min at 150 rpm. After that, the mixtures were centrifuged at 10,000 rpm for 10 min, and the filtrate was collected for analysis. The concentration of the dye in the solutions (before mixing with phosphorylated xylan and after centrifugation) was measured using a UV-2550 spectrophotometer  at a wavelength of 596 nm. The removal of the dye from the solution was calculated according to the following equation:Co and C are the COD concentrations of the dye solutions before and after treating with phosphorylated xylan, mg/L, respectively.The chemical oxygen demand (COD) test is commonly used to indirectly measure the amount of organic pollutants found in wastewater. In this set of experiments, the samples were treated with potassium dichromate under acidic conditions with silver as a catalyst at 150 \u00b0C for 2 h in a thermoreactor . The solutions were cooled for 20 min in the digester then stored in the dark for 30 min . The conThe effect of the reaction temperature on the DS and charge density of phosphorylated xylan is illustrated in F-value, and p-values) were estimated and are presented in p-value, A, B, C, AB, AC, BC, A2, B2, and C2 were less than 0.05, implying that the correlation factors were significant [2 of 0.9790 and 0.9395 for DS and charge density were in agreement with the adjusted R2 of 0.9970 and 0.9914, respectively. In this case, the F-value of B (Temperature) was the smallest of the three factors, indicating the negligible effect of temperature when compared to other factors (time and xylan/STMP molar ratio) in increasing the DS and charge density of phosphorylated xylan. Furthermore, it was clear that time had a major effect on the DS and charge density of phosphorylated xylan [The results in nificant . The preed xylan . Mw) and number-average molecular weight (Mn) of the phosphorylated xylan sample were 23,500 g/mol and 18,600 g/mol, respectively, whereas those of unmodified xylan were 20,800 g/mol and 11,850 g/mol, respectively. A slight increase in the molecular weight of xylan due to the grafting phosphate group was also observed in another work [Mw to Mn, may be due to the grafting of the phosphate group to xylan. The degree of polymerization of xylan was decreased from 90 to 81, which was probably due to the degradation of the xylan chain in alkaline conditions [The properties of phosphorylated xylan with a DS of 0.79 and charge density of \u22123.40 mmol/g, which were produced under the conditions of 80 \u00b0C, 4 h, and with a xylan/STMP molar ratio of 1/3, are listed in her work . The decnditions .5 unit formulas of xylan and phosphorylated xylan were also calculated based on the elemental analysis. It was clear that the carbon content decreased from 42.5 to 19.1%, the oxygen content increased from 47.1 to 55.9%, and the hydrogen content decreased from 5.8 to 2.21%, which was attributed to the high content of oxygen and the low content of carbon and no hydrogen in \u2013OPO3Na2 grafted to xylan backbone. The content of phosphorus increased from 0.03% of unmodified xylan to 23.10% of phosphorylated xylan. The phosphorus here was clearly from the \u2013OPO3Na2 segment in phosphorylated xylan as the unmodified xylan only had unnoticeable phosphorus content (0.03 wt %). These changes confirmed the successful phosphorylation of xylan using STMP. In addition, the phosphorous content measured using ICP-AES, 31.81%, was used to calculate the DS through Equation (1), and was 0.783, which was very close to the 0.79 value obtained using the ascorbic-acid method.Elemental analysis of the xylan and phosphorylated xylan was carried out to determine the content of carbon, hydrogen, oxygen, and phosphorous in xylan and phosphorylated xylan and the data is listed in \u22121 were associated with xylan. A broad absorbance peak at 3427 cm\u22121 corresponded to the hydroxyl stretching vibration [\u22121 was assigned to the symmetric C\u2013H stretching vibration, and that at 1648 cm\u22121 contributed to the absorbed water in the samples [\u22121 was assigned to C\u2013O stretching in C\u2013O\u2013C linkages [\u22121 was assigned to the \u03b2-glucosidic linkage between the xylose units, indicating that the xylose residues were linked by \u03b2-form bonds [\u22121 and 1164 cm\u22121 suggested the presence of arabinosyl units, which were only attached at position 3 of the xylopyranosyl constituents [\u22121 and 1042 cm\u22121 related to the C\u2013H and C\u2013O bond stretching frequencies. Compared with the spectrum of xylan, the spectrum of phosphorylated xylan appeared to have three new peaks at 1293 cm\u22121, 1105 cm\u22121, and 995 cm\u22121, respectively. The new adsorption peak at 1293 cm\u22121 was ascribed to the stretching vibration of the P=O band. The peak at 1105 cm\u22121 originated from the stretching vibration of the C\u2013O\u2013P band [\u22121 was ascribed to the O\u2013P\u2013O band [FTIR spectra of unmodified and phosphorylated xylan are illustrated in ibration . The abs samples . The strlinkages ,34. The rm bonds . The lowtituents . The regO\u2013P band ,17. The P\u2013O band . These nThermal analysis of unmodified xylan and phosphorylated xylan are illustrated in w/w). The viscosity of xylan and phosphorylated xylan solutions decreased with increasing shear rate at the same concentration, exhibiting a pseudoplastic or shear-thinning behavior in the range of shear rates tested. The effect of dye concentration on the efficiency of phosphorylated xylan is illustrated in Based on the results in + cation is added to the dye solution, it generates the electrolyte effect, which reduces the dye removal efficiency of phosphorylated xylan. If multivalent cations such as Fe2+ are present in the dye solution, there is competition between the cation and the dye segments to bind to the anionic sites of the phosphorylated xylan. These chelated units exert a partially positive charge, which promotes electrostatic repulsion between the phosphorylated xylan and the dye in the solutions [Salts are generally available in dyeing wastewater and may affect the efficiency of phosphorylated xylan in dye removals from the solution. olutions .This study indicates the effective interaction between phosphorylated xylan and a cationic dye. It is worth mentioning that the cationic dye was used only as a model of small molecules for the fundamental investigation in the current study. However, the practical application of phosphorylated xylan for dye removals may not be considered to be a stand-alone process since this process may generate phosphorylated xylan/dye sludges with a high water content that may be challenging to dispose of at industrial scales . TherefoSimultaneous adsorption and flocculation have been reported to be effective in removing organic components from wastewater ,46. PhosMw) of 23,500 g/mol. FTIR, ascorbic acid analyses, and ICP-AES analyses confirmed that the phosphate groups were successfully grafted to the xylan backbone. The thermal stability of xylan was reduced through phosphorylation. The phosphorylated xylan was used as a flocculant, and the results confirmed that 96.5% of the dye and 93.6% of COD were removed from the dye solution (100 mg/L) with phosphorylated xylan at a concentration of 350 mg/L. Furthermore, phosphorylated xylan was most effective in dye removal at pH 9 and 30 \u00b0C. The experimental and theoretical analyses confirmed that the charge neutralization was the main interaction mechanism between the dye and phosphorylated xylan. The presence of salt slightly hampered the effectiveness of phosphorylated xylan in dye removal.In this study, phosphorylated xylan with the maximum DS of 0.79 and charge density of \u22123.40 mmol/g was produced under the optimal conditions of 80 \u00b0C, 4 h, and a xylan/STMP molar ratio of 1/3. This product had a molecular weight ("}
+{"text": "Borehole transient electromagnetic (TEM) techniques have been proven to be efficient for nondestructive evaluations (NDEs) of metal casings using eddy-current properties. However, physical limitations and bad borehole conditions restrict the use of eddy-current sensors, which makes downhole casing inspections very different from those of conventional NDE systems. In this paper, we present a uniform linear multi-coil array-based borehole TEM system for NDEs of downhole casings. On the basis of the borehole TEM signal model, a numerical multi-coil array approach using the Gauss\u2013Legendre quadrature is derived. The TEM response can be divided into two independent parts related to the transmitting-receiving distance (TRD) and the observation time and casing thickness. Using this property, the signal received by the multi-coil array is weighted to cancel the influence of the TRDs of the different array elements to obtain the optimal response according to the linearly constrained minimum variance criterion, which can be shown to be identical to that of achieving the maximum signal-to-noise ratio. The effectiveness of the proposed method was verified by applying the uniform linear multi-coil array to a borehole TEM system for NDEs of oil-well casings. Field experiments were conducted, and the results demonstrate the effectiveness of the proposed method. Transient electromagnetic (TEM) techniques have gained much attention over the past several decades owing to their use in a wide range of applications in geophysical prospecting, such as mineral and petroleum exploration , geotechMuch research has previously been undertaken with respect to the design and use of eddy-current sensors to improve the NDE performance for downhole casings. Considering the limited space and the high temperatures, which result in a weak TEM response and low SNR, multi-turn coil approaches have been proposed to enhance the signal strength and to improve the inspection performance. In Ref. , the eddAt present, there have been minimal previous attempts to eliminate the influence of TRD on downhole measurements, which is difficult to suppress using the traditional multi-turn coil sensor with single-receiver ,15,16,17In this paper, we present a uniform linear multi-coil array-based borehole TEM system for NDEs of downhole casings. Using a Gauss\u2013Legendre quadrature-based numerical approach to the borehole TEM signal model, we apply a weight to the signals received by the multi-coil array according to the linearly constrained minimum variance (LCMV) criterion . It is sThe rest of this paper is organized as follows. The borehole TEM signal model based on a linear uniform multi-coil array with a coaxial transmitting coil and multiple receiving coils is presented in M receivers with an inter-element spacing of \u0394z and TRDs ranging from z1 to Mz, which are comprised of coaxial coils wound around a soft magnetic core in a cylindrically layered medium. The multi-cylindrically layered structures of the borehole TEM system including the electrical and geometrical parameters of the jth layer  are illustrated in M receivers are located in the second layer, with their number of turns given by NT and NR, respectively, where each receiver has the same number of turns. In this paper, M receivers are utilized instead of the traditional single receiver sqp denote the sqpth element of the vectors F and WTX(z), respectively. The simulation results of the RMSE are shown in In addition, the weighted array outputs of the third and fourth cases with \u0394M, we find that the width of the null of the RMSE curves becomes much narrower. Taking M = 8 as an example, even though the neighboring receivers with small inter-element spacing can be weighted to obtain a better performance, the tolerance of the proposed method for the array geometric error will also be reduced so that a high accuracy array fabrication is required, where even a 1-mm error of the inter-element spacing in the fabrication may introduce a much larger error than that of M = 6 or M = 4. Therefore, a reasonable number of receivers should consider not only the SNR and the numerical approximation, but also the array geometric error to achieve a better NDE performance. The optimization of the receiver number and the inter-element spacing of the multi-coil array need to be investigated further in future studies.With increasing A uniform linear multi-coil array-based borehole TEM system was proposed to improve the NDE performance for downhole casings. We presented a Gauss\u2013Legendre quadrature-based numerical approach for the borehole TEM signal model. It was shown that the TEM response is coupled to the TRD, which greatly influences the NDE performance. Moreover, on the basis of the proposed numerical approximation, the received signals of the uniform linear multi-coil array were weighted according to the LCMV criterion to cancel the influence of the TRD, where the optimization and performance analyses of the proposed array weighting were also investigated. Simulations and experiments for a standardized oil-well casing inspection demonstrated the effectiveness of the proposed system."}
+{"text": "The prevalence of allergic rhinitis has increased significantly globally over the last two decades. Detection of sensitizing aeroallergens plays a crucial role in the diagnosis and management of this troublesome disease. This study aims to investigate the spectrum of aeroallergens sensitization in patients with allergic rhinitis in a tertiary care hospital.A descriptive cross-sectional study conducted in the Department of Otorhinolaryngology of our hospital between January 2016 to December 2019. Ethical approval was taken from the Institutional Review Committee (No: 210/19). Patients diagnosed with allergic rhinitis were enrolled using the convenience sampling technique. Data entry and analysis was done using IBM Statistical Package for Social Sciences version 20.0.Among 170 patients, altogether 103 (60.6%) patients yielded positive responses on the skin prick test. The most prevalent aeroallergens were Lepidoglyphus 86 (50.60%), Dermatophagoides pteronyssinus 85 (50%), Dermatophagoides farina 82 (48.20%), Thyrophagus 50 (29.40%), Blomia 46 (27.10%), Acarus 43 (25.30%), cat dander 26 (15.30%), dog dander 24 (14.10%), cow and buffalo dander 20 (11.8%), ragweed 20 (11.8%), grass pollen 18 (10.60%) and mugwort 17 (10%).This study highlights that the frequency of aeroallergens based on skin prick test in patients presenting to a tertiary care hospital which showed the dominance of house dust mites, dog and cat hair, pollen, and grasses. Reduced exposure and training of patients about protection against these agents will possibly help in controlling the severity of allergic rhinitis in this region. The study was approved by the Kathmandu University School of Medical Sciences Institutional Review Committee (No: 210/19). Patients diagnosed as AR according to the AR and its Impact on Asthma (ARIA guidelines 2019) and residing in Kavre district were enrolled in our study. Patients with Chronic rhinosinusitis, nasal polyposis, benign or malignant tumors of the nose and paranasal sinuses, or other known cases of non-allergic rhinitis like occupational rhinitis were excluded. Patients with pregnancy and drug-induced rhinitis, wheezy bronchitis, and bronchiectasis were also excluded from the study. Similarly, patients with the parasitic infestation, patients under treatment with antihistamines, steroids, and antileukotreines within 7 days period, patients under 18 years of age, severe eczema, dermographism, and those with a history of previous life-threatening anaphylaxis were not included. Convenience sampling was done and the sample size was calculated using the formula,n = required sample sizeZ = 1.96 at 95% Confidence Interval (CI)p = prevalence,50% from previous studiese = margin of error, 8%Where,Taking a 10% non-respondent rate, the sample size becomes 165. However, 170 patients were enrolled in the study. The medical records containing demographic data, presenting symptoms, symptoms of co-morbidities, general medical history, drug use, occupational and environmental exposure, family history of allergy, and smoking were collected.15 SPT was performed after patients had stopped taking long-acting antihistamines for more than 1 week and short-acting antihistamines and sympathomimetic drugs for 5 days before the test. In the present study, allergens were selected based on the plant species existing in the Kavre district and other possible allergens. The aeroallergens included in the test battery were Lepidoglyphus destructor (storage mite), Dermatophagoides pteronyssinus, Dermatophagoides farinae, Thyrophagus, Blomia, Acarus, cat dander, dog dander, cow and buffalo dander, horse dander, rat dander, Artemisia douglasiana (ragweed), Poaceae (grass pollen), Artemisia vulgaris (mugwort), Hordeum vuigare (barley), Fraxinus (ash) and Ficus religiosa (bodhi) tree pollen, Betula (birch) pollen, Piantago (plantain), Alternaria alternata, Corylus (hazel), and Aspergillus.SPT was performed according to European guidelines.A small amount of allergen extract was placed on the volar aspect of the forearm and introduced into the skin with a lancet. The lancet was penetrated at a low angle and its tip was lifted gently to raise the epidermis, without inducing any bleeding. To avoid false-positive results, the drops were placed at least 3 cm apart from each other. The test areas were numbered with a skin marker. A positive control  and negative control  were also included in the test. A separate lancet was used for each test. The test solution was wiped off immediately after the SPT with the help of an absorbent paper towel on the skin prick area and carefully pressing it on the skin, without blending the different dilutions. The mean wheal diameter was read at 15 minutes. Wheel diameter of more than 3 mm was considered positive. A positive histamine reaction (\u22653 mm) and a negative saline control reaction (<3 mm) was considered for the validity of SPT. Positive response to at least one of the allergens was accepted as the presence of sensitization.Data entry and analysis was done using IBM Statistical Package for Social Sciences version 20.0.Lepidoglyphus 86 (50.60%), Dermatophagoides pteronyssinus 85 (50%), Dermatophagoides farina 82 (48.20%), Thyrophagus 50 (29.40%), Blomia 46 (27.10%), Acarus 43 (25.30%), cat dander 26 (15.30%), dog dander 24 (14.10%), cow and buffalo dander 20 (11.8%), ragweed 20 (11.8%), grass pollen 18 (10.60%) and mugwort 17 (10%)  patients yielded positive response on SPT. The most prevalent aeroallergens were 17 (10%) .There were 101 (59.40%) females and 69 (40.60%) males. The mean age was 31.84 years. The minimum age was 18 years, and the maximum was 66 years .Dermatophagoides farinae, Lepidoglyphus destructor, Thyrophagus, Blomia, Acarus which belong to a family of House Dust Mite (HDM)s were the predominant aeroallergens followed by dog and cat dander.In the current study, we revealed some interesting findings regarding the aeroallergens sensitization spectrum in patients with AR. We observed that Dermatophagoides pteronyssinus, 17 This could be related to the high humidity and ambient temperatures which have been reported as optimal conditions for HDM propagation.18 Kavre district is located at a height of 1007 meters to 3018 meters from sea level and constitutes of 23.70%of the area belonging to the tropical region and 65.30% of the area constituting of the subtropical zone. The characteristics of the local climate are optimal for HDM propagation. Lifestyle may be another potential contributor to the high prevalence of sensitization to HDMs. Most of the Nepalese use cotton pillows, mattresses, and carpets which act as sources of HDM. At the same time, the exacerbating air pollution caused by automobile exhaust also contributes to the high detection of HDMs.19 Our study is in contrast with a study performed by Mohammadi et al.20 where the authors found that the most common allergens in that area were trees, weeds, grasses, and Dermatophagoides pteronysinus respectively. This could be explained by the fact that the spectrum of aeroallergens is significantly diverse in different countries and even in different parts of a country.21Our findings correspond with previously published studies in literature.Our study findings also illustrate the prevalence of plant pollen in Kavre. A possible explanation could be thatthere is a larger percentage of cultivated land in Kavre district. Hence, a greater proportion of people in this particular area are involved in farming for household or agricultural purposes, cutting grasses for feeding cattle and collecting firewood for cooking purposes which exposes them to plant-borne aeroallergens in particular pollen seasons.22 In our study, animal dander was also identified as an important risk factor for the development of AR. This finding is the following study by Wang J et al and in contrast with a study performed by Mohammadi et al.23 In Nepal, as in many other developing countries, many households keep cats and dogs as indoor pets. This could increase the chances of being exposed to dog and cat dander which act as allergens. This factor must be taken into account since avoiding contact with cat-dog dander allergens could probably help in the prevention of AR.Previous studies have shown that the more contacts one person has with pet allergens, the more possible he or she develops a symptom of airway hyper-reactivity.24In the present study, the total positive rate of SPT was found to be significantly higher in women than in men. This finding may be due to our study population, which itself consisted of more women which are supported by a study in Turkey.23 and Sattar HA et al.25 However, no convincing clarifications have been found in previous literature to suggest that sex leads to differential exposure to aeroallergens26 and no convincing clarifications have been given in previous literature regarding this aspect.This is in contrast with the study performed by Wang J et al 27 where the authors suggested that the sensitization of all allergens tended to increase and reached its highest degree in young adults. Conversely, studies from other countries reported no difference in the prevalence of positive SPT in AR between younger and older patients.29Our results demonstrated that the most frequently affected age group was between 20-40 with the highest peak in mid-twenties. Our findings are supported by a study by Boulet et al,To the best of our knowledge, this is the first study addressing the prevalence of common aeroallergen sensitization patterns in patients with AR in the Kavre region. However, there are several limitations to this study. The study was conducted by retrospective analysis. This study was primarily a single-center study. Hence, we hope that more multicenter studies will be conducted in the future to reach a more accurate and comprehensive conclusion. We could not include some less common inhalant allergens in the SPT battery. Although in theory a patient may be solely sensitized to that particular aeroallergen not included in the test battery.This study demonstrated that the frequency of aeroallergens based on SPT in Kavre district showed the dominance of house dust mites, dog and cat hair, pollen, and grasses. Reduced exposure and training of patients about protection against these agents will possibly help in controlling the severity of AR in this region."}
+{"text": "Numerous tissue-derived factors have been postulated to be involved in tissue migration of circulating monocytes. The aim of this study was to evaluate whether a defined hypoxic gradient can induce directed migration of na\u00efve human monocytes and to identify responsible autocrine/paracrine factors.Monocytes were isolated from peripheral blood mononuclear cells, transferred into chemotaxis chambers and subjected to a defined oxygen gradient with or without the addition of CCL26. Cell migration was recorded and secretome analyses were performed.Cell migration recordings revealed directed migration of monocytes towards the source of hypoxia. Analysis of the monocyte secretome demonstrated a reduced secretion of 70% (19/27) of the analyzed cytokines under hypoxic conditions. The most down-regulated factors were CCL26 (\u2212\u200999%), CCL1 (\u2212\u200995%), CX3CL1 (\u2212\u200995%), CCL17 (\u2212\u200985%) and XCL1 (\u2212\u200983%). Administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26.Hypoxia induces directed migration of human monocytes in-vitro. Autocrine/paracrine released CCL26 is involved in the hypoxia-mediated monocyte migration and may represent a target molecule for the modulation of monocyte migration in-vivo. Peripheral blood monocytes representing about 4\u20138% of all circulating leukocytes, possess only a limited life span in the blood circulation and undergo spontaneous apoptosis after 24\u201348\u00a0h . HoweverVarious pathological conditions like bacterial infections, ischemic diseases , neoplasia, chronic inflammation, and autoimmune diseases (e.g. rheumatic arthritis) are typically associated with the tissue infiltration of circulating monocytes , 6. TherAs tissue inflammation is associated with hypoxia , severalThe study was approved by the local Ethics Committee of the University Medical Center Schleswig\u2013Holstein, Kiel, Germany (protocol identification: D519/18). Peripheral blood monocytes were obtained from leukapheresis products from the Department of Transfusion Medicine  and isolated by Ficoll-Paque PLUS  density gradient centrifugation and selective adherence to cell culture surfaces according to an established protocol .The hypoxia system employed in our study represents an easy to handle application, which also can be utilized by other researchers for the study of migratory behavior of various cell types under defined oxygen gradients. For the described experiments a \u00b5-slide migration chamber system  was applied. The chamber includes a 1\u00a0mm wide observation area, where cells are seeded and cellular movements can be recorded. The observation area is connected with reservoir chambers enabling the formation of chemical gradients by the addition of chemotactic substances into the respective filling ports  and its temporal decline after the addition of GO and CAT was measured by using a flexible pO2 probe , that was placed in different positions throughout the chamber  were loaded into the observation area  according to the manufacturer\u2019s protocol. Initially, 6\u00a0\u00b5l of cell suspension  were removed by exchanging the culture medium. Hypoxia was induced by placing the cell culture inserts equipped with a gas-permeable membrane  into the respective well. Inserts were filled with DMEM-F12 culture medium containing 600 U/ml GO and 36,000 U/ml CAT, resulting in a rapid decrease of pO2 to levels of approximately 40\u00a0mmHg within the monocyte culture [pO2 to levels were confirmed by using a flexible pO2 probe ]. Additionally, cells were cultured under hypoxic and normoxic conditions with CCL26 at a final concentration of 0.3\u00a0\u00b5M. After 1 and 5\u00a0h of hypoxia, the cell culture inserts were removed and monocyte supernatants were collected and stored at \u2212\u00a080\u00a0\u00b0C. For protein extraction cells were lysed using RIPA buffer containing 150\u00a0mM sodium chloride, 1% NP-40, 1%, sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) and, 50\u00a0mM Tris\u2013HCl . Cells were scraped off the bottom using a rubber policeman, disrupted with a syringe, and sonicated on ice for 10\u00a0min. The remaining solution was centrifuged 15\u00a0min at 14,000\u00d7g at 4\u00a0\u00b0C. Supernatants were frozen at \u2212\u00a020\u00a0\u00b0C until use.To maximize the yield of protein and culture supernatants for subsequent biochemical analyses, additional hypoxia experiments were performed in parallel in 6-well plates employing our established hypoxia enzyme insert system with minor modifications . As monoAnalyses of the cytokines and chemokines secreted by monocytes under hypoxic and normoxic conditions were performed using Proteome Profiler Human Chemokine Arrays  according to the manufacturer`s protocol provided with the assay kit. 1\u00a0ml of pooled cell culture supernatant derived from N\u2009=\u20095 independently performed experiments was applied to the respective array membranes. Expression levels of 31 cytokines/chemokines were simultaneously evaluated by densitometric analyses of the arrays using the ImageJ 1.41 software (NIH). For each spot on the membrane, the optical density was determined and the cut off signal level was set to 10% of the mean intensity of the respective reference spots. Based on these criteria regulation of 27/31 cytokines/chemokines could be analyzed and quantified while 4 cytokines/chemokines  showed an optical density below the cut off and were excluded.Concentrations of protein samples were determined with Roti-Quant assays . 20\u00a0\u03bcg of total protein were mixed with 4\u00d7 Laemmli buffer  and incubated for 3\u00a0min at 95\u00a0\u00b0C. Samples were separated by 8% SDS-PAGE and transferred to a PVDF membrane . After 2\u00a0h of blocking with TBST buffer containing 3% of bovine serum albumin (Carl Roth) at room temperature, the membranes were incubated overnight at 4\u00a0\u00b0C with specific primary antibodies, anti-CCL26 , and anti-RAGE . Signals from peroxidase-conjugated secondary antibodies  were detected using the ECL kit . Protein bands on the membranes were visualized with the western blot imaging system Fusion FX  and intensities of the respective protein bands were analyzed using the ImageJ software 1.41 (NIH).All values are expressed as mean\u2009\u00b1\u2009standard deviation (SD). Data of cell migration studies were analyzed by using circular statistics (Rayleigh test) available in the software \"Chemotaxis and Migration Tool\" (Ibidi). All other data were analyzed with the software Graph Pad Prism version 5.01 for Windows  and tested for normality using the Kolmogorov\u2013Smirnov test. If normality was not present, data were transformed (arcsine of the square root of x) before using parametric tests. Statistical comparisons of two groups were performed using the Student\u2019s t-test or One-sample t-test. A p-value\u2009<\u20090.05 was considered significant.2 at both sides of the observation area . Hypoxic conditions were stable throughout the whole experiment lasting for at least 6\u00a0h  in combination with our recently described enzymatic hypoxia model , 15. TheMonocytes seeded into the observation area of the migration chamber were subjected to oxygen gradients for 5\u00a0h. Recordings and analyses of monocyte movements revealed a statistically significant directed migration towards lower oxygen concentrations  or ischemic diseases  , 6. BesiSo far, the generation of stable and precise oxygen gradients in cell cultures is mainly limited to the use of special microfluidic devices. These systems are capable of generating oxygen gradients for cell culture experiments using the principle of oxygen diffusion, oxygen scavenging by chemicals, or electrolytic reactions . HoweverUntil now, the role of molecular oxygen as chemoattractant or chemorepellent for human cells is only rudimentary investigated and the underlying cellular, as well as molecular mechanisms remain unclear. Nevertheless, several authors have proven the role of oxygen as a chemotactic factor, primarily for tumor cells. Interestingly, depending on the cell line employed, both the migration of tumor cells towards regions of higher oxygen levels as well as the attraction of cells to hypoxic areas have been reported \u201325. To tCytokines are low molecular weight proteins that are secreted by various cell types either constitutively or upon inflammatory stimuli and regulate complex physiological processes such as leukocyte recruitment and activation , 26. GenHypoxia alone is sufficient to induce directed migration of human monocytes. CCL26 seems to be a key player in hypoxia-mediated monocyte migration and may represent a clinical target for modulating monocyte migration in ischemia associated illnesses.Additional file 1: Figure S1. Red dots indicate the spatial representation of the mean endpoint after the evaluation of cell movements of at least 20 cells per experiment (N\u2009=\u20094\u20135). A: oxygen gradient (hypoxia); B: no oxygen gradient (normoxia). P-values were calculated using circular statistics (Rayleigh test). Black lines inside the quadrants show the linear correlation of the center of mass in each experimental group. Figure S2. Detailed description of the array proteins. Figure S3. Red dots indicate the spatial representation of the mean endpoint after the evaluation of cell movements of at least 20 cells per experiment (N\u2009=\u20093). A: no oxygen gradient (normoxia) in combination with a gradient of CCL26; B: oxygen gradient (hypoxia) with homogeneously distributed CCL26. P-values were calculated using circular statistics (Rayleigh test). Black lines inside the quadrants show the linear correlation of the center of mass in each experimental group. Figure S4. Upper panel: Detection of RAGE by Western blotting in cell lysates of monocytes subjected to hypoxic and normoxic conditions (3 representative samples out of N\u2009=\u20097 are shown). Columns show the mean of N\u2009=\u20097 experiments, bars denote SD. Exp, experiment; H, hypoxia, N, normoxia. Lower panel: Colocalization of RAGE and CCL26 in monocytes (3 representative samples out of N\u2009=\u20097 are shown). Yellow color is indicative of cellular RAGE/CCL26 colocalization. Scale bars represent 3\u00a0\u00b5m. Exp, experiment."}
+{"text": "TM (DD) reconstruction algorithm presents a reconstruction implementation without any dependence on the tube voltage. In comparison, it allows a calibration curve that is directly proportional to the RED, reducing the need of more than one calibration curve. This could potentially optimize CT acquisitions and reducing the dose given to the patient. Three different phantoms were used to evaluate the DirectDensityTM algorithm in simple and anthropomorphic geometries, as well as setups with metal implants. Scans with the DD algorithm were performed for 80, 100, 120, and 140\u00a0kVp. As reference a scan with the standard 120\u00a0kVp scan was used. Radiotherapy photon plans were optimized and calculated on the reference image and then transferred to the DD images, where they were recalculated. The dose distributions obtained this way were compared to the reference dose. Differences were found mainly in pure air and high density materials such as bones. The difference of the mean dose was below 0.7%, in most cases below 0.4%. No indication was found that the algorithm is corrupted by metal inserts, enabling the application for all clinical cases. This algorithm offers more variability in CT parameters for radiation therapy and thus a more personalized image acquisition with a high image quality and a lower dose exposure at a robust clinical workflow.In radiation therapy, a Computed Tomography (CT) image is needed for an accurate dose calculation. To allow such a calculation, the CT image values have to be converted into relative electron densities. Thus, standard procedure is to calibrate the CT numbers to relative electron density (RED) by using a phantom with known composition inserts. This calibration curve is energy and CT dependent, therefore most radiotherapy CT acquisitions are obtained with 120\u00a0kVp, as each tube voltage needs an additional calibration curve. The commercially available DirectDensity Beforehand, a relation between CT numbers and relative electron density (RED) or mass density (MD) has to be established in the form of a calibration curve in the treatment planning system (TPS) as described by e.g., Schneider et al.The commercially available reconstruction algorithm \u2018DirectDensity\u2019 (DD)  promises a feasible workflow as it constructs the RED information directly from the raw CT data.Van der Heyden showed in a patient study for 33 patients that the mean dose difference between HU based dose calculated plans and those calculated on the DD image sets were found to be smaller than 1%.The present study analyzes the effect of the DD images on the dose distributions for a phantom with materials with high, medium and low density, exemplary for the density range present in the human body. As analyzing tools, a simple dose difference is accompanied by dose profiles, allowing a distinct analysis where the differences occur.Additionally, CT scans with metal implants are compared as well as the combination of the DD algorithm with a metal reduction algorithm,Finally, the potential CT dose reduction benefits that could be utilized when dose acquisition parameters  are optimized for image quality, are presented.22.ATo understand the procedure behind the DirectDensity images and understand problematic setups and cases, a brief introduction of the algorithm is given. A more detailed description is given by Ritter.S), a bone image (IB) is generated. In the projection plane, a model based material decomposition is applied to the original sinogram (S\u00b5), separating all materials present in the human body into either water or bone. With an underlying physical attenuation model, the effective water thickness (dW) for all materials can be obtained. The combination of the effective bone thickness (dB) gained from the information of the bone sinogram (SB) and the effective water thickness (dW) together with the RED of water and bone gives an electron density line integral (SRED). Thus, in a final step a RED proportional image (IDD) via filtered back projection of the RED sinogram is gained.Figure The connection between the image values of the final DD image and the RED is given in eq. (1).2.BTo obtain the RED information from the CT scan, calibration curves for 80, 100, 120, and 140\u00a0kVp tube voltage were generated with the Gammex 467 Tissue Characterization Phantom  for the HU and the DD reconstruction. All scans were taken with a constant tube current of 250\u00a0mAs on the SOMATOM Confidence . The reconstruction with 3\u00a0mm slice thickness was executed with the B40s and E30s convolution filters. For the calibration, the mean CT value of each tissue mimicking insert was plotted against the RED of the material. The mean CT value for each insert was obtained in ImageJ . The CT values were averaged over a sphere of 2\u00a0cm diameter, centered in the middle of the insert. As the calibration curves in Fig. 2.CAll phantoms displayed in Fig. The dose was calculated in the TPS Eclipse with the Anisotrop Analytical Algorithm (AAA) , a 3D pencil beam superposition convolution algorithm. For this algorithm, only RED but not MD needs to be assigned to the CT voxels for a correct dose calculation.To compare the different dose distributions, a dose difference between each plan on the DD reconstruction with the HU dose distribution as reference was created. Because the dose distributions were calculated on identically positioned phantom CT scans, which were performed without moving the phantom, the gamma criteriaAdditionally, dose\u2010volume histograms (DVH) were used to evaluate dose distributions in specific regions like the PTV and surrounding OARs as contoured in Fig. 2.C.1To evaluate the dose distribution in simple geometries a slightly modified version of the planar IMRT phantom Gammex 473 , scanned with 250\u00a0mAs, was used. The phantom, consisting of solid water slabs, was extended by a cork as well as two plaster inserts as shown in (a) in Fig. To also evaluate a more realistic setup, the anthropomorph male Alderson Radiation Therapy (ART) Phantom  was additionally investigated. Scans were conducted for the pelvis (scanned with 250\u00a0mAs) and thorax region (scanned with 140\u00a0mAs) as presented in part (d) and (e) of Fig. On the IMRT phantom, a single 6\u00a0MV open field was planned in Eclipse with a TrueBeam model . The plan was computed on the HU image and then copied to all DD images, whereas the assigned monitor units (MU) were held constant. For the Alderson phantom, a 3D conformal and a volumetric modulated arc therapy (VMAT) plan was optimized for a simulated prostate and lung carcinoma treatment.2.C.2One of the main advantages of the implementation of the DD algorithm in the clinic is the reduction from many CT calibration curves to only one. Therefore, the effect of metal implants in combination with the DD reconstruction was tested, as the DD reconstruction could define the metal as high density bone due to the applied threshold. If this is the case, an inadequate interpretation of metal could wrongfully influence the reconstruction around these implants. For this study, a third phantom was used. The CBCT Electron Density Phantom , as shown in (b) in Fig. For a more anthropomorphic setup, the ART head was scanned with and without a brass insert simulating a dental prosthesis at 200\u00a0mAs. As differences due to the artifacts induced by metal ought to appear, an additional reconstruction with a metal reduction algorithm  was also evaluated. The iterative Metal Artifact Reduction (iMAR) algorithm reduces the artifacts caused by metal implants via beam hardening correction, sinogram inpainting and frequency splitAll RED estimations were performed with the presented calibration curves limited to a maximum RED of 1.7. Thus differences for higher RED ought to appear.2.C.3To evaluate the possible dose savings when the DD algorithm is implemented, the tube\u2010current as well as the voltage were varied. The pelvis and thorax of the ART phantom was scanned again using CARE kV and CARE Dose4D . The CARE Dose4D algorithm modulates the tube current on the basis of a topogram33.A3.A.1First, the relative electron density of different materials in the IMRT phantom is compared. As the dose computation relies on the right mapping of the RED, differences in the mean RED will most likely result in dose differences. Table 3.A.280 .Despite the differences observed in the depth dose curves, there is a good agreement between the DD dose distribution and the HU reference dose for the evaluation of the 0.5% dose difference with passing rates over 98.3%. Having a closer look at the location of the failed points, it can be noticed that for the lower tube voltages more points in the plaster regions fail, while for the higher tube voltages this shifts to the air region. Because there are two plaster inserts, far more points for the DD3.A.3Figures The passing rates of the 0.5% dose difference for the anthropomorphic ART phantom, again, show the good agreement between the DD dose distributions when compared to the dose calculated on the HU reconstruction. The VMAT plans show an agreement of over 99.3%, while a slightly lower agreement is found in the 3D\u2010conformal plans . An exception is the 3D\u2010conformal thorax plan, where a large dose is deposited in the trachea leading to passing rates around 90% for 0.5% and over 98% for 1% dose difference.As the DVH show no visible differences, the mean dose was the point chosen to evaluate. The results are shown in Table 3.BTable However, a calibration for high density materials could be added, diverging from the straight line from eq. . If this3.CThe use of the CARE kV and CARE Dose4D options allow the CT to acquire a high quality image with a potentially reduced dose exposure by choosing the kV and adapting the mAs to the given situation. While CARE Dose4D can be used with the conventional HU calibration, CARE kV can only be enabled in radiation therapy when the DD algorithm is implemented.A comparison between the CT parameters estimated during the acquisition with and without the CARE options is given in Table The passing rate for the dose difference between the dose calculated on these scans and the HU dose are in the same order of magnitude as all other DD scans. The image quality see Fig.  was foun4TM algorithm was implemented and validated. With a simple phantom, the consistency of the RED conversion for the conventional HU to RED conversion and the Direct Density to RED conversion was tested. The results show small discrepancies that indicate that there will be discrepancies in the dose distributions. These were evaluated for different radiation modalities and setups as well as simple and also anthropomorphic geometries. A direct correlation was observed between an overestimated RED and an overestimation in dose and vice versa. Air was found to produce the biggest dose discrepancies. The highest RED difference in air was +3.3%. In general, dose was overestimated in the DD air regions in comparison to the HU dose distributions. In air, decreasing the x\u2010ray tube voltage lead to a decreased RED deviation and thus to a decreased dose difference. For plaster as bone substitute, a maximum RED difference of \u22121.7% was noticed. Here, an increased tube voltage results in an increasing agreement.The Siemens DirectDensityAlmost all evaluated phantoms showed a good passing rate of over 99% at 0.5% dose difference (the one exception reached 98% at 1% dose difference). This matches the results of Ref. The differences for the mean dose (see Table Additionally, we showed that the DD reconstruction can also be used when metal implants are present. However, the metal density is dramatically underestimated in all cases.It is advisable to focus on one tube voltage when metal implants are present and extend the calibration curve for this specific tube voltage. As this study is limited to the selected materials, a general statement about the correctness of materials foreign to the body is critical as other chemical compounds might have an influence. In these cases, however, the HU calibration is probable to also give a wrong output.TM algorithm in the clinical routine in radiotherapy departments besides a robust workflow: The first side effect that could be exploited is the comparability with other institutions as the CT characteristics are reduced. Thus, the RED curve needed for the implementation are more alike than a standard Hounsfield lookup table. A comparison to the Siemens RED curve and to the curves used by Ritter and Van der HeydenTM could be used with differences of less than 1% in the mean dose. If acceptable, this would benefit the patient by canceling the additional dose of another planning CT.Two major benefits could result from the implementation of the DirectDensityThe second major benefit is the patient dose reduction by the implementation of DD with CARE kV and CARE Dose4D. This can reduce the dose significantly without reducing the image quality, allowing a more patient specific CT acquisition.A problem that could occur with the implementation are the danger of CT scans with an unsuitable tube voltage, resulting in an unusable scan and thus a rescan with additional dose to the patient.5TM images and on the standard HU images. The results for dose distributions calculated on CT scans containing metal implants showed a larger difference but are still in good agreement, opening up the possibility to implement the direct density reconstruction algorithm for all clinical protocols. The DirectDensityTM implemented in the clinical routine allows a robust workflow while moving CT acquisition in radiotherapy to a dose optimized and thus a more personalized medicine.A CT reconstruction method for a direct translation between image values and relative electron density was implemented and found to provide sufficient image quality and dose calculation accuracy. The evaluated dose distributions showed only small differences between the dose calculated on the DirectDensityWe have no conflict of interest to declare."}
+{"text": "Reaching the 90\u201390-90 targets requires efficient resource use to deliver HIV testing and treatment services. We investigated the costs and efficiency of HIV services in relation to HIV testing yield in rural Karonga District, Malawi.Costs of HIV services were measured over 12\u2009months to September 2017 in five health facilities, drawing on recognised health costing principles. Financial and economic costs were collected in Malawi Kwacha and United States Dollars (US$). Costs were calculated using a provider perspective to estimate average annual costs (2017\u00a0US$) per HIV testing episode, per HIV-positive case diagnosed, and per patient-year on antiretroviral therapy\u00a0(ART), by facility. Costs were assessed in relation to scale of operation and facility-level annual HIV positivity rate. A one-way sensitivity analysis was undertaken to understand how staffing levels and the\u00a0HIV positivity rate affected HIV testing costs.HIV testing episodes per day and per full-time equivalent HIV health worker averaged 3.3 (range 2.0 to 5.7). The HIV positivity rate averaged 2.4% (range 1.9 to 3.7%). The average cost per testing episode was US$2.85 (range US$1.95 to US$8.55), and the average cost per HIV diagnosis was US$116.35 (range US$77.42 to US$234.11), with the highest costs found in facilities with the lowest daily number of tests and lowest HIV yield respectively. The mean facility-level cost per patient-year on\u00a0ART was approximately US$100 (range US$90.67 to US$115.42). ART drugs were the largest cost component averaging 71% (range 55 to 76%). The cost per patient-year of viral load tests averaged US$4.50 (range US$0.52 to US$7.00) with cost variation reflecting differences in the tests to ART patient ratio across facilities.Greater efficiencies in HIV service delivery are possible in Karonga through increasing daily testing episodes among existing health workers or allocating health workers to tasks in addition to testing. Costs per diagnosis will increase as yields decline, and therefore, encouraging targeted testing strategies that increase yield will be more efficient. Given the contribution of drug costs to per patient-year treatment costs, it is critical to preserve the life-span of first-line ART regimens, underlining the need for continuing adherence support and regular viral load monitoring. Renewed global efforts are being made to bring the HIV epidemic under control, notably through the release of the UNAIDS 90\u201390-90 targets in 2014 .At the individual level, the benefits of timely initiation of ART among those who test HIV positive include improved life expectancy and lower risks of opportunistic infections . At the To understand the affordability of HIV services requires an assessment of its costs which is critical to estimating budgetary requirements. At the same time, these data contribute to the understanding of how to improve planning and allocate existing resources to maximise value for money or efficiency. While large scale, multi-site cost data are generally required to capture the variability in costs and efficiency , informaIn Malawi, the HIV epidemic remains generalised with an estimated adult HIV prevalence of 10.6% by 2016 . SeventyWhile these achievements are impressive, new strategies, including better targeting of HIV testing and care and treatment, will be needed to achieve the 90\u201390-90 targets in Malawi. However, further expansion of targeting HIV services is dependent on resource availability. Assessing the resource requirements and therefore, affordability, requires an understanding of the costs of service delivery, data which can also help to ensure better and more efficient use of existing resources and to improve planning and budgeting. To date, cost studies in Malawi have exclusively examined either the costs of HIV testing or the costs of ART, or have estimated costs at a time when previous HIV policy guidelines were implemented Table s 16\u201318].\u201318.16\u201318In 2016, the Ministry of Health (MoH) in Malawi released updated guidelines on HIV testing and on the clinical management of HIV in adults and children in efforts to move closer to its goal of achieving universal access to ART , 20. Then\u2009=\u20095) providing HIV services to the HDSS population in order to assess HIV service delivery . IsA standardised Microsoft Excel-based instrument was used to record resource use and, where available, price information from each facility. Data were collected retrospectively for the 12\u2009months to September 2017. Most data collection took place during October and November 2017. Resources were categorised into capital inputs  and recurrent inputs .Service delivery data were captured from two sources 1) HMIS; and 2) from facility records . Routine data on persons tested for HIV were extracted from facility registers. Data extracted from ART patient cards were ART regimen and number of tablets by month of visit. In facility E, patient utilisation data were obtained from monthly summaries compiled by clinical staff.Two types of costs were estimated: 1) financial costs, defined as the actual expenditure incurred on resource inputs , and 2) economic costs, defined as the value of all resource inputs, including donated or subsidised items, which were valued at their market price . A micro-costing approach, a method which identifies and values each input consumed in delivering a service , was appCapital inputs: Physical space and office equipment used to deliver HIV services was audited and measured during visits to each facility. Shared usage across testing and treatment services was identified from interviews with staff. The cost of space used was estimated from rental costs for equivalent nearby spaces. Prices for office equipment  were obtained from the Karonga District Health Office and from medical suppliers in the country. Capital equipment prices were annualised over 25\u2009years. Training costs, provided to health workers in the previous 2 years, were estimated using information provided by the District Health Office, (staff per diems and travel expenses) and by national non-governmental organisations running HIV training programs (unit \u201cper participant\u201d training costs). Staff training costs were annualised over 2 years, in line with MoH guidelines that all registered/certified HIV service providers are to receive training at 2 year intervals [ntervals , 20.Recurrent inputs: Staff delivering HIV services were interviewed to illicit the amount of time that workers spent on different HIV services and salaries were apportioned accordingly. Salaries (wages and benefits) were collected from either the facilities (two faith-based facilities) or from the District Health Office. Per diems for lay counsellors were collected from the staff.Prices of HIV test kits and other clinical and non-clinical supplies  were obtained from medical suppliers based in Lilongwe. Drug costs (ART and Cotrimoxazole) were obtained from The Global Fund ART pricing list for 2017 . LaboratFacility survey data were analysed using STATA v15. Facility-level cost and outcome calculations were undertaken using Microsoft Excel 2007. Total annual costs and average (unit) costs were calculated for each facility and separately for HIV testing and for HIV care and treatment services. All costs were adjusted to 2017 United States dollars (US$) using an average exchange rate over the 12\u2009month study timeframe  . BecauseHIV testing services: The cost per testing episode  and per HIV case detected were\u00a0calculated by dividing the total facility costs of running testing services in 1 year by the number of testing episodes, and the number of new HIV-positive individuals identified over the same timeframe .HIV care and treatment: The annual cost per patient-year for ART care was calculated by dividing the total annual costs of providing care and treatment services by the number of HIV patients receiving care and treatment.The levels of technical efficiency (relationship between inputs to the service outputs) and economic efficiency  of HIV testing and HIV care and treatment were estimated as:A one-way sensitivity analysis, varying one parameter at a time holding all others constant, was undertaken to understand how staffing levels and the annual HIV positivity rate affected the costs of HIV testing services. Cost variations were explored by applying a +/\u2212\u200910% variation range to staffing levels and the annual positivity rate, and by applying annual HIV positivity rates observed in studies from other settings in Malawi.The mean number of health workers who provided HIV testing services across the five facilities was 7.4 (range 3 to 13) yielding an average of 3.3 full time equivalent (FTE) per facility , of which approximately 3.0  were counsellors  Tables\u00a0 & s4.TaA total of 12 health workers were trained in HIV testing in the past 2 years . No health workers were trained in HIV testing during these 2 years at the other facilities. HIV testing surveillance or audits were conducted on a quarterly basis in all five facilities, and supervision visits were conducted every month.Over the 12\u2009months to September 2017, a total of 18,509 HIV testing episodes  were conducted. The mean number of testing episodes per facility was 3702 (range 520 to 9544). The average number of new HIV positive cases across all five facilities was 91 , yielding an average annual HIV positivity rate of 2.4% (range 1.9 to 3.7%).Based on the number of days per week that facilities conducted HIV tests , the number of HIV testing episodes conducted per day and per FTE HIV testing staff averaged 3.3 . No clear trend was observed over the 12-month period in the number of HIV tests undertaken by month and per facility  to $18,582  Table\u00a0. The cosRecurrent costs, in particular personnel followed by the cost of test kits, comprised the majority of total HIV testing costs Fig. . Health When staff inputs were varied by +/\u2212\u200910%, the mean cost per person diagnosed with HIV changed by +/\u2212\u20095% and ranged from $110.49 to $122.20 , and the mean number of FTE per facility was 2.5  (Tables\u00a0n\u2009=\u2009453) identified over the timeframe. The total number of viral load tests was 678 (range 7 to 362).The number of ART current patients totalled 2110 across the five facilities (range 187 to 1021). The total number of newly initiated ART patients over the 12\u2009months was 412 (range 13 to 223), which equates to 91% of the new HIV-positive cases (The proportion of patients on paediatric ART averaged 5.6% (range 3.6 to 6.8%), and all were on the first-line 3TC/AZT/NVP regimen. Among the 1992 adult ART patients, 92.4% were on the first-line regimen TDF/3TC/EFV 300/300/600, with a further 5.6% on a different first-line regimen. Only 2% of adult ART patients were on second-line regimens. On average, ART was dispensed to patients every 2.9\u2009months or 4.1 times per patient-year .The total annual HIV care and treatment costs ranged from $19,003 (Clinic A) to slightly over $100,000  Table . The meaRecurrent costs contributed the most to total costs, of which ART drugs were the largest cost component averaging 71% across the five facilities . This translated to an average ART drug cost of $70.73 US per patient-year across the five facilities (range $63.89 to $75.04). Cotrimoxazole drug costs contributed, on average, a further 6% to total costs, or $5.89 per patient-year. Personnel comprised the second largest cost component (after drugs) averaging 11% (range 4 to 18%) or $10.79 (range $4.55 to $21.07).The variability in the average annual per patient cost of viral load laboratory tests, which ranged from $0.52 (Clinic A) to $7.00 , reflected the differences across the facilities in the ratio of viral load test per ART patient over the 12\u2009months which ranged from a low of 4% (Clinic A) to 50% .Per patient-year costs of capital inputs averaged $4.06  across the facilities; range $1.31  to $13.74  driven by the cost of ART training. National and district supervision costs averaged $1.77 per patient-year or 2% of total HIV care and treatment costs.This district-level case study, to cost the delivery of facility-based HIV services in the context of Treat-All, highlights potential areas for cost-saving and has implications for future resource allocation in terms of how these services are delivered in this setting. These findings are particularly timely, given the current flat-lining of external funding and the imminent approach of the deadline for the 90\u201390-90 targets, and indicate key areas for strategy reform in HIV testing.HMIS data reveal that the average annual HIV positivity rate has been declining year on year from 4.4% (2014) to 2.0% (2018) in these five facilities, suggesting that identifying new HIV cases through existing facility-based approaches may be close to reaching saturation. Our estimate of the mean cost per HIV case diagnosed (which ranged from\u00a0US$77.42 to US$234.11) is\u00a0higher than documented elsewhere in Malawi, and is\u00a0likely driven by the comparatively lower HIV prevalence found in the area , 16. OurWhile facility-based testing has been a mainstay of Malawi\u2019s achievement in diagnosing 73% of PLHIV by 2016, our findings suggest refocusing resources to differentiated HIV testing services to identify PLHIV who do not know their status. This would increase the diagnosis rate and reduce the cost per diagnosis. Alternative testing strategies such as home- and community-based testing, HIV self-testing, and index tracing are likely to become more necessary for diagnosing first-time testers and the hardest-to-reach persons including men and key populations , 29.Our findings also confirm that as the facility-based HIV positivity rate declines and the cost per HIV diagnosis rises, staff time becomes an increasingly important contributor to costs. With the number of HIV testing episodes per FTE health worker per day well below the national guideline of a maximum of 15 per day, our results support policies to phase out the use of dedicated HIV counsellors in facilities, and to\u00a0move towards greater role diversity among existing health workers providing other HIV or general health services. This recommendation, however, should take into account the numbers and levels of different health workers within a facility.We estimated the average per patient-year cost of core HIV treatment services to be within a relatively narrow range across the facilities (US$90.67 to US$115.42), supporting the existing centralisation policies of ART delivery in small rural clinics. Seventy-one percent of annual treatment costs was ART drugs, which is similar to findings elsewhere in Malawi and in other sub-Saharan African countries . PreservLimitations to this study include not costing some supporting HIV service components, including community-based testing, early infant diagnosis, or facility-based index testing. However, use of these testing mechanisms was very low during the study period. With respect to HIV treatment, we did not include the management and treatment of HIV-related illnesses, prevention services  or home visits for patients who miss appointments. Further, we did not include patient costs which may act as a barrier to care. These components should be considered in future cost studies to ensure more holistic estimates of HIV service delivery costs. A second limitation, as found elsewhere , was thaNevertheless, this study provides detailed cost estimates following standard methodologies that add to the evidence base around HIV testing and treatment. It also identified patterns of resource use related to the different services, indicating a re-thinking of resource allocation at the facility level maybe required to improve efficiency and reduce the unit cost of HIV testing. The sampled facilities are typical of rural health facilities in Malawi and the prices of important contributors to cost are set at the national level, suggesting our estimates are a good basis for making cost estimates elsewhere in\u00a0the country. Finally, this study was completed before the Covid 19 pandemic which may have implications for achieving the UNAIDS targets.Moving towards differentiated testing strategies in this setting can lead to efficiency improvements and therefore, freeing up resources for achieving the 90\u201390-90 goals. Among the recommendations is the increased sharing of personnel across activities at facilities. This study also highlights the importance of preserving the life-span of first-line regimens and therefore, underlines the need for higher coverage of regular viral load monitoring. The results show that these types of cost analysis continue to be a useful tool in identifying areas for informing planning, achieving efficiency and freeing up resources in an era of stagnating funds.Additional file 1. : Supplementary Tables."}
+{"text": "To reduce hospitalization costs, it is necessary to prevent avoidable hospitalization as well as avoidable readmission. This study aimed to examine the relationship between clinic physician workforce and unplanned readmission for ambulatory care sensitive conditions (ACSCs).n\u2009=\u2009127,209). The primary outcome was unplanned readmission for ACSCs within 30 or 90\u2009days of hospital discharge. A hierarchical logistic regression model was developed with patients at the first level and regions  at the second level.The present study was a retrospective database research using nationwide administrative claims database of acute care hospitals in Japan. We identified patients aged \u226565\u2009years who were admitted with ACSCs from home and discharged to home between April 2014 and December 2014  of clinic physicians per 100,000 population were significantly associated with decreased odds ratios for 30-day and 90-day ACSC-related readmissions. This association did not change even when sensitivity analyses was conducted.Among patients who had history of admission for ACSCs, greater clinic physician workforce prevented the incidence of readmission because of ACSCs. Regional medical plans to prevent avoidable readmissions should incorporate policy interventions that focus on the clinic physician workforce. The Japanese population is the most rapidly aging in the world ; approxiThe concept of ambulatory care sensitive conditions (ACSCs) is often used in the context of avoidable hospitalization . ACSCs aReadmission is a common phenomenon and imposes a burden on the medical system . In EnglMost previous studies that have examined the relationship between primary care physician per population and hospitalization for ACSCs have revealed lower hospitalization rates for ACSCs in areas with greater access to primary care . HoweverThe Diagnosis Procedure Combination (DPC) system is a case-mix classification system used in Japan for reimbursements to acute care hospitals under the public medical insurance scheme. We used data obtained from the DPC database which contains administrative claims data and discharge clinical summaries. The data were collected by the DPC research group from voluntarily participating hospitals, which account for approximately 50% of acute care hospitals in Japan . The datWe identified patients who fulfilled the following criteria: 1) patients aged \u226565\u2009years at the time of admission; 2) patients admitted unexpectedly with ACSCs between April 1 and December 31, 2014; 3) patients with no history of hospitalization within the past year; 4) patients admitted from home and discharged to home. Patients were followed up until March 31, 2015. For individuals who had \u22652 admissions during the study period, the first admission was considered as the index hospitalization. Patients with missing data pertaining to independent variables were excluded.The primary outcome variable was unplanned readmission for ACSCs within 30\u2009days or 90\u2009days of hospital discharge. ACSCs were those defined by Bardsley et al. . Using 3The patient-level variables were: age; sex; comorbidities; body mass index (BMI); Barthel index at discharge; surgery under general anesthesia, epidural anesthesia or spinal anesthesia; length of hospital stay; and schedule of implementation of home care program after discharge. Age was grouped into four categories: 65\u201374\u2009years, 75\u201384\u2009years, 85\u201394\u2009years, and\u2009\u2265\u200995\u2009years. Comorbidities were those defined by Elixhauser et al. . Only thThe region-level variables used were the full-time equivalents (FTEs) of clinic physicians per 100,000 population, the FTEs of hospital physicians per 100,000 population, the number of hospital beds per 100,000 population, and the population density of inhabitable areas (population/inhabitable area ratio) in the secondary medical service areas of residence of each subject. The secondary medical service areas are subprefectural regions comprising of several municipalities . These gA total of 127,209 patients from 1162 hospitals and 344 secondary medical service areas were included in the analysis. Figure\u00a02, low ADL function at discharge, length of stay, schedule of implementation of home care program after discharge, uncomplicated diabetes, complicated diabetes, cardiac arrhythmia, congestive heart failure, chronic pulmonary disease, solid tumor without metastasis, renal failure, valvular disease, liver disease, rheumatoid arthritis/collagen vascular diseases, and hypothyroidism . Secondary medical areas are subprefectural regions comprising of several municipalities. Abbreviations: FTEs, full-time equivalents. Table S2. Results of hierarchical logistic regression showing correlates of 30-day and 90-day ACSC-related readmissions. Abbreviations: ACSCs, ambulatory care sensitive conditions; FTEs, full-time equivalents; CI, confidence interval; BMI, body mass index."}
+{"text": "The Voronoi entropy for random patterns and patterns demonstrating various elements of symmetry was calculated. The symmetric patterns were characterized by the values of the Voronoi entropy being very close to those inherent to random ones. This contradicts the idea that the Voronoi entropy quantifies the ordering of the seed points constituting the pattern. Extension of the Shannon-like formula embracing symmetric patterns is suggested. Analysis of Voronoi diagrams enables the elements of symmetry of the patterns to be revealed. Symmetry considerations play a key role in modern science ,2,3, giviP is the fraction of polygons with k sides or edges  in a given Voronoi diagram [i = 1 (corresponding to k = 3) to the largest coordination number of any available polygon, e.g., to i = 4 (corresponding to k = 6) if a polygon with the largest number of edges is a hexagon. In our recent paper, we demonstrated that the labeling of the value vorS with the wording \u201cVoronoi entropy\u201d was misleading and confusing [It is generally accepted that the Voronoi diagrams allow the orderliness of the 2D distribution of points to be quantified with the so-called Voronoi entropy, defined according to the Shannon-like formula as: diagram ,11,12. Tonfusing . In actuonfusing ,14,22. Ihttps://www.physics.uci.edu/~foams/do_all.html). The procedure was repeated 20 times, and the average value of the Voronoi entropy and its standard deviation was established as We analyzed Voronoi diagrams built for sets of points demonstrating different elements of symmetry . In the first stage, the random sets of 200 and 1000 points located inside a circle of a given diameter were generated by the MATLAB software script. The Voronoi tessellation for the set of 200 random points is depicted in r groups ,24. It iIn the second stage, the mirror images of the 20 initial patterns were built and placed on the initial patterns, as shown in In the third stage, the point symmetry (inverse) images of the 20 initial patterns were built and put on the initial patterns, and their Voronoi entropy was calculated . The average value of the Voronoi entropy inherent to patterns similar to those depicted in In the fourth stage, the initial images were rotated for the angles n-polygons in the initial patterns and in the transformed ones remained the same. Thus, the value of the Voronoi entropy, calculated according to Equation (1), was also expected to be the same, as demonstrated in Hence, we conclude that the studied symmetry transformations did not change the Voronoi value of the patterns. This was true for the inversion, mirror reflection, and rotational symmetry operations. This result was quite trivial, as summarized in The Voronoi entropy did not change dramatically under the symmetry transformations because almost all of the random patterns already appeared over the initial 2D distribution of points shown in vorS is calculated according to Equation (1), N is the total number of the symmetry elements inherent to the pattern, including the trivial symmetry operation  contains Equation (1) as a particular case. Indeed when N = 1 and n = 0, the 2D pattern has the trivial axis of symmetry only and we return to Equation (1), namely: N = 2) of that calculated for the random mosaics shown in In order to resolve the puzzle and to quantify ordering appearing in various patterns, we propose to redefine the generalized Voronoi entropy for all kinds of patterns (symmetric and non-symmetric ones) as follows:p points and the following question: Does it demonstrate the elements of symmetry? We show that the Voronoi tessellation may help answer this question. In the first stage, we constructed the Voronoi diagram of the pattern. Edges and vortices of the diagram formed the simply connected graph. The number of edges E of the graph is given by the Euler formula:P is the number of Voronoi polygons, V is the number of the vertices of the graph (in other words of the Voronoi diagram), and L. The number of edges (say n of them) may have the same length L. Thus, the probability to find the edge with the length L is given by: Now consider the arbitrary 2D pattern containing m, namely: N appearing in Equation (2). The procedure enabling the finding of elements of symmetry is presented in N, defined as the total number of the symmetry elements inherent to the pattern appearing in Equation (2).If all of the edges constituting the graph are of different lengths, its Voronoi entropy equals It is generally agreed that the Shannon-shaped Voronoi entropy quantifies the ordering of 2D point patterns ,25. We h"}
+{"text": "For this, we conducted a series of atomistic density functional theory (DFT) calculations on H-passivated NCs of Ge-rich GeSi random alloys, with Ge atomic concentration varied from 50 to 100% and diameters ranging from 1 to 4\u00a0nm. As a result of the dominant confinement effect in the DFT computations, a composition invariance of the line shape of the bandgap diameter dependence was found for the entire computation range, the curves being shifted for different Ge concentrations by \u0394E(eV)\u2009=\u20090.651(1\u2009\u2212\u2009x). The shape of the dependence of NCs bandgap on the diameter is well described by a power function 4.58/d1.25 for 2\u20134\u00a0nm diameter range, while for smaller diameters, there is a tendency to limit the bandgap to a finite value. By H-passivation of the NC surface, the effect of surface states near the band edges is excluded aiming to accurately determine the NC bandgap. The number of H atoms necessary to fully passivate the spherical GexSix1\u2212 NC surface reaches the total number atoms of the Ge\u2009+\u2009Si core for smallest NCs and still remains about 25% from total number of atoms for bigger NC diameters of 4\u00a0nm. The findings are in line with existing theoretical and experimental published data on pure Ge NCs and allow the evaluation of the GeSi NCs behavior required by desired optical sensor applications for which there is a lack of DFT simulation data in literature.We present a detailed study regarding the bandgap dependence on diameter and composition of spherical Ge-rich Ge On the other hand, the integrated photonics based on elements Si-Ge-Sn from group IV is experiencing a pronounced increase in the research activity in the field. Thus, the SiGe light detectors can extend their sensitivity in short-wave infrared range3. However, there is a low efficiency of light emission and band-edge absorption in SiGe large crystals due to the indirect bandgap character and necessary participation of phonons to the optical transitions. The efficiency can be improved by quantum confinement in NCs or by Sn alloying or by strain engineering to obtain direct bandgap in (Si)GeSn8. Versatile techniques were developed for embedding Ge and GeSi NCs in oxides for fabrication of optoelectronic devices benefiting from the advantages of materials and technology, namely the compatibility with CMOS technology and cost-effective fabrication, while being environmentally friendly. Also, they are a very advantageous alternative to III\u2013V semiconductors for optoelectronic devices. The most interesting applications based on Ge and GeSi NCs are in optoelectronics and nanophotonics, i.e. photodetectors11, LEDs12, non-linear optics applications13 and energy harvesting devices15.SiGe being one of the most studied semiconductor alloy continues to be in front of the developing researches for many microelectronics and optoelectronics applications. For high-speed SiGe CMOS technology, the SiGe heterojunction bipolar transistors achieved record performances18, enabling bandgap engineering along with composition19, shape20 and strain22 leading to tuning of optical and photoelectrical properties of NCs.Quantum confinement effect was evidenced in small Ge and GeSi NCs23. Another way of bandgap tuning is by tailoring its level of directness, as recently demonstrated direct bandgap light emission in Ge and GeSi nanowires with hexagonal structure24, and GeSi quantum dots25 and also by infrared detection extended to longer wavelengths in NCs of direct bandgap GeSn alloys26.It was shown that two mechanisms compete in achieving no-phonon radiative transitions in NCs of Si and Ge that are indirect bandgap semiconductors in bulk. One mechanism is related to the relaxation of momentum conservation law due to the spatial confinement and Heisenberg uncertainty principle, being dominant in both Si and Ge NCs, and the other mechanism is the inter-valley coupling between direct and indirect states induced by the interface of the NC with the embedding matrix28. Moreover, by alloying Ge with Si, GeSi NCs are more thermally stable than Ge NCs by impeding the fast diffusion of Ge during nanocrystallization by annealing29. Alloy GeSi NCs benefit from the complete miscibility of Ge with Si over the whole composition range, while maintaining the same crystalline structure20. The miscibility and intermixing of Ge and Si in GeSi random alloys can be associated to the strong self-diffusion that is theoretically explained by the vacancies formation and their contribution to the diffusion processes31. The vacancies formation energy reduces for high Ge concentration30 increasing the diffusion coefficient as experimentally proved32. By employing DFT computation it was shown that the local electronegativity of the defects is strongly dependent upon the nearest neighbor environment33.Strong quantum confinement effect is expected in Ge NCs as the Bohr exciton radius is 24\u00a0nm, thus facilitating the bandgap tuning by tailoring the NCs size in relatively large NCsTheoretical and numerical studies in the frame of DFT are valuable tools to complement experimental data and better understand the results, providing new insights in the development of nanomaterials with targeted properties.2 is dependent on their size34. A study of structural and electronic properties of hydrogenated Si and Ge nanowires and Si, Ge, Si/Ge NCs (0.8 to 2.4\u00a0nm) shows that encapsulated Ge NCs could act as optical absorption centers in the infrared region35. The study on the structural stability of H-passivated GeSi NCs by calculating formation enthalpies of different GexSiyHz isomers reveals that most stable GeSi:H NCs are the ones with the lowest formation enthalpy and widest bandgap36. The theoretical works from literature report results on small diameter range Ge NCs (1\u20132\u00a0nm), only, in contrast to experiments dealing with larger GeSi NCs39. Thus, there is a lack of theoretical DFT investigation on SiGe NCs of commonly experimentally observed spherical shape and for a wide range of compositions and sizes.With respect to GeSi nanostructures, it was shown based on calculated density of states (DOS) that the absorption edge of small Si and Ge NCs (0.6\u20131.0\u00a0nm) embedded in SiOIn this work, we present first principles electronic DOS and energy gap DFT calculations on spherical H-passivated GeSi NCs with large ranges of Ge content (50\u2013100%) and NCs diameter (1\u20134\u00a0nm). A composition invariance of the line shape of the bandgap diameter dependence was found for the whole diameter and composition computation range. For comparison with experimental bandgap values and to be used for the design and characterization of optoelectronic devices based on GeSi NCs, the bandgap diameter dependences of different GeSi NC compositions are extrapolated to larger NCs by considering the asymptotic bulk values.x\u2009=\u2009100% Ge case, Fig.\u00a0For DFT calculations, spherical NCs of Ge and GeSi with different Ge contents  were constructed using in-house software. The initial coordinates of Ge and Si atoms in NCs correspond to the bulk cubic Ge lattice (space group Fd-3m) with a lattice constant of 5.66\u00a0\u00c5. GeSi NC is built in a similar way as Ge NC, but Ge atoms are randomly substituted by Si atoms to obtain the desired Ge concentration. The generated GeSi spheres have diameters in the 1.25\u20133.96\u00a0nm range. For the 40, as seen in the surface detail of Fig.\u00a0The surface of both Ge and GeSi NCs was passivated with H atoms (annihilating dangling bonds effects in Ge:H and GeSi:H NCs). In the Ge NC, the H atoms are positioned in the vertices of Ge tetrahedron or out of tetrahedron depending on the number of Ge dangling bonds Fig.\u00a0d. Similax\u2009=\u200950% is exemplified in Fig.\u00a0The geometric construction of energetically relaxed H-passivated GeSi NC with 23, but also to ensure the computation convergence. The diameter dependences of the number of H atoms and the number of Ge and Si atoms in each NC . The accuracy assessment of LDA and generalized gradient approximation (GGA) methods is made by band structure computation of bulk Ge and Si. As can be seen in Figure The calculated energy gap 43, spectral photocurrent42 and scanning tunneling spectroscopy (STS)44. The standard deviation of the experimental results with respect to the fit has a reasonable value of \u03c3\u2009=\u20090.12\u00a0eV, taking into account that the experimental NCs usually have a quite broad size distribution.If the 0.63\u00a0eV theoretical gap is considered, the fit results are quite similar to that of the asymptotic experimental value of 0.66\u00a0eV. The fit curve extrapolated to higher diameters (15\u00a0nm) is shown in Fig.\u00a046, tight binding (TB)45, 47. One can remark that the power law dependence .Related to the effect of passivation on the bandgap, it was experimentally shownThe type of passivation is of a great importance for the optoelectronic properties of the NCs. In experiments, NCs are often embedded in a dielectric matrix, the surface not being H-passivated necessarily. By H-passivation of NC surface, the effect of surface states near the band edges is excluded aiming to accurately determine NC bandgap. The H-passivation of spherical NCs is easily performed using our in-house developed code for the construction of NCs and has the advantage to fully passivate the free Ge bonds at NCs surface, as well as to remove the surface states from the bandgap region that is similar to the effect that takes place in amorphous hydrogenated Si. The software can also be used for O-passivation by replacing H atoms, but in this case the O atoms are placed only at sides where two Ge free bonds can be passivated by a single O atom, the rest of free Ge bonds on the NC surface remaining passivated by H atoms. We expect O\u2013Ge bonds to produce stronger deformation of the Ge lattice at the NCs surface, resulting in some changes of the bandgap in respect to the case of the passivation with H only. DFT computations in progress will show the effect of the type of passivation atoms on the NCs bandgap.xSix1\u2212 NCs provide an additional parameter, namely the composition for bandgap control over a spectral range wider than that of Ge NCs. We focus our attention on Ge-rich GexSix1\u2212 NCs because of the interest to keep the optical absorption edge and bandgap related cut-off wavelength of spectral photocurrent at longer wavelengths to be compared with reported experimental results up to 1700\u00a0nm wavelengths29. Thus, in DFT calculations of DOS and energy gap, we consider Ge at.% concentrations of 95, 90 and 75 at.% Ge in H-passivated GeSi NCs. The results are compared with those for stoichiometric GeSi (50:50) and pure Ge NCs by varying the NC diameter d from 1.25 to 3.96\u00a0nm as presented in Table xSix1\u2212 NCs, the Ge concentration cannot be kept strictly constant for the whole range of diameters, the standard deviation being about 0.3%  NC is compared to that for Ge NCs in Fig.\u00a0A, \u03b1 and Egbulk are given in inset. The experimental dependences of Egbulk (dependent on Ge concentration) for asymptotic values are taken from the Ioffe database50  curves are vertically shifted by subtracting 0.651(1\u2009\u2212\u2009x) term dependent on Ge concentration x, the data are well superposed over the curve for Ge NCs, shown in Fig.\u00a0Eg of GexSix1\u2212 NCs as a function of diameter d and Ge\u00a0concentration x can be approximated by Eg\u2009=\u2009EgGe(d)\u2009+\u20090.651(1\u2009\u2212\u2009x) in which EgGe(d) is the bandgap diameter dependence for pure Ge NCs. This invariance of the shape of the computed bandgap valid for the whole explored diameters (1.6\u20133.9\u00a0nm) and composition range  instead of Egbulk  of cubic Ge(Si). The spheres have been filled with Ge and Si atoms in the cubic Ge coordination and subjected to lattice relaxation after the H-passivation of the NCs surface. The number of H atoms necessary to fully passivate the NC surface reaches the number of Ge core atoms for smallest NCs and still remains of about 25% for 4\u00a0nm size. The computed DOS for different concentrations and diameters has been used for obtaining the NC bandgap as a function of the diameter for different Ge concentrations. It was found that the shape of Eg is almost independent on composition for the whole investigated diameter-composition range being described by Eg\u2009=\u2009EgGe(d)\u2009+\u20090.651(1\u2009\u2212\u2009x), in which EgGe(d) is the bandgap of Ge:H NCs. For diameters from 1.6 to 4\u00a0nm, the shape of the bandgap dependence on diameter can be fitted by a power function EgGe(d)\u2009=\u20090.66\u2009+\u20094.58/d1.25, and shows a tendency of upper bandgap\u00a0limitation for smaller diameters. The extrapolation to the bulk bandgap value of Eg fit curves can be used for the design and characterization of optoelectronic devices based on GeSi NCs.Spherical H-passivated Ge-rich Ge51 in order to take advantage of the localized basis functions  leading to linear scaling of computation time with the dimensions of the atomistic model. The computations were performed with LDA for the exchange\u2013correlation energy functional.All atomistic simulations were performed using the SIESTA codexSix1\u2212 NC (for each considered Ge concentration x) were generated by considering a cubic simulation cell of 10\u00a0nm edge length in which the NC surrounded by vacuum is centered. The simulation cell acts as the unit cell in periodic boundary conditions computations. This configuration is necessary because the overlap of electronic wave functions belonging to neighboring NCs must vanish.The atomic coordinates of spherical Ges2 4p2 of Ge, 3s2 3p2\u2014Si and 1s1\u2014H were employed, respectively. The calculations were done using a double-zeta basis size, i.e. two basis functions for each numerical atomic orbital, with a 150 Ry mesh cut-off, a limit of 50 steps for self-consistent field loop with a convergence tolerance of 10\u20133 for the elements of the density matrix and 10\u20134 for the elements of the Hamiltonian matrix. The convergence criteria refer to the simultaneous self-consistency of the density matrix and the Hamiltonian matrix. The self-consistency of the density/Hamiltonian matrix is achieved when the maximum difference between the output and the input on each element of the density/Hamiltonian matrix in a SCF loop is smaller than the respective tolerance. The passivation of NC surface is mandatory for excluding surface localized states, but also to ensure the convergence of the DFT-SIESTA computation. According to folding zone theory, for NCs the calculations are done in \u0393 point, only.LDA pseudopotentials for valence electrons 4Total energy was computed by diagonalization of the effective Kohn\u2013Sham Hamiltonian, for 300\u00a0K electronic temperature of the occupation function (Fermi\u2013Dirac distribution). For geometrical relaxation of NCs, we employed the conjugate gradient method with a maximum atomic displacement of 0.1\u00a0\u00c5 and the stop criteria of either 50 relaxation steps or a force tolerance of 0.1\u00a0eV/\u00c5. DOS was calculated for each NC, per unit energy and per unit volume, ignoring electronic spin, as Supplementary Information."}
+{"text": "PHT phosphate transporter genes, global transcriptional regulators such as those of the SPX family and genes involved in galactolipid and sulfolipid biosynthesis such as MGD2/3, PECP1, PSR2, PLD\u03b61/2 and SQD2. Breeding for enhanced PUE in watercress will be accelerated by improved molecular genetic resources such as a full reference genome sequence that is currently in development.Watercress is a nutrient-dense leafy green crop, traditionally grown in aquatic outdoor systems and increasingly seen as well-suited for indoor hydroponic systems. However, there is concern that this crop has a detrimental impact on the environment through direct phosphate additions causing environmental pollution. Phosphate-based fertilisers are supplied to enhance crop yield, but their use may contribute to eutrophication of waterways downstream of traditional watercress farms. One option is to develop a more phosphate use efficient (PUE) crop. This review identifies the key traits for this aquatic crop (the ideotype), for future selection, marker development and breeding. Traits identified as important for PUE are (i) increased root surface area through prolific root branching and adventitious root formation, (ii) aerenchyma formation and root hair growth. Functional genomic traits for improved PUE are (iii) efficacious phosphate remobilisation and scavenging strategies and (iv) the use of alternative metabolic pathways. Key genomic targets for this aquatic crop are identified as:  Nasturtium officinale R. Br.) is a semi-aquatic plant that grows in flowing shallow freshwater and is found across Europe, Asia, the Americas, the Caribbean, New Zealand and Australia  is vital for plant survival; it forms the phosphodiester bonds that link nucleotides in nucleic acids and is critical for the structure of proteins and carbohydrate polymers, for powering cells through the release of phosphate from ATP and for regulating several metabolic pathways , 7. SympNinety percent of the global demand for phosphorus is used for food production, however, rock phosphate is a limited resource with estimates that reserves could be exhausted in the next 50\u2013100\u00a0years \u201314. In a2PO4\u2212, HPO42\u2212 and PO3\u22124) [Phosphates in soils are classified into inorganic P (Pi) and organic P (Po) and fractionised based on their solubility , 22. Po d PO3\u22124) . The avad PO3\u22124) \u201329.Like soil, P in aquatic systems is also divided into different fractions based on solubility and reactivity in aquatic systems, with dissolved orthophosphate the most bioavailable , 31. P iSince P retention in sediments is high, P delivery into freshwater systems is largely governed by release from point sources such as sewage treatment works (STWs), leaking septic tanks, and from excess fertiliser application \u201335. GlobNaturally, phosphate levels in chalk aquifers  are less than 20\u00a0\u03bcg/l, however, inputs of phosphate rapidly increase these concentrations above P targets downstream of watercress farms . In the One important strategy to tackle this problem of eutrophication is through plant breeding. By breeding watercress varieties with improved phosphorus use efficiency (PUE), the impact of watercress farming on eutrophication could be minimised. To date, no breeding for nutrient use has been conducted in watercress even though P release represents a clear issue in watercress production.Phosphate use efficiency (PUE) is defined as the capacity for biomass production using the P absorbed , 41. HerTo date, knowledge on P acquisition by aquatic plants only covers the effectiveness of plants for phytoremediation , rather than breeding for PUE in aquatic crops such as water chestnut (Eleocharis dulcis), water spinach (Ipomoea aquatica), lotus (Nelumbo nucifera) and watercress. Present information does not cover morphological or genetic components to improve PUE in aquatic species, and with new plant species emerging as suggested model organisms, watercress is offered as a model crop for aquatic systems \u201348. The Zea mays), common bean (Phaseolus vulgaris) and Arabidopsis thaliana with RSA shown to be a highly plastic trait, changing in response to the availability of water, nutrients and hormone signalling [Root structural architecture (RSA) defines the spatial configuration of the root system and variation in RSA can reflect efficient phosphate uptake by plants . Much ofgnalling \u201353. For gnalling .Reduced root growth angle (RGA): is one of the most important RSA traits for improving P acquisition in many soil-grown species. In maize and common bean, lower RGA  is associated with increased P accumulation and improved growth in P deficient soil where P is concentrated in the topsoil [ topsoil \u201357. HoweIncreased lateral root density: (arising from the pericycle of mature roots) also enhances P acquisition by allowing plants to explore outside P-depleted zones. Using maize recombinant inbred lines (RILs) with contrasting lateral rooting phenotypes, significant differences in phosphorus acquisition, biomass accumulation and relative growth rate were observed under low phosphorus availability [lability , 59. IncAdventitious roots: from above ground structures can enhance topsoil foraging by up to 10% in stratified soil [Solanum dulcamara) plants under long-term submergence, and as mentioned, adventitious roots are responsible for a higher proportion of P acquisition in watercress than basal roots [ied soil . These ried soil . In aquaied soil , 63. Theal roots , 64. ThuRoot cortical aerenchyma:  are an adaptation to waterlogged soils and reduce the risk of asphyxiation and their formation has been suggested to increase P uptake in deficient soils [nt soils . Severalnt soils \u201368. Modent soils . Howevernt soils . Most aqnt soils . For aquRoot hair density and root hair length: root hair density increases up to 5 times in low P conditions [nditions \u201373. Usinnditions . Root handitions . Root handitions \u201378. RootSynergism of root phenotypes should also be considered. A modelling approach in Arabidopsis showed that the combined effects of root hair length, root hair density, tip to first root hair distance and number of trichoblast files (responsible for forming root hairs) on P acquisition was 3.7-fold greater than their additive effects .For aquatic species such as watercress, the root ideotype for determining optimal P acquisition remains unknown. Although, absorption of P through the shoots is still debated, root uptake is generally regarded as the mode of P uptake in aquatic plants \u201381. WateIn addition, the root cap can account for 20% of the phosphate absorbed by the roots of Arabidopsis . Therefo. Watercress with higher PHT1 expression may result in improved biomass accumulation in P deficient water, but this has yet to be tested.Plants are reliant on phosphate transporters to acquire P from the environment and transport P between tissues, and this includes for aquatic plants. The PHT1 (PHOSPHATE TRANSPORTER 1) family is the most widely studied group of P transporters and is primarily responsible for P uptake but also has a role for P transport between tissues \u201387. A brAdditional traits that are important in other crops are organic acid (OA) exudation and phosphatase activity. Since these control release of P from organic forms in the soil, they are less relevant to watercress cultivation where P released from bound sources would be rapidly lost to the watercourse. However, phosphatases that remobilise P from intracellular sources have been identified in Arabidopsis so similar phosphatases could enhance internal P utilisation in watercress , 92.Alongside phosphate acquisition, PUE also refers to more efficient P utilisation associated with re-translocation and recycling of stored P, that relies on effective P transportation within the plant, P scavenging, and use of alternate biochemical pathways that bypass P use \u201395. Re-tAlternative P use strategies includes substituting phospholipids in cell walls with sulfolipids and galactolipids. Several enzymes in the glycolytic pathway depend on P so bypass enzymes such as pyrophosphate-dependent phosphofructokinase (PPi-PFK), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) can be recruited to use pyrophosphate (PPi) for a P donor and conserve limited ATP pools , 97, 98.Both aspects of PUE (acquisition and utilisation) rely on accurate sensing of the P state within the plant and external environment to alter global gene expression and ensure appropriate responses to upregulate P uptake and P use pathways , 103.Many agricultural traits, such as PUE, are under the control of multiple rather than single genes, defined as quantitative traits and can be mapped on the genome as quantitative trait loci (QTL) and used to identify co-located candidate genes . QTL mapQTL for overall PUE metrics as well as QTL for more specific architectural root traits associated with low P tolerance have been identified in several economically important crops including soybean, soybean (Glycine max), rice (Oryza sativa), maize and common bean \u2013109. RSALPR1) were identified which explained 52% of the variance in primary root length [Brassica napus) primary root length decreases, lateral root length and density increases with declining P concentration [Studies on other Brassicaceae species are likely of most genetic relevance for QTL mapping in watercress, however QTL associated with other species such as soybean, rice, sorghum and wheat are summarised in t length . In rapentration . Severalntration . HoweverGenes involved in phosphate acquisition and utilisation identified in other species for PUE are likely to be candidate genes in aquatic crops like watercress. A set of 95 core phosphate starvation-inducible genes, whose expression changes >2 fold under P deficiency have been identified in Arabidopsis, resulting in the identification of candidate genes for PUE in several other crops including wheat, maize, oat (Avena sativa) rice and white lupin  under P deficiency \u2013136. TheAUXIN-INDUCED IN ROOT CULTURES 12 (AIR12) involved in auxin-induced production of lateral roots and PHOSPHATE DEFICICENCY RESPONSE 2 (PDR2) which is part of growth changes in the plant apical meristem under P deficiency [PDR2 is a major component of the P starvation response and functions together with LPR1 and its close paralog LPR2 as a P-sensitive checkpoint in root development by monitoring environmental P concentration, altering meristematic activity and adjusting RSA [Specific genes involved in root architecture are targets for enhanced PUE. Although RSA traits are highly quantitative, a BLAST to the rapeseed reference genome revealed 19 candidate genes related to root growth and genetic responses to low P in Arabidopsis . These gficiency , 138. PDting RSA , 140.PHR1 (PHOSPHATE STARVATION RESPONSE 1) and PHL1 (PHR-LIKE 1) code for transcription factors that play critical roles in the control of P starvation responses [PHO2 gene, responsible for P allocation between roots and shoots and affects expression of other PSR genes such as PHT transporters [PHR [ZAT6, WRKY75, BHLH32 and OsPTF1) and other regulatory elements (such as SIZ1 and ARP6) are summarised in Genes involved in transcriptional control are multi-functional under P deprivation; some have overlapping roles in RSA development, P signalling and P utilisation. They are discussed together here despite partial involvement in P utilisation. esponses . PHR1 mesporters . SPX traers [PHR \u2013145. Thetaa1 (involved in auxin synthesis) and aux1 (involved in auxin transport) have impaired root hair growth in low P [TIR1 increases under low P availability which results in increased sensitivity to auxin and production of lateral roots [ROOT HAIR DEFECTIVE 6-LIKE-2 (RSL2) and ROOT HAIR DEFECTIVE 6-LIKE-4 (RSL4) are responsive to P deficiency and promote root hair initiation and elongation. ARF19 is a key transcription factor promoting auxin-dependent root hair elongation in response to low P [HPS1 (HYPERSENSITIVE TO PHOSPHATE STARVATION 1) is involved in regulating the sucrose transporter SUC2 and hps1 mutants exhibit significant P-starvation responses under P-sufficient conditions [et al. (2021) used RNA sequencing (RNA-seq) approaches to identify responses to submergence in watercress and found several ABA biosynthesis and catabolism genes associated with stem elongation [Auxin, sugars and other hormones such as cytokinins, ethylene, abscisic acid (ABA), giberellins and strigolactones are implicated in phosphate-induced determination of RSA so genes involved in these pathways may be significant candidates , 147. Unin low P . Expressal roots . Mutantsto low P . HPS1  genes that encode phosphate transporters responsible for transport of P anions are well characterised and are grouped into four families . PHT proteins other than PHT1 are involved in the uptake, distribution and remobilisation of P within the plant, however, PHT1 in the plasma membrane is the most important [OsPHT1;9 and OsPHT1;10 overexpressing rice plants have reduced biomass under high phosphate compared to wild-type plants [PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR (PHF1), are also important for proper functioning of P transporter genes. Homologs of PHT1 transporter genes, transcriptional factors including the SPX gene family, and genes involved in RSA determination such as PDR2 and LPR1/2 could be candidate genes for improving phosphate acquisition in watercress, but these genes have yet to be identified in aquatic crops.For P acquisition, the mportant \u2013153. Phomportant . For exae plants . AccessoPHR1 and PHL1. Here we target genes primarily involved in utilisation, including those responsible for P transport within the plant, alternate metabolic pathways, and internal P-scavenging. Using an Arabidopsis Affymetrix gene chip, changes in global gene expression have been analysed in response to P deprivation [PHT1 genes and PHO1;H1 (involved in P loading into xylem vessels) [The transcriptional regulation of PUE is complex: there is some overlap with genes involved in both phosphate acquisition and phosphate utilisation, such as the global transcriptional regulation by rivation . The expvessels) . Genes iPHT1 genes are induced in other tissues such as leaves, anthers, pollen and seeds, suggesting PHT1 involvement in diverse processes such as root-to-shoot distribution [Pho1 mutants exhibit P deficiency in the shoots due to lack of P loading into the xylem vessels [PHT transporters also play a role in P utilisation through re-translocation of P within the plant. Five of the 13 maize ribution . PHO1 is vessels , 156.. PAP26 functions in PUE by scavenging P from intracellular and extracellular P-ester pools, to increase P availability in the plant. Homologs of PAP26 should be investigated in watercress.Phosphatases are important for remobilisation of fixed P. Eleven genes encoding different purple acid phosphatases were reported to be upregulated under P starvation in Arabidopsis . The AraPPC1, PPC2, PPC3). PPC1 is expressed in roots and flowers, PPC2 in all organs and PPC3 in only roots [PPCK1 and PPCK2 genes are additional important components for P-bypassing [Plants also respond to P starvation by utilising alternative metabolic pathways. Genes involved in lipid metabolism and biosynthesis represent the largest group of core PSI (phosphate starvation inducible) genes in Arabidopsis, demonstrating their importance in PUE under P starvation . Three gly roots , 160. PEypassing , 161.et al. (2005) were involved in lipid biosynthesis pathways in Arabidopsis. This includes the genes MGD2 and MGD3 whose expression changed 11-fold and 48-fold under P deficiency, respectively. These genes encode major enzymes for galactolipid biosynthesis and are involved in replacing phospholipids in cell membranes with non-phosphorus lipids such as galactolipid digalactosyldiacylglycerol (DGDG) [PECP1 is involved in the liberation of P from phospholipids and is upregulated under P deprivation, with up to 1785-fold increases in expression reported in roots [PSR2 encodes a phosphatase involved in galactolipid biosynthesis and whose expression increases 174-fold in P deprived seedlings [PECP1 and PSR2 have similar roles in the dephosphorylation of phosphocholine (PCho) in the galactolipid synthesis pathway. However, despite their massive upregulation, it has been observed that inactivation of PECP1 and PSR2 does not alter plant growth or plant P content under P-deprivation so PCho is not likely a major source of P under limiting conditions [PLD\u03b61 and PLD\u03b62 encode phospholipases D zeta 1 and 2 that hydrolyse major phospholipids such as phosphatidylcholine which yields phosphatidic acid (PA) and PA phosphatase (PAP) and releases DAG  and P [SQD2 is the primary gene in this pathway and encodes an enzyme that catalyses the final step in the sulfolipid biosynthesis [GDPD1 is involved in the formation of glycerol-3-phosphate (G3P) from phospholipid products (such as glycerolphosphoglycerol), that can be dephosphorylated to release P [Membrane phospholipids constitute approximately 20% of the total P in the leaves of P-sufficient plants . This rel (DGDG) . PECP1 iin roots \u2013166. PSReedlings . Both PEnditions , 169. PLs) and P \u2013172. Phoynthesis . GDPD1 ielease P .PHT1 genes responsible for P redistribution, within the plant, genes involved in P scavenging (i.e. PAP26), genes implemented in metabolic pathways that bypass P use including galactolipid biosynthetic pathways  and those involved in sulfolipid biosynthesis (e.g. SQD2) could be candidate genes for improving phosphate utilisation in watercress.Homologs of Watercress root research is virtually completely absent in the literature, with no studies on root responses to phosphate availability. Nevertheless, the finite nature of rock phosphate and the fact that watercress cultivation methods have the potential to result in environmental damage (associated with fertiliser input to waterways), are clear drivers, as with soil-grown crops, to breed for watercress with improved PUE. Uncovering phenotypic traits and the molecular basis for PUE are important early steps in breeding for phosphate use efficiency.et al. (2017) independently used RNA-seq approaches to assemble the watercress transcriptome de novo. They identified 33 candidate genes related to glucosinolate biosynthetic pathways using Arabidopsis glucosinolate genes to search for homologous sequences in the watercress transcriptome [No commercial breeding programs exist for watercress worldwide, but germplasm collections are emerging \u2013219. Devcriptome .2 individuals was established by Voutsina (2017) using parents with contrasting nutrient and growth phenotypes. Genotype-by-sequencing of this mapping population enabled the construction of first genetic linkage map for watercress and identified 17 QTL for morphological traits of interest, antioxidant capacity and cytotoxicity against human cancer cells [Additionally, a watercress mapping population comprising 259 Fer cells . HoweverScreening this mapping population and wider germplasm for root traits may reveal individuals with extreme PUE phenotypes, which could allow the development of markers and QTL associated with this complex trait. RNA-seq approaches could ultimately lead to the identification of candidate genes. Together these findings will assist in developing commercial cultivars with a reduced need for phosphate, and a reduced negative environmental impact. et al., 2015), plus PHR1, the global regulator of P starvation responses. Annotated transcripts were obtained from transcriptomic studies of watercress by Voutsina et al., 2016; Jeon et al., 2017; M\u00fcller et al., 2021 and matches for these candidate genes were assessed by searching for genes using AGI (Arabidopsis Gene Identifiers) [PHT1;4, SPX1, PHR1, MDG3, PEPC1, PLD\u03b61/2, PSR2 and SQD2, with all corresponding e-values ranging from 0 to 3.00E-32. Additionally, M\u00fcller et al. identified homologous transcripts for MDG2. Voutsina et al. had transcripts corresponding to PHT1;3, and MDG2, and Jeon et al. had hits for PHT1;2 and PHT1;3. No matching transcripts were found for PHT1;1 in any of the three studies. Where FDR values  were\u2009<\u20090.05, changes to expression patterns were noted. Interestingly, M\u00fcller et al. also observed varying levels of upregulation of PHT1;4 and PHR1 following submergence, which may suggest a link between phosphate starvation and submergence responses.To assess whether homologs of the candidate PUE genes identified in this study exist in watercress, available watercress transcriptome data was mined for 13 key PUE genes selected from tifiers) , 222. AcWatercress is a non-model leafy green crop, grown using traditional aquatic systems across the world, but also increasingly seen as a suitable crop for vertical hydroponic indoor systems. It is ranked as the top \u201cpowerhouse\u201d food, with the highest nutrient density, including high concentrations of essential vitamins, minerals and phytonutrients . HoweverPHT homologs) and (vii) genes involved in P utilisation, particularly those with roles in P remobilisation  and P-bypass enzymes (e.g. PPC1), may prove good candidate genes for PUE.Breeding new varieties with improved phosphate use efficiency (PUE) is thus a priority for both traditional outdoor and indoor systems. Prebreeding for PUE in aquatic crops such as watercress should target a suite of traits , includiBarriers for root trait breeding include the difficulties in evaluating roots in the substrate, their phenotypic plasticity in response to numerous environmental factors and limited genomic knowledge for watercress. Recent developments in high-throughput phenotyping and whole-genome sequencing-based genotyping will accelerate QTL identification, the discovery of individual genes involved in PUE and lay the foundations for development of new watercress varieties with improved low P tolerance."}
+{"text": "Escherichia coli (DEC) in environmental waters of Johannesburg, South Africa. Samples were collected and cultured on selective media. An 11-plex PCR assay was used to differentiate five DEC, namely: enteroaggregative (EAEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The antibiotic resistance profile of isolates was determined using the VITEK\u00ae-2 automated system. The virulence profiles of 170 E. coli tested showed that 40% (68/170) were commensals and 60% (102/170) were pathogenic. EPEC had a prevalence of 19.2% (32/170), followed by ETEC 11.4% (19/170), EAEC 6% (10/170) and EHEC 3% (5/170). Hybrid DEC carrying a combination of simultaneously two and three pathogenic types was detected in twenty-eight and nine isolates, respectively. The antibiotic susceptibility testing showed isolates with multidrug resistance, including cefuroxime (100%), ceftazidime (86%), cefotaxime (81%) and cefepime (79%). This study highlighted the widespread occurrence of DEC and antibiotic resistance strains in the aquatic ecosystem of Johannesburg. The presence of hybrid pathotypes detected in this study is alarming and might lead to more severe diseases. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant strains in this area.This study was undertaken to determine the virulence and antibiotic resistance profiles of diarrhoeagenic  Escherichia coli (E. coli) is an anaerobic Gram-negative, typically rod-shaped bacterium considered as part of the normal flora of the gut of humans and warm-blooded animals, and can also be found in the environment, e.g., in water and soil [E. coli is classified into harmless commensals, pathogenic and extra-intestinal pathogenic strains [Escherichia coli (DEC) strains are among the causative agents of diarrhoea outbreaks and other serious waterborne and foodborne infections in humans [E. coli (AIEC), (ii) diffusely adherent E. coli (DAEC), (iii) enteroaggregative E. coli (EAEC), (iv) enterohaemorrhagic E. coli (EHEC), (v) enteroinvasive E. coli (EIEC), (vi) enteropathogenic E. coli (EPEC) and (vii) enterotoxigenic E. coli (ETEC) [Diarrhoea is one of the common causes of morbidity and mortality among infants and children in most developing countries . It is eand soil ,5. Accor strains . Pathogen humans ,8. Seveni (ETEC) ,10,11. Ti (ETEC) ,13.pAA), for fimbriae production, which contains the aggregative adherence fimbriae type R (aggR) [ipaH) and invasive antigen locus  genes, which encode the virulence regulon, the key for severe/invasive intestinal infections and dysentery [E. coli adherence factor plasmid (EAF) that carries the bfpA gene, which encodes for the bundle-forming pili [eaeA gene) and the bfpA gene, while atypical EPEC (aEPEC) are strains that do not contain the bfpA gene [EAEC is a pathotype associated with persistent diarrhoea in humans and has been identified as possessing a plasmid of aggregative adhesion (R (aggR) . EHEC isR (aggR) ,15. EIECysentery ,17. EPECing pili . TypicalfpA gene . ETEC isfpA gene .E. coli [E. coli virulence genes are generally found on plasmids, transmissible by means of conjugation [Hybrid DEC harbouring a combination of virulence genes have emerged worldwide and have been reported as a public health concern ,22. ThisE. coli  or to thjugation . The firjugation . Since tjugation  and in tjugation , EPEC/EHjugation  and STECjugation ,22.E. coli is also widely recognised due to its role in spreading antibiotic resistance in the water environment [E. coli bacteria, including conjugation and horizontal gene transfer among the isolates [The widespread occurrence of waterborne infections and the increase in antibiotic-resistant strains have become global health concerns. According to the Centres for Disease Control and Prevention, at least 2.8 million people acquire an antibiotic-resistant infection, and more than 35,000 people die each year in the United States alone . In addiironment . Severalironment ,29,30. Uironment . Studiesisolates . HoweverIn South Africa, only a few studies have reported the virulence and antibiotic resistance profile of DEC isolated in the environment. Therefore, this study aimed to determine the virulence profile of five DEC with special attention to the hybrid strains with the potential genetic combinations and their antibiotic resistance profile in the environmental water of Johannesburg, South Africa.n = 101) were collected from nine water sources between August 2020 and February 2021. These included: (i) surface water from Jukskei River (n = 46) and Kliprivier River (Eikenhof) (n = 33), (ii) run-off water, upstream and downstream from the six Hennops river sites (n = 19), and (iii) sewage water from a stream and a well (n = 3), all in the Johannesburg, Gauteng Province, South Africa , before sample collection. Environmental water samples . The membrane filters were aseptically placed directly onto HiChrome\u00ae Coliform chromogenic media  and incubated  at 37 \u00b0C for 18 to 24 h. Bluish colonies were selected as presumptive E. coli  and were sub-cultured onto M\u00fcller-Hinton agar plates  and re-incubated at 37 \u00b0C for 24 h. Pure colonies were Gram-stained and identified using the VITEK-2\u00ae automated system . Escherichia coli isolates were inoculated into Luria Bertani (LB) broth , grown overnight (at 37 \u00b0C for 24 h) and stored long-term at \u221280 \u00b0C in a biofreezer  in a 50% (v/v) sterile glycerol solution  Ltd., Gauteng, South Africa) until further analysis. \u00ae Laboratories Pvt. Ltd., Maharashtra, India) at 37 \u00b0C overnight and the total genomic DNA was extracted using the silica/guanidium thiocyanate  method adapted from the article previously published [Two mL of presumptive isolates were grown in LB broth ), 2 \u03bcL of MgCl2, 1 \u03bcL of Q-solution, 4.0 \u03bcL of PCR-grade water and 2 \u03bcL of sample DNA. The following conditions were used during PCR amplification performed in a Bio-Rad MyCyclerTM Thermal cycler : an initial activation at 95 \u00b0C for 15 min, followed by 35 cycles consisting of denaturation at 94 \u00b0C for 45 s, annealing at 55 \u00b0C for 45 s, extension at 68 \u00b0C for 2 min and final extension at 72 \u00b0C for 5 min.The 20 \u03bcL Hotstart multiplex PCR reaction consisted of 10 \u03bcL of Qiagen master mix , 1 \u03bcL of the primer mixture (forward and reverse)  agarose gel , and stained with 5 \u00b5L of a 10 mg/mL stock solution of ethidium bromide  using TAE buffer . Gel electrophoresis was performed at 90 volts for 120 min. A 100 bp molecular weight marker  was included as a reference in all gels. The gel images were visualised and captured using a Gel DocTM EZ system .The PCR-amplified products were separated on a 2.5%  ATCC 25922, enteroinvasive (EIEC) ESCCOS ATCC 43893, enteroaggregative (EAEC) ESCCO 14, enterohaemorrhagic (EHEC) ESCCO 21, enterotoxigenic (ETEC) ESCCO 22 and enteropathogenic (EPEC) S-ESCCO 16 Pl. All these strains were purchased from the National Health Laboratory Service (NHLS), South Africa, and were confirmed in a previous study [eaeA, eagg, Asta, bfp, gapdh, ial, lt, mdh, sta, stx1, stx2). The malate dehydrogenase (mdh) housekeeping gene was used as an internal control and the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene was used as an external control. The gel images were analysed by reading the presence of bands detected. All data obtained were recorded and exported into a Microsoft Excel sheet for analysis.The control strains used in this study included us study . The posEscherichia coli and some commensal isolates were selected and tested for antibiotic susceptibility using the VITEK\u00ae2 automated system . Briefly, a bacterial suspension with an optical density (turbidity) of 0.5 McFarland was prepared in saline (0.85%)  from an overnight bacterial culture incubated  for 18 h. The antibiotics included in the VITEK\u00ae2 automated system panel were: ampicillin, amikacin, amoxicillin/clavulanic acid, cefepime, cefotaxime, cefoxitin, ceftazidime, cefuroxime, cefuroxime-Axetil, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, piperacillin/tazobactam, tigecycline, tobramycin and trimethoprim/sulfamethoxazole. The minimum inhibitory concentration (MIC) for each isolate tested was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2021 guidelines [All pathogenic idelines .\u00ae Coliform chromogenic media  identified 288 presumptive E. coli, which appeared bluish, as from the manufacturer. The PCR assay confirmed 170 E. coli isolates with the detection of the mdh housekeeping gene. The presence of the gapdh gene used as an external control excluded any possible PCR inhibition in all samples. The 11-gene multiplex PCR assays indicated that 40% (68/170) were commensals E. coli (ComEC) and 60% (102/170) were positive for at least one pathogenic E. coli type. In total, 101 environmental water samples were collected and analysed in this study. The HiChromeE. coli (EPEC) was the most detected in this study, with 19.2% (32/170) of isolates, of which atypical EPEC (aEPEC) harbouring only the eaeA gene accounted for 18% (30/170) while typical EPEC (tEPEC) harbouring both the eaeA and bfp genes accounted for 0.6% (1/170), and the bfp gene alone was present in 0.6% (1/170). Enterotoxigenic E. coli was detected in 11.4% (19/170) of isolates, with only a limited number of isolates harbouring both lt and sta genes , while sta alone was present in 9% (15/170) and lt alone in 1.2% (2/170). EAEC harbouring the eagg gene was present in 6% (10/170), and EHEC was detected in 3% (5/170), of which 1.2% (2/170) of isolates harboured stx1 and stx2 each, whereas both stx1 and stx2 were detected in 0.6% (1/170). EIEC harbouring the ial gene was not detected in this study. E. coli pathotypes and their virulence genes detected.Enteropathogenic Hybrid pathogenic types forming two pathotypes harbouring a combination of virulent genes were detected in 28 isolates, including EAEC/EPEC , EAEC/ETEC , EPEC/ETEC , EPEC/EHEC  and EHEC/ETEC . The complete distribution of virulence genes forming a hybrid of two pathotypes is illustrated in E. coli isolates forming three pathotypes. Singleplex PCRs were run on all hybrid isolates as confirmatory tests and the results showed the presence of the single genes detected by different pathogenic types , EHEC/ETEC/aEPEC  and EAEC/EHEC/ETEC . ic types .E. coli isolates (n = 100) using the VITEK\u00ae2 automated system  showed isolates with multidrug resistance . Antibiotic resistance patterns of selected sistance . All isosistance . At leasE. coli (DEC) can pass from the environment to humans, causing severe diseases. Studies have reported waterborne diarrhoeal disease claiming two million deaths worldwide each year, mostly in children below 5 years of age [Diarrhoea outbreaks are a persistent problem with significant economic and potential public health impacts worldwide. In most developing countries, people continue to use environmental water (river water and stream) for domestic activities such as bathing, washing clothes, cooking and drinking . It has s of age .E. coli were identified using HiChrome\u00ae Coliform selective chromogenic media  and the VITEK\u00ae-2 automated system . Studies have reported these techniques to generate many errors and they can accommodate the growth of other species, leading to the misidentification of colonies, especially in environmental samples [E. coli were isolated, and the prevalence of commensal and DEC identified was 40% (68/170) and 60% (102/170), respectively. Among the DEC identified, EPEC was the most common single enteropathogen, with 19.2% (32/170) of cases. Studies have shown that infections due to EPEC are usually endemic in developing countries [eaeA, and 1.2% (2/170) were typical EPEC (tEPEC), of which 0.6% harboured both eaeA and bfp genes and 0.6% harboured the single bfp gene. The importance of distinguishing typical and atypical EPEC is that tEPEC causes infections mostly in infants, while aEPEC has been reported to cause infections in both children and adults [This study was carried out to determine the virulence profile of DEC and their antibiotic resistance profile in the environmental water of the Johannesburg region, South Africa. In total, 101 samples were included in this study, from which 288 presumptive  samples . As suchountries ,39. The ountries ,40 and eountries ,41. In td adults . The prod adults  in Vhembd adults .This study detected ETEC in 11.4% (19/170) of isolates. The genes that encode both heat-liable (LT) and heat-stable (ST) enterotoxins in ETEC are generally found on plasmids, transmissible and causing severe diarrhoea . Our finThis study has identified 6% (10/170) of EAEC pathotypes in environmental water. Infection due to the EAEC pathotype is dangerous in immuno-compromised individuals and children  and has stx1 and stx2 gene, while one isolate harboured both stx1 and stx2. The detection of the stx gene in river water is of concern because the colonisation of the human large intestine with EHEC stx even in low proportions can result in potentially fatal complications, such as haemolytic uremic syndrome (HUS) [The current study detected the EHEC pathotype in 3% (5/170). Two isolates harboured each single me (HUS) . Previoume (HUS) , 15.08% me (HUS)  and 8.3%me (HUS) .ial virulence genes was not detected in environmental water. This is not surprising because this pathotype is mostly reported as causing dysentery in humans and sometimes in animals [A similar study conducted in the USA has reported 14% of EHEC in water samples . In Geor animals ,51.E. coli (EHEC/ETEC) have previously been reported associated with diarrhoeal disease in humans and animals [Our study revealed hybrid pathotypes with virulence combinations among the isolates. This might be due to the mechanism of conjugation in virulence-associated genes which define a pathotype and are carried on mobile genetic elements (plasmids) . Hybrids animals ,52. In t animals  and in B animals , respect animals . The pre animals  identifi animals  and Mexi animals . All theeaeA + stx1 + stx2 + lt + sta were detected in two isolates. This indicates that hypervirulent strains are circulating in our area. Further confirmatory tests such as sequencing and comparative genomics would need to be performed on these strains to characterise and determine their phylogenetic position. Similarly, in Finland, three hybrid pathotypes were detected among E. coli strains harbouring up to six gene products [Interestingly, in this study, three different DEC strains were simultaneously detected in isolates. Hybrids of aEPEC + EHEC + ETEC harbouring products ; while iproducts  detectedE. coli is one of the pathogens that cause common hospital-acquired and community-acquired infections worldwide. Consequently, treatment is becoming increasingly difficult due to high rates of antimicrobial resistance [E. coli isolates was determined and resistance to multiple antibiotics was identified in the environmental water using the VITEK\u00ae2 automated system . Resistance to the most common antibiotics used for the treatment of infections due to DEC, including last-resort antibiotics, was observed among the isolates. The most abundant resistances were against cefuroxime (100%) and both ampicillin and ceftazidime (94%), followed by cefotaxime (88%), cefepime (84%), amoxicillin/clavulanic acid (82%) and imipenem (70%). These findings are higher as compared to previous study findings in the KwaZulu-Natal (Durban), Western Cape (Stellenbosch) and Eastern Cape provinces of South Africa [In recent times and in modern medicine, the increasing microbial drug resistance has been identified as the biggest public health challenge and listed as one of the public health priorities among CDC fights ,27,56. Asistance . In thish Africa ,30,48. Th Africa . To prevh Africa ,58.\u00ae-2 automated system to assess drug susceptibility. This phenotypic method is mostly used in routine diagnosis laboratories to determine the bacterial susceptibility/resistance to antimicrobials (which sometimes generates errors). It has also been reported that some of the VITEK\u00ae-2 cards\u2019 susceptible MIC breakpoints differ from one another as recommended for therapy of some infections per CLSI [\u00ae-2 automated system does not detect the presence of antibiotic-resistant genes as performed in scientific research laboratories. Further tests including sequencing would need to be performed on these isolates for the detection of extended-spectrum beta-lactamases, carbapenemases and plasmid-mediated colistin antibiotic resistance genes.One of the limitations of this study was the use of the VITEKper CLSI . FurtherE. coli bacteria, which are environmental and public health concerns. This present study highlighted the widespread occurrence of potentially DEC and antibiotic resistance genes in the aquatic ecosystem of Johannesburg, South Africa. Among the enteropathogens tested, EPEC was the most dominant, followed by ETEC, EAEC and EHEC. The presence of hybrid pathotypes detected in this study can pose a potential public health risk to consumers of untreated water in the region. The single-step 11-gene multiplex PCR system used in this study is potentially a quick, powerful and useful method for routine monitoring, virulence gene screening and risk assessment of water quality in developing countries. This study reported the presence of numerous multidrug-resistant strains among the isolates, of which resistance to the most common antibiotics used for the treatment of infections due to DEC was observed. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant"}
+{"text": "Technology designers often exclude individuals aging with diverse needs, capabilities, and disabilities from engaging in the design process, which can hinder the usability and usefulness of emerging technologies. In this symposium, investigators report on research and development efforts aimed at understanding the needs of, and developing supportive technologies for, people aging with long-term disabilities. This symposium features projects from the Rehabilitation Engineering Research Center on Technologies to Support Aging-in-Place for People with Long-Term Disabilities (RERC TechSAge), which is an interdisciplinary collaboration between Georgia Tech and the University of Illinois at Urbana-Champaign. First, Bayles et al. will discuss findings from the Aging Concerns, Challenges, and Everyday Solution Strategies II study, focused on Deaf older adults\u2019 use of technologies as solution strategies for common everyday challenges. Next, Mitzner et al. will highlight the development of an evidence-based group exercise intervention (Tellewellness Tai Chi for Arthritis) aimed at promoting both physical exercise and social interaction for older adults with long-term mobility disabilities. Kadylak et al. will describe how voice-activated digital assistants can support older adults aging with mobility disabilities by reporting on findings from a longitudinal demonstration project with older adults in assisted and independent living communities. Exploring the potential for smartbathroom technology to promote aging in place, Sanford et al. will discuss how smartbathroom sensor data can be analyzed and vizualized to identify ways to communicate insight from sensor data to improve training of occupational therapy practitioners. Susy Stark from Washington University will serve as the discussant for the symposium."}
+{"text": "Para phenylenediamine (PPD) is a common component of hair dye as well as temporary tattoos and is a well-known cause of type 4 hypersensitivity reactions from topical exposure. While there have been several cases reported in the literature describing toxicities following ingestion, there are a paucity of reports of severe systemic disease following topical exposure. Cases of PPD ingestion have been reported to present with angioedema-like reactions, often progressing to rhabdomyolysis and renal failure. To our knowledge, there have only been two reported cases of severe reactions following topical exposure to PPD. We present a case of a 59-year-old man with topical exposure to hair dye who presented with an angioedema-like reaction shortly after topical exposure to PPD containing hair dye that rapidly progressed to rhabdomyolysis, renal failure, and eventually death. Para phenylenediamine (PPD) is a common component of hair dye and henna tattoos and is known to be associated with type 4 hypersensitivity reactions . AlthougA 59-year-old man with a past medical history of atrial flutter status-post ablation, hypertension, osteoarthritis, and depression presented to the emergency department (ED) with a three-day history of a burning, erythematous rash involving the neck, forehead, and cheeks.\u00a0He denied any history of allergies or taking new medications. He did report applying a new hair dye to his scalp and beard. No other environmental exposures were identified. Apart from an elevated blood pressure, all other vital signs were within normal limits. He was diagnosed with allergic contact dermatitis and was discharged home with a Solu-Medrol dose pack, ranitidine, loratadine, and instructions to return to the ED if symptoms worsened.\u00a0Six days later, he returned to the ED with worsening asymmetric facial swelling (right > left), difficulty breathing, dysphagia with upper and lower lip swelling, and an erythematous rash with wheals on his bilateral upper extremities and neck. Labs were notable for a leukocytosis of 16.5 cell/L with an unremarkable basic metabolic panel and venous blood gas. Bedside laryngoscopy was performed by ENT showed normal mucosa, no pooling of secretions, and no laryngeal edema. He was admitted for angioedema and started on dexamethasone, famotidine, diphenhydramine. His lisinopril was discontinued out of an abundance of caution in the setting of angioedema of unknown etiology.\u00a0On hospital day 1, he became febrile, tachycardic, and tachypneic with hypoxia. Repeat laryngoscope examination showed the increased base of tongue fullness, pooling of thick clear secretions, and laryngeal edema.He was naso-tracheally intubated for airway protection and was started on broad spectrum antibiotics, including vancomycin and cefepime for possible sepsis. A computed tomography scan of his neck showed extensive soft tissue swelling and edema with extension to the anterolateral neck. Dexamethasone, famotidine, and diphenhydramine were continued during his entire. Blood cultures grew methicillin-resistant Staphylococcus aureus (MRSA) in two bottles and his antibiotics were narrowed to vancomycin.\u00a0He was started on continuous renal replacement therapy (CRRT) for worsening renal failure.\u00a0Dermatology was consulted for possible PPD allergy from recent hair dye use. PPD patch test was not performed as he was on systemic steroids and the yield was thought to be insignificant. On hospital day 3, he developed upper and lower lip mucosal lesions with necrotic erosions and overlying hemorrhagic crust. He was started on Valtrex for presumed  herpes simplex virus 1 (HSV-1) infection. On hospital day 5, he showed significant clinical improvement with discontinuation of vasopressors and transition to intermittent hemodialysis. However, on hospital day 6, he became febrile to 107 F with a worsening metabolic profile including refractory hyperkalemia with prolonged PR and QRS intervals unresponsive to medical therapy and CRRT. His creatine phosphokinase was elevated to >22,000 units/L, and he developed bullous lesions with serous drainage of his right lower extremity as well as erythema and induration of his left axilla. Given his clinical deterioration and concern for compartment syndrome, emergent bedside fasciotomy was performed with no evidence of necrotic muscle. Shortly after the procedure, the patient went into cardiac arrest and expired.PPD has been a common component of hair dye and henna tattoos for more than 100 years. There is a well-described PPD hypersensitivity reaction that typically presents as a type 4 hypersensitivity reaction after topical exposure that involves pruritus, erythema, and papules involving areas of dye exposure . A retroThere have only been two reported cases of severe reaction to topical application of PPD. In both of these cases, the patients experienced severe anaphylaxis and died. The first patient received typical treatment for anaphylaxis but, despite this, died . In the Currently, there is no specific antidote or treatment for patients suffering from either topical or ingested PPD poisoning. Airway management is key, especially in those suspected of having laryngeal edema. A rapid evaluation with laryngoscopy along with prompt intubation can decrease mortality. Prompt initiation of steroids and antihistamines to target mast cell degranulation is key as PPD poisoning is thought to be a type 1 hypersensitivity unlike the type 4 hypersensitivity reaction associated with topical use. Rhabdomyolysis as a direct effect of PPD-induced necrosis of skeletal muscle can be managed with aggressive hydration, and dialysis should be considered if the patient is believed to be experiencing heme-pigment induced acute renal failure ,10. PlasPrompt recognition of the diagnosis of PPD toxicity through history is essential. As there are no evidence-based treatments available, supportive care as outlined above remains the mainstay of treatment. Plasma exchange transfusion can be considered\u00a0a possible last-line therapy for patients who fail to respond to supportive care.\u00a0Our patient\u2019s presentation is incredibly unique in that topical exposure to PPD presented like ingestion of PPD."}
+{"text": "Ocular toxoplasmosis caused by Toxoplasma gondii is an infectious disease which is widely distributed around the world and can present with various clinic manifestations. We are here reporting an unusual case presented with epiretinal membrane (ERM), i.e., macular pucker.A 16-year old male patient visited our outpatient clinic complaining of decreased vision for about 8 years in his left eye. The best-corrected visual acuity (BCVA) was 20/20 OD and 20/400 OS. There was sensory exotropia in his left eye. No inflammatory cells or flare were found in his anterior chamber or vitreous cavity OU. An ERM involving his left macular area was found on his dilated fundus exam, which was confirmed by Optical Coherence Tomography (OCT). The ERM was found to involve his left macular area with his foveal ellipsoid zone absent. The right eye was found to be within normal limit.After a thorough discussion with the patient and his parents about treatment options and surgical benefits, risks and alternatives, we performed vitrectomy, peeled off the ERM and collected the vitreous sample for parasite testing during the procedure. Patient\u2019s blood also was drawn for serological testing. Vitreous sample analysis and serological tests confirmed ocular toxoplasmosis OS as his final diagnosis. Unfortunately, the BCVA of this patient was not improved after the surgery, but the exotropia disappeared.ERM is an unusual clinical presentation of ocular toxoplasmosis. We may add Toxoplasma gondii infection as a differential diagnosis when encountering ERM cases. Toxoplasmosis is an infectious disease caused by the obligate intracellular parasite Toxoplasma gondii, which is widely distributed around the world. About 30% of people in the world are infected with toxoplasmosis . Cats arA 16-year old male patient visited our outpatient clinic with a chief complaint of decreased vision for about 8 years in his left eye. He had a history of close contact with cats and dogs, with no history of any other systemic illness, trauma or surgery. The best-corrected visual acuity (BCVA) was 20/20 in his right eye and 20/400 in his left eye. The intraocular pressure was normal in both eyes. There was sensory exotropia in his left eye. No inflammatory cells were found in his anterior chamber or vitreous OU. The fundus exam of his left eye showed a white oval-shaped ERM with the size of of 2.0 papillary diameter, some pigmented chorioretinal scars, and yellow-white round retinochoroidal lesions on the temporal side of the macula . His intraocular pressure was still within normal limits. Unexpectedly, his left exotropia resolved after the surgery. The lens and vitreous remained unremarkable. Postoperative fundus examination  or postnatally acquired . CongeniIn our case, the ERM covering the macular area of the patient\u2019s left eye was accompanied by severe visual loss with sensory exotropia. After thorough preoperative examinations and detailed discussions of benefits, risks and alternatives with the patient and his parents, a middle and posterior vitrectomy combined with ERM peeling surgery was performed. One month after the surgery, the patient\u2019s BCVA was still 20/400, but the exotropia vanished and the eye position was notably improved.Currently, intraocular fluid and serologic testing of Toxoplasma gondii DNA, IgG and IgM are commonly used to diagnose Toxoplasma gondii infection. The patient\u2019s vitreous humor and blood specimens were tested for Toxoplasma DNA, IgG and IgM. The results of intraocular fluid showed that Toxoplasma DNA was zero, Toxoplasma IgG was 6.95\u2009IU/ml (reference range less than 4\u2009IU/ml). His serum Toxoplasma IgG was 20\u2009IU/ml (reference range was less than 20\u2009IU/ml), and the GWC value of Toxoplasma was 36.72 (reference range 0\u2009~\u20092). Because Toxoplasma gondii is an obligate intracellular parasite, the positive rate of Toxoplasma DNA detection in intraocular fluid is usually low, about 30\u201340% . AlthougThe classical treatment of ocular toxoplasmosis is triple therapy with pyrimethamine, sulfadiazine and prednisone . For immERM, especially thick ERM, secondary to ocular toxoplasmosis as the main presentation is rare. Intraocular fluid and serologic testing can provide sufficient evidence for the diagnosis of toxoplasmosis. Vitrectomy combined with ERM peeling is an effective treatment for ERM secondary to ocular toxoplasmosis. This case serves as a reminder that the possibility of Toxoplasma gondii infection should be taken into consideration in pediatric ERM patients."}
+{"text": "Antimicrobial resistance (AMR) can be induced by overuse or misuse of antimicrobials. Few researches were involved in consumers' knowledge and attitude toward antimicrobial use (AMU) in food production. This study was designed to investigate the knowledge and awareness, perception, and attitude of Chinese consumers toward AMU in food production. Their behavior, purchase intention of antimicrobial-free food products, and confidence in information sources were also investigated.t-test and one-way analysis of variance (ANOVA) test, and the difference in intention was performed by Chi-square test, when compared with demographic factors.As a descriptive cross-sectional study, an online electronic survey questionnaire was conducted between February 25 and March 8, 2022, involving 1,065 consumers in China. Factor analysis was conducted to identify underlying patterns of the attitudes and information sources. Spearman correlations were employed to determine the relationship between knowledge, attitudes and the intention to pay extra. The differences in knowledge and attitudes were performed by independent The findings showed that even though 75.0% of them heard of AMR, and 48.2% knew the definition of AMR, the level of consumers' knowledge of AMU in farming production and food regulations in China was not high (48.9% of participants replied correctly). About half viewed AMU and AMR as a potential risk to their health. Of these participants, 61.3% claimed that they were more likely looking for specific information about AMU on food packaging, and 58.3% changed their eating or cooking habits due to the concern. In addition, 79.8% were willing to pay extra for antimicrobial-free food products. Information sources from professionals and authorities were considered more accurate than those from media, the internet, word of mouth, and others.Chinese consumers had insufficient knowledge and neutral attitudes about AMU in farming production and food regulations in China. A large proportion of the participants were willing to purchase antimicrobial-free food products. Most of them obtained related information from the media. This study highlighted the importance of updated education and effective communication with consumers in China. It helps to develop the reliable foodborne AMR surveillance system along food chain and improve government communication and consumer awareness. The emergence and spread of food pathogens resistant to antimicrobial drugs, especially those multi- and pan-drug resistant bacteria also known as \u201csuperbugs\u201d has created alarming concerns over public health due to significant economic and health impacts . AMR is Since the 1940s, veterinarians and animal scientists have demonstrated that adding small amounts of antimicrobials (<200 g/ton feed) to feed or water could promote animal growth and higher feed conversion rates, reduce production diseases and improve food animal welfare, which had created a livestock production standard that has resulted in lower production costs and lower meat prices . In 2013Association between interventions to reduce AMU in farm animals and decrease in the level of antimicrobial-resistant bacteria in livestock was identified . Owing tConsumers, as one of the important stakeholders, play a significant role in the food supply chain . It is iA scientific report across the EU conducted by European Food Safety Authority (EFSA) showed that European consumers were aware of antimicrobials and AMR, however, the level of understanding of AMU in farming production was not high . SimilarThere were some differences in concerns and intentions regarding AMU in food animals from country to country. Most Chilean consumers considered AMU as a potential risk to their health, so they believed that it was a necessary practice to avoid AMU and were more likely to purchase antimicrobial-free products certified by trusted authorities . NeverthAs few studies focused on the investigation of consumers' knowledge, attitude, and behavior toward AMU in food production, and there were conflicts and gaps of information among previous studies, further research is needed to identify more convincing evidence and potential reasons contributed to this issue, especially after different levels of limitation were enforced due to changes in regulations. In China, as one of the largest consumer markets globally, research was not done on the Chinese consumers' knowledge, attitude, and behavior since China released the updated restriction on AMU in food production in 2019. This study aimed to investigate the knowledge and awareness, perception and attitude of Chinese consumers toward AMU in food production, and to evaluate their behavioral changes, purchase intention, and confidence in information sources of antimicrobial-free food products.The online survey questionnaire, consisted of seven sections and 28 questions , was impThe survey was piloted in a sample of 15 participants to ensure that it was easy to understand in the Mandarin version and that there were no technical problems in consent instructions, order and category of questions, and response duration. Then the questionnaire was revised and finalized according to the suggestions before the formal data collection.The online survey was conducted between February 25 and March 8, 2022. The questionnaire was distributed by Tencent Questionnaire and Wechat app as a web link to invite public consumers to participate in the survey. The minimum recommended sample size was 385 participants based on the standard formula for sample size calculation of simple random sampling, with a 5% margin of error, a confidence interval of 95%, and the current population in China. Given potential missing data, sample size was added by 20% extra to reach 481 samples. The survey was completed by 1065 respondents, which met the required number of samples.t-test was replaced by t'-test, and one-way analysis of variance (ANOVA) test was replaced by Welch test.Descriptive statistics included frequencies or percentages of categorical variables, and means \u00b1 standard deviations (SD) and 95% confidence interval (CI) of continuous variables. In terms of Shapiro-Wilk test, histogram, Q-Q plot, and the numbers of samples, all the continuous variables were considered as near normal distribution. Levene test was used to determine the homogeneity of variances. If variances showed heterogeneity, t-test when compared with \u201cgender,\u201d and by ANOVA test and Bonferroni post-hoc test for other demographic groups. Factor analysis with Direct Oblimin rotation was conducted to identify underlying patterns of the attitudes. Initial eigenvalues were used to screen the number of factors. Kaiser-Meyer-Olkin (KMO) measure and Bartlett's test of Sphericity were used to verify the adequacy of factor analysis. The internal reliability of each scale was then tested to evaluate the contribution of each item to the factor with Cronbach's \u03b1 coefficient. Spearman correlations between the identified scales were employed, to determine the relationship between the mean scores of attitudes. The differences in attitudes among demographic characteristics were also evaluated. Spearman correlations were employed to determine the relationship between the percentage of the intention to pay extra. The difference in intention was performed by Chi-square test for all demographic groups. Similarly, factor analysis with Direct Oblimin rotation was conducted to identify information sources. The confidence scales of information sources were constructed by computing the mean value of the loading items for each factor. Measure of sampling adequacy and internal reliability test were also performed. The dependence value of Cronbach's \u03b1 coefficient was defined as acceptable when it was \u22650.70. P < 0.05 was considered statistically significant. Data analysis was conducted in IBM SPSS Statistics for Mac version 26 . GraphPad Prism for Mac version 8.0  was used to create charts.The differences in knowledge and awareness were performed by independent Demographic characteristics were presented , includiGeneral awareness of AMR or antibiotic resistance was high. Of respondents, 75.0%  claimed that they had heard of antibiotic resistance or AMR, 48.2%  knew what AMR was, 18.4%  felt that they did not know it, while 33.4%  were not sure about the definition of AMR.However, the knowledge and awareness of AMR and AMU in food production was not high. Respondents gave 8.8\u00b13.7 95%CI 8.6\u20139.0) correct answers for 18 statements from Q9 to Q11, which meant that participants were able to reply correctly with 48.9% (8.8/18) on average to these statements ,C. For tI 8.6\u20139.0P < 0.001) (P < 0.001), senior high school (both P < 0.001), and college (both P < 0.001). Participants in the \u201cpostgraduate and higher degrees\u201d group were also significantly more knowledgeable than those who were bachelors (P = 0.044). When compared with work status, those who were \u201cemployed full-time (\u226530 h per week)\u201d had significantly higher awareness than those who were \u201cunemployed\u201d (P= 0.008). In addition, there was a significant difference among multiple income groups. Those \u201cprefer not to say\u201d had lower knowledge level than respondents whose total income was \u201c\u00a5 500,000\u2013\u00a5 1,000,000 per annum\u201d (P = 0.014). Those with total household income \u201c\u00a5 200,000\u2013\u00a5 500,000 per annum\u201d had higher awareness of AMR and AMU in food production, compared to respondents with household income \u201cunder \u00a5 50,000 per annum\u201d (P < 0.001), and \u201c\u00a5 50,000\u2013\u00a5 100,000 per annum\u201d group (P = 0.007). There was no statistically significant difference in comparisons among \u201cgender,\u201d \u201cage,\u201d \u201chousehold adults\u201d and \u201chousehold children\u201d .It was discovered that there was a significant difference in the knowledge between the education levels  . Those wF = 3.2, P = 0.007). The respondents who were employed full-time gave 1.6 \u00b1 1.0 (95%CI 1.6-1.7) correct answers which was significantly higher than those who were retired, 1.2 \u00b1 0.9 (95%CI 1.0-1.4), (P = 0.011). There was no significant difference among different groups of \u201cgender,\u201d \u201cage,\u201d \u201chighest level of education,\u201d \u201chousehold income\u201d, \u201chousehold adults,\u201d and \u201chousehold children\u201d .Knowledge and awareness of current situations of AMU in food production in China were also low. Respondents gave 1.6\u00b11.0 (95%CI 1.5\u20131.6) correct answers for 4 statements, which meant that participants were able to answer correctly to 38.9% (1.6/4) on average of these statements . Over haThe percentage scores of participants for each statement were reported . ResultsP < 0.01), meaning that the participants who were more concerned about the issue were less likely to perceive that insufficient measures were being taken to limit the overuse of antimicrobials.The exploratory factor analysis of perception and attitude was yielded with a two-factor solution which explained 82.3% of the variance. Factor one consisted of three concern statements related to AMR and AMU in food production, which was renamed the scale of antimicrobial-use attitude. Factor two included one perception related to current situations of that in China, which was renamed the scale of China-situation attitude . The intF = 9.2, P < 0.001) . Similarly, the younger respondents who were 18\u201330 years old perceived to a less extent that insufficient measures were being taken to control the overuse of antimicrobials, compared to those aged 41-50 (P < 0.001), and 51\u201360 (P < 0.001). Differences could also be observed among the household income groups both in the scales of antimicrobial-use attitude and China-situation attitude. For the scale of antimicrobial-use attitude, the respondents whose household income was \u201c\u00a5 200,000\u2013\u00a5 500,000 per annum\u201d replied with higher concern than those with \u201cunder \u00a5 50,000 per annum\u201d (P = 0.026) and \u201c\u00a5 100,000\u2013\u00a5 200,000 per annum\u201d (P = 0.033). For the scale of China-situation attitude, the respondents whose household income was \u201c\u00a5 50,000\u2013\u00a5 100,000 per annum\u201d perceived to a less extent that insufficient measures were being taken to control the overuse of antimicrobials than the groups with household income at \u201c\u00a5 200,000\u2013\u00a5 500,000 per annum\u201d (P = 0.003) and \u201c\u00a5 500,000\u2013\u00a5 1,000,000 per annum\u201d (P = 0.015). Similarly, the respondents whose household income was \u201cnot stable/not sure\u201d perceived to a less degree that insufficient measures were being taken to control the overuse of antimicrobials than the groups \u201c\u00a5 200,000\u2013\u00a5 500,000 per annum\u201d (P = 0.027) and \u201c\u00a5 500,000\u2013\u00a5 1,000,000 per annum\u201d (P = 0.019). When compared with the work status, the participants who were students were less worried about AMU in food production than those who were employed full-time (P = 0.005). Likewise, students perceived less that insufficient measures were taken to control the overuse of antimicrobials than those who were retired (P = 0.028). When compared with the highest education level, the respondents who had completed postgraduate and higher degrees perceived to a higher extent that insufficient measures were taken to control the overuse of antimicrobials, compared to others who completed the education of senior high school (P = 0.002), college (P < 0.001), and bachelor (P = 0.001). However, there was no significant difference in comparison among \u201cgender,\u201d \u201chousehold adults,\u201d and \u201chousehold children\u201d for both the scales .In addition, it was found that the scales of antimicrobial-use attitude and China-situation attitude were affected by age (< 0.001) . The midOf the participants, 61.3%  claimed that they were more likely to look for specific information about AMU on food packaging when they purchased food products, and 58.3%  changed their eating or cooking habits due to the concern of AMR and AMU in farming . In addir = 0.142, P < 0.01)  of the respondents were willing to pay a higher price for antimicrobial-free food products, whereas 20.2%  were not willing to pay extra. Most respondents viewed AMR and AMU as potential risks to their health, as the Spearman correlation showed a statistical significance in difference between the attitude and the willingness to pay ( < 0.01) . Among t < 0.01) . Of 215 2 = 32.9, P < 0.001), work status , and household income  (P = 0.001), 89.0% (97/109) of 41\u201350 years old (P = 0.001), and 92.5% (74/80) of 51\u201360 years old (P < 0.001), were willing to pay higher prices for antimicrobial-free food products, all significantly >74.6% (482/646) of participants who were 18\u201330 years old willing to pay a higher price. When compared with the work status, 83.1% (513/617) of the full-time employed respondents showed the willingness to pay more for antimicrobial-free food products, while 71.1% (189/266) of the students showed willingness (P < 0.001). When compared with household income, 91.0% (152/167) of the subjects in the \u201c\u00a5 200,000\u2013\u00a5 500,000 per annum\u201d group showed the willingness to pay more for antimicrobial-free food products, significantly >67.9% of the participants in the \u201cunder \u00a5 50,000 per annum\u201d group (P < 0.001), 79.4% (224/282) in the \u201cunder \u00a5 50,000 per annum\u201d group (P = 0.001), 73.3% (33/45) in the \u201cNot stable/Not sure\u201d group (P = 0.002), and 67.0% (63/94) in the \u201cPrefer not to say\u201d group (P < 0.001). Of the subjects, 84.0% (226/269) in the \u201c\u00a5 100,000\u2013\u00a5 200,000 per annum\u201d group answered they were willing to pay, significantly more than 67.9% (93/137) in the \u201cunder \u00a5 50,000 per annum\u201d group and 67.0% (63/94) in the \u201cPrefer not to say\u201d group (both P < 0.001). However, there was no significant difference compared among \u201cgender,\u201d \u201chighest level of education,\u201d \u201chousehold adults,\u201d and \u201chousehold children\u201d .It was discovered that there were significant differences in the willingness to pay compared among age (\u03c7< 0.001) . For ageThe percentage score of participants for each information source were reported . Most reThe exploratory factor analysis of information sources yielded a two-factor solution which explained 58.2% of the variance. Factor one consisted of seven sources, i.e., social media, Internet, media , farmers, social training, food companies/supermarkets, family and friends, which was renamed as the scale of media, Internet, word of mouth, others. And factor two included five sources, i.e., scientists, health professionals/doctors, veterinarians, education from universities and academic institutions, and the national food safety agencies/governments, which was renamed as the scale of professionals and authorities. The internal consistency for each factor was assessed with Cronbach's \u03b1 coefficient, whose \u03b1 were 0.86 and 0.84, respectively for factor 1 and factor 2 .r = 0.494, P < 0.01), indicating that those who were confident in the professional sources were also confident in the media sources and vice versa. The top three trusted sources were scientists , health professionals/doctors , and national food safety agencies/governments , while the most untrusted sources were farmers . However, 28.9%  of the respondents answered neutral/don't know/not sure about media, Internet, word of mouth, others, and it might be due to the lack of knowledge and awareness of this issue.Not all sources were considered accurate information about AMR and AMU in food production. Mean\u00b1SD (95%CI) scale scores and factor correlations were presented . Five ouIn this study, consumers were clearly aware of AMR, however, the knowledge of AMU in farming production was not high, which matched the results in the surveys conducted in Europe and Chile . We alsoPrevious studies indicated that there were some differences in general concern about AMU in livestock animals as a potential risk to public health from country to country. For instance, most Chilean consumers considered AMU as a potential risk to their health , while DIn terms of the behavioral changes of consumers, it was discovered in this study that more than half of the participants claimed that they were more likely to look for specific information about AMU on food packaging when they purchased food products. They changed their eating or cooking habits due to the concern of AMR and AMU in farming. This should urge food companies to provide detailed information to the consumers about the antimicrobials and chemicals used during production and certify that drug residues are within policy and regulation limit.It was identified in previous studies that consumers in the US, Canada, Germany, and Chile were willing to pay a higher price for food products free of antimicrobials, depending on their knowledge and attitude , 26, 33.In terms of the research on risk communication from the perspective of authorities and food companies, there are three essential and necessary components when communicating information with consumers . First, With the aspect of communication and information sources, most respondents obtained information on AMR in farm animals from the media, followed by the Internet and family and friends in this survey although sources from professionals and authorities were considered more accurate. This result was similar to a previous report that the majority of consumers preferred scientists and food safety agencies as the more reliable sources of information compared to the food industry and the media . HoweverThis study was the first to investigate the knowledge, and attitudes of Chinese consumers toward AMU in food production, and evaluated their behavioral changes, purchase intention, and confidence in information sources of antimicrobial-free food products. However, it also had limitations. Given the survey method based on an online questionnaire, only participants with access to technology equipment were reached. Furthermore, the samples were imbalanced, e.g., most participants were 18 to 30 years old (60.7%), and 31.5% of respondents were from Zhejiang Province, therefore the answers may be geographically and demographically biased because the participants were not enough to be representative of Chinese consumers as a whole. Nonetheless, this study provides a valuable contribution to the exploratory investigation of knowledge, attitude, and behavior of Chinese consumers. Further research is needed to widen population representation.This study demonstrated that most Chinese consumers were aware of AMR, however, the knowledge of AMU in farming production was not high. They were observed to have insufficient awareness in terms of the related food regulations in China as well. Almost half of the participants viewed the issues of antimicrobial residues in food products and AMR from live farm animals as potential risks to human health. More than half of the respondents claimed that they had been more likely to look for specific information about AMU on food packaging when they purchased food products, and had changed their eating or cooking habits due to the concern of AMR and AMU in farming. A large proportion of the participants were willing to pay a higher price for antimicrobial-free food products. Most of them obtained information on AMR and AMU in farm animals from the media, whereas information sources from professionals and authorities were considered more accurate and reliable than those from media, the Internet, word of mouth, and others.The identified evidence in this research highlighted the importance of updated education for the public and effective communication with the consumers, which could help improve foodborne AMR surveillance system along food chain and communication strategies. However, this study was limited to a biased younger population in a more developed province, Zhejiang, in China. Further studies should expand to other populations and regions on AMR and AMU in food production.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Zhejiang University School of Public Health. The patients/participants provided their informed consent to participate in this study.Study design: QD, XD, YZ, and MY. Collection of data: QD, JG, YZ, and MY. Analysis and interpretation of data: QD, DH, YZ, and MY. Drafting the manuscript: QD, YZ, and MY. All authors have read, revised, and approved the final version of the manuscript.This research was financially supported by the Fundamental Research Fund for the Zhejiang Provincial Universities (grant number 2021XZZX029), the Key Laboratory of Intelligent Preventive Medicine of Zhejiang Province (grant number 2020E10004), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (grant number 2019R01007), and the Key Research and Development Program of Zhejiang Province (grant number 2020C03002) China.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": "The excitation wavelength independent emission and emission wavelength independent excitation spectra along with the analogous PL decay kinetics of bulk and NPLs suggest the intrinsic nature of broadband emission. The experimental low sound velocity (\u223c1090 m s\u22121) and associated low bulk and shear moduli  indicate the large anharmonicity and significantly soft lattice structure, which trigger the broadband STE emission in 2D NPLs of RbPb2Br5. Strong electron-longitudinal optical (LO) phonon coupling results in broadband STE emission in 2D RbPb2Br5 NPLs.Self-trapping of excitons (STE) and concomitant useful broadband emission in low-dimensional metal halides occur due to strong electron\u2013phonon coupling, which exhibit potential applications in optoelectronics and solid-state lighting. Lattice softness and high anharmonicity in the low-dimensional structure can lead to transient structural distortion upon photoexcitation that should promote the spatial localization or trapping of charge carriers, which is essential for STE. Herein, we report the ligand-assisted reprecipitation synthesis of ultrathin (\u223c3.5 nm) two-dimensional (2D) metal halide, RbPb 2Br5 nanoplates, trigger strong electron\u2013phonon coupling and consequently highly Stokes shifted broadband emission originates from self-trapped excitons.The presence of large anharmonicity and lattice softness in low dimensional metal halide, RbPb Only a few potential alternatives are available to replace Cs in the all-inorganic AB2X5-type structures.10,11 Rb, as a neighbouring alkali metal of Cs, is suitable to form AB2X5-type metal halides. However, layered RbPb2Br5 has rarely been investigated except for a few electronic structural studies on its single crystals.11,12 Simple synthesis and fundamental understanding of the optical properties of RbPb2Br5 in the form of 2D nanoplates/nanosheets or nanocrystals are still elusive.Metal halides have fascinated researchers due to their countless stimulating properties and potential applications in optoelectronics, photodetection and photovoltaics.13 Strong electron\u2013phonon coupling causes elastic lattice deformation and consequent STE results in broadband emission with a large Stokes shift, which showed promise for solid state lighting.14 Thereby, it would be exciting to study the optical properties and electron\u2013phonon coupling in 2D layered RbPb2Br5 both in the form of bulk and ultrathin nanostructures.Dimensionality reduction facilitates the disconnection of the metal halide octahedra and the lattice becomes progressively softer. In such systems, self-trapped excitons (STEs) form due to local structural distortion upon photoexcitation.2Br5, which displays broadband self-trapped excitonic (STE) emission. We have further synthesized controlled bulk polycrystals of RbPb2Br5 by solvent free mechanochemistry. Photoluminescence excitation (PLE) and PL spectra of RbPb2Br5 NPLs and bulk polycrystals are independent of emission and excitation wavelengths, which reveal that broadband emission originates from relaxation of the same excited state. We have observed a significant Stokes shifted (\u223c400 nm) emission with a long PL lifetime in the microsecond (\u03bcs) range. The temperature-dependent (15\u2013350 K) PL studies confirm the strong electron-longitudinal optical (LO) phonon coupling in 2D RbPb2Br5 NPLs. Large lattice anharmonicity and low sound velocity (\u223c1090 m s\u22121) indicate the structure to be elastically very soft with significantly low bulk and shear moduli. The soft crystal lattice and strong electron\u2013phonon coupling trigger the broadband STE emission in 2D NPLs of RbPb2Br5.Herein, we present simple ligand-assisted reprecipitation (LARP) synthesis of 2D ultrathin few-layer nanoplates (NPLs) of layered metal halide, RbPb2Br5via the ligand-assisted reprecipitation (LARP) method at room temperature  along with organic ligands  was swiftly injected into chloroform under vigorous stirring. After 20 s, the as-synthesized product was collected and washed with chloroform for further measurement. Further, we have synthesized micrometre sized particles (\u223c1 g) of RbPb2Br5 by implementing solid-state mechanochemical grinding with appropriate stoichiometric ratios of RbBr and PbBr2  of RbPb2Br5 adopts a tetragonal crystal structure with the I4/mcm space group, which consists of intercalated Rb+ ions sandwiched in between the 2D layers of [Pb2Br5]\u2212 polyhedral units  patterns of the as-synthesized bulk and NPL samples have been indexed to the tetragonal phase of RbPb2Br5 (space group I4/mcm) (15 The PXRD pattern of the as-synthesized NPLs obtained from the LARP route shows diffraction peaks corresponding to mainly (002), (004), (006) and (008) planes, which clearly indicates the 2D growth along the <110> direction. The characteristic layered peak appears at 2\u03b8 = 11.66\u00b0 corresponding to the (002) plane in both RbPb2Br5 bulk polycrystals and NPLs. We have studied the environmental and the thermal stabilities of the mechanochemically synthesized bulk RbPb2Br5 powders. The bulk polycrystals display excellent crystallinity and stability even after 250 days of being kept under ambient conditions  .15 The P2Br5via the LARP method with and without the addition of capping ligands (OA and OLAm), and the comparison of the PXRD patterns reveals that the directional growth takes place only in the presence of OA and OLAm 7\u2013CH PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>CH\u2013(CH2)8\u2212] from both OA and OLAm.18,19 The \u2013CH2 bending modes at \u223c1460 and \u223c1374 cm\u22121 have also appeared from the hydrocarbon chains of OA and OLAm. Additionally, the spectrum of NPL solution shows a typical vibrational signature of the carboxylic acid group (\u03bdOC) at 1722 cm\u22121 along with the \u03bdN\u2013H stretching mode at \u223c3300 cm\u22121 from the amine moiety. The peak broadening of N\u2013H stretching mode indicates the successful coordination of OLAm to the surface of nanostructures , which further reveals thin 2D nanoplate morphology  and the height profile measurements reveal a thickness of \u223c3.5 nm of the NPLs , which crphology . The latf 5\u20138 \u03bcm . Furtherf 5\u20138 \u03bcm . Thereby2Br5 obtained by the LARP method in the presence of OA and OLAm confirms the 2D NPL morphology (I4/mcm symmetry demands interplanar spacings for the (hkl) and (h-kl) family of lattice planes to be identical. Interestingly, the HRTEM analysis reveals that d110 and d1\u201310 are non-equivalent by 0.13 \u00c5. The corresponding FFT analysis also exhibits a deviation of 0.11 \u00c5 between (110) and (\u2212110) interplanar lattice spacings. Additionally, the (200) and (020) planes show a deviation of 0.08 \u00c5 in the interplanar distances  and (\u2212110) interplanar lattice spacings by 0.07 \u00c5  pattern of the RbPb2Br5 NPLs additionally confirms the single crystalline nature (2Br5.The transmission electron microscopy (TEM) image of the as-synthesized RbPbrphology , which irphology . The parrphology , inset. rphology . The tetistances , inset. e nature . The dif2Br5 NPLs, we have performed Raman spectroscopic investigation at room temperature. The distinctive vibrational modes appear from the PbBr8 polyhedron in RbPb2Br5 NPLs  and 2.87 \u00c5 (six shorter bonds), respectively.24 While the covalent bond distance between Pb and Br is about 2.66 \u00c5, close to the sum of covalent radii of Pb (1.46 \u00c5) and Br (1.20 \u00c5),25 the Pb\u2013Br ionic interaction distance is found to be 3.25 \u00c5.26 The Rb\u2013Br distance is 3.59 \u00c5 in RbPb2Br524 which is close to the sum of the ionic radii of Rb+ (1.66 \u00c5) and Br\u2212 (1.96 \u00c5).26 Thereby, RbPb2Br5 exhibits a chemical bonding hierarchy which induces anharmonicity and softness in the crystal lattice.To understand the lattice dynamics in 2D RbPbBr5 NPLs . Two str\u03b3), which is defined as:27where \u03c9 is the frequency of the given phonon and V is the unit cell volume. \u03b3 indicates how the bond stiffness is affected by the change in the interatomic bond distances and the probability of phonon\u2013phonon interaction is proportional to the square of \u03b3. The value of \u03b3 is estimated to be 1.6 from the experimental measurement of the sound velocity of RbPb2Br5 NPLs (see methods in the ESI). The longitudinal (vl) and transverse sound velocity (vt) were measured to be 1744 m s\u22121 and 980 m s\u22121, respectively, which are reasonably low in comparison to other earlier reported metal halides  was estimated using the formula:which exhibits a low value of 1090 m s\u22121. The high value of \u03b3 in RbPb2Br5 NPLs represents the presence of high lattice anharmonicity.27 The elastic moduli of the sample were estimated by using the following equations:where B and G are the bulk and shear moduli respectively. \u03c1 refers to the density of the sample. The estimated bulk and shear moduli  are found to be lower compared to other all-inorganic halide perovskites revealing a significantly softer lattice  and 4.27 eV (290 nm), respectively, with a long absorption tail in the lower energy region  recombination.14,34,40\u201343 The large Stokes shift is caused by the energy loss in the process of excited state structural distortion.34The optical properties of the RbPbon onset . At roomon onset . The broon onset . Significrystals . The lar2Br5 NPL sample shows an absolute PLQY of \u223c13%, which is reasonably higher compared to that of the earlier reported broadband STE-emitting Pb based 2D halide perovskites , as well as thermal energy at room temperature (26 meV).49 This high Eb value is beneficial for radiative recombination.50 However, this value is lower compared to that of the 0D perovskites because of the weaker confinement.51To probe the underlying mechanism of broadband emission in RbPbthe NPLs . Temperaperature . Such a 2Br5 NPLs, we further determined the Huang\u2013Rhys electron\u2013phonon coupling parameter (S) and effective phonon energy (\u0127\u03c9phonon) using the Toyozawa model,42,52where S represents the distortion of the self-trapped state with respect to the ground state, \u0127 is the reduced Planck constant and \u03c9phonon is the phonon frequency. Through fitting of the experimental FWHM with the above model, we found an effective phonon energy of 56.10 meV with a high S of 15 4Cu2Br6,55 which showed STE emission due to strong electron\u2013phonon coupling.To get an estimation of the electron\u2013phonon coupling in RbPb S of 15 . Similar14 Thereby, we fitted the FWHM to the Fr\u00f6hlich longitudinal optical (LO) phonon broadening model developed by Rudin et al.  is the PL line width and \u03930 originates due to the scattering of charge carriers from disorder which is temperature-independent and results in inhomogeneous broadening. \u0393ac and \u0393LO originate due to the scattering of charge carriers from the acoustic phonon and Fr\u00f6hlich scattering of the longitudinal optical (LO) phonon, respectively, whereas, \u03b3ac and \u03b3LO are the corresponding coupling constants.57 From the fitting, we found the optical phonon energy (ELO) as 17 meV (137 cm\u22121) which is in good agreement with the Raman mode observed near 130 cm\u22121 in RbPb2Br5 NPLs 2CdxPbx1\u2013Br459 and Cs3Bi2I6Cl3,60 and also the 3D double perovskite Cs2AgBiBr653 but much larger than that of the popular 3D lead halide perovskite, (FA/MA)Pb(I/Br)357  of 10.03 \u03bcs  measurements to gain more insight into the excitonic recombination dynamics in RbPb10.03 \u03bcs . Additio10.03 \u03bcs  and infe2Br5 were synthesized by the halide mixing strategy via mechanochemistry. The PXRD patterns of RbPb2Cl2Br3 and RbPb2Br4I reveal that both the materials possess similar tetragonal crystal structures to RbPb2Br5   to RbPb2Br5 (\u223c695 nm) to RbPb2Br4I (\u223c765 nm)  by mechanochemical grinding, as confirmed from its PXRD pattern .15 The solid-state electronic absorption spectrum of as synthesized CsPb2Br5  broadband PL emission with no overlap between their excitation and emission spectra. Furthermore, the NPLs exhibit a long lifetime component in the microsecond range. A soft crystal lattice along with high anharmonicity boosts structural distortion during photoexcitation where electrons can be easily trapped. As a result, 2D all-inorganic metal halide RbPb2Br5 exhibits strong electron\u2013phonon coupling and STE with broadband emission. Furthermore, we have tuned the band gap in the range of \u223c2.9\u20133.6 eV by adjusting the halide composition in the soft lattice of RbPb2Br5. Thus, our findings give a new direction towards the fundamental understanding of the emission process in low dimensional halides with the influence of chemical bonding, lattice anharmonicity and lattice dynamics which show potential prospects in a new generation of solid-state lighting and displays.In summary, we have synthesized ultrathin NPLs of 2D layered RbPbAll data are available in the manuscript and in the ESI.\u2020K. B. conceived the idea and designed the study. J. P., A. D., K. K. and K. B. carried out the synthesis, structural, optical, and sound velocity measurements, and other analyses. C. helped in the synthesis of NPLs. J. P. wrote the first draft; and A. D., K. K. and K. B. contributed to editing the manuscript.The authors declare no conflict of interest.SC-013-D2SC02992H-s001"}
+{"text": "Mycobacterium tuberculosis remains the gold standard in mycobacteriology laboratories, constrained by the very high risk of contamination; therefore, contamination rate is an important key performance indicator (KPI) for laboratory monitoring and evaluation processes.Culture of This study aimed to investigate the factors that contribute to elevated contamination rates in the Sudan National Tuberculosis Reference Laboratory.A laboratory-based retrospective study was applied; a TB culture register-book was carefully reviewed and data from 2 January 2019 to 31 December 2019 were entered, cleaned, and analyzed using IBM SPSS 20. A multivariate logistic regression model was performed to examine two dependent variables, the massive contamination, and the single tube contamination against predictors of reason for cultivation, type of specimen, experiment team, and the quarter of cultivation.p value 0.007; adjusted odds ratio AOR 3.570 (1.415\u20139.005). The correlation between the single tube contamination and the massive contamination is significant; p value 0.01.It has been found that in 2019 contamination rates were frequently higher; the highest rates were recorded in January and November, 28.2 and 25.2%, respectively. August is an exception with an accepted contamination rate of 4.6%. Of 1,149 specimens requested for culture, 945 (82.2%) samples were eligible to be included in multivariate logistic regression analysis. The team conducting the experiment was significantly associated with a high single tube contamination The study concludes that high culture contamination is the greatest risk to the quality of laboratory service and can end in either the loss of specimens or delay in the decisions of initiating patient treatment. In addition, the low quality or incompleteness of data increases the uncertainty and undermines the measurement of key performance indicators. Sudan is a moderately tuberculosis (TB) burdened country. In 2014 the prevalence was 159/100,000; it is higher among adults, in urban settings, and in the male population, although 11\u201313% of all notified cases are children. In 2017, the estimated TB incidence was 77/100,000, for a total of 31,000 cases and a mortality rate of 12/100,000. Comparatively, TB case notifications remained steady for the last decade in a range of . In 2016, the total notification was 21,091 cases. Among them, 19,305 (91.5%) were new, while 1,786 (8.5%) were retreatment cases. Of the new cases, 6,520 (30.9%) were smear-positive, 6,167 (29.2%) were smear-negative, 4,951 (23.5%) were extra-pulmonary, and in 1,667 (7.9%) sputum was not done. Last year there were three presumptive cases of extensively drug resistant tuberculosis (XDR-TB); only one of them was confirmed and the notified multi drug resistant tuberculosis (MDR-TB) cases were 220 .In Sudan, the GeneXpert network was designed and consists of three levels; it was managed and supervised centrally by the National Tuberculosis Reference Laboratory (NTRL), connected with 18 state laboratories located in capital cities, equipped with one machine or more. Based on MDR notifications, it was expanded to serve 72 sites in localities. An external quality assessment (EQA) system was planned but not yet implemented. The GeneXpert network was employed as the earliest diagnostic tool in the diagnostic algorithm which prioritizes screening and early identification of rifampicin resistant cases among TB suspect and MDR-TB high-risk groups, such as TB retreatment cases, MDR-TB contact, HIV patients, and prisoners. To scale up the programmatic management of drug resistant tuberculosis; sputum smear microscopy was only dedicated for the monitoring of treatment of first-line anti TB drugs (FLDs). So far, culture is the gold standard diagnostic method in the mycobacteriology laboratory.M. tuberculosis  is considered the reference mycobacterial growth medium . Solid erculosis . Cultivarculosis .The accuracy of technical manipulation of analytical and pre-analytical processes either negatively or positively impacts the prone of mycobacterial culture contamination. Soft decontamination procedures due to the constraints of less time or lower concentration might increase the possibility of contamination occurrence. In addition, poor quality of specimens, delay in transportation to the laboratory, as well as poor quality of culture media and incubation conditions might be addressed as possible causes of contamination . In this\u00ae MTB/RIF assay  and Line Probe Assay GenoType MTBDRplus for rifampicin and isoniazid resistance , and GenoType MTBDRsl for XDR detection.Sudan National Tuberculosis Reference Laboratory (NTRL) is the sole reference TB laboratory in the country; it stands at the top of the hierarchical network of four regional TB culture laboratories, 18 smear microscopy quality assurance (EQA) laboratories, and 327 tuberculosis management units (TBMUs). The diagnostic menu includes a variety of conventional laboratory tests: Ziehl\u2013Neelsen (ZN) and Phenolic-Auramine fluorescence microscopy (FM), egg-based cultures, first line, and second-line drug susceptibility testing (DST). In addition, the WHO endorsed molecular techniques XpertSudan National Tuberculosis Reference Laboratory is adopting the Petroff method for sputum decontamination and homogenization. Standard operating procedure (SOP) describes the addition of an equal volume of 4% sodium hydroxide solution (4% NaOH) to the sputum sample, gentle shaking, and homogenization for 15 min. Neutralization of alkaline solution includes three without-centrifugation options: the use of hydrochloric acid (HCl) with phenol red indicator, use of phosphate buffer saline (PBS) pH 6.8, or direct inoculation onto two tubes of Ogawa acidified medium. L\u00f6wenstein-Jensen medium (LJ) Slants were incubated at 37\u00b0C for 8 weeks and inspected regularly for the recording of negative results. Culture contamination and the grades of positive results  were recorded immediately when visible mycobacterial growth appear.The culture laboratory register book was carefully reviewed. It includes data about specimens, patients, and results of smear and culture. Data were categorized, double entered onto statistical package for social sciences (IBM SPSS statistics 20), checked, and cleaned. Category \u201cNot recorded\u201d was elaborated for not filled data from the original institution that requested the culture, unknown data, or data not transcribed from the request form inside the reference laboratory. Specimens that were requested and not received or not processed were excluded. Furthermore, information about sample collection, sample reception, and sample processing dates were not completely filled in the culture register book; therefore, these variables were also excluded from this study. Dried culture tubes indicate ignorance of early inspection of culture slants, undermines the good microbiological technique (GMT), and also ends to the loss of specimen; therefore, dried tubes were considered as contaminated. In case of two different direct and indirect smears positive results from the same specimen, or any other difference between positive results of each single culture tube, the highest grade was considered. For example, if the result was (++) and the result of the other tube was (+) the result of culture was recorded as (++).p values were calculated, and a correlation between both types of contamination was determined.In this study, the proposed predictors include the reason for cultivation, type of specimen, the team conducting the experiment, and the quarter of cultivation which referred to three successive months in the year ; each predictor that had complete data was illegible for statistical analysis. These predictors were examined separately against two dependent variables: the massive contamination  and contamination of single tube (contamination of only one or two cultivated tubes). A multivariate logistic regression model was applied to examine statistical associations at 95% confidence intervals, the reference category is first ordered ascending. Odds ratios, upper bound, lower bound, and This study is based on secondary data, and it did not involve working with any type of human subjects, clinical specimens or interventions, patients\u2019 interviews, or clinical trials; however, the proposal has been scientifically and ethically approved by the national public health laboratory ethical committee. The publication permission and approval were obtained from the general directorate of the National Public Health Laboratory (NPHL).From 2 January 2019 to 31 December 2019, a number of 1,149 specimens were requested for culture in the National Tuberculosis Reference Laboratory, of which 1,125 (97.9%) were received and processed and have had a laboratory culture result. The majority of specimens (881\u201378.3%) were requested for treatment monitoring, whereas 83 (7.4%) were baseline specimens and the reason for culture request of 161 (14.3%) were not recorded. In addition, 1,040 (92.4%) specimens were sputum, 30 (2.7%) were other than sputum, and 55 (4.9%) were not recorded. Experiments were conducted by five principal operators in six work teams and the range of workload for culture is 51\u2013159 specimens per month. Only 496 (43.2%) of patients\u2019 demographic information is complete and shown in After following the exclusion criteria, of the 1,125 registered samples, a total of 945 (84%) samples were eligible to be included in statistical analysis; of them, 656 (73%) were smear-negative, 255 (16.7%) were smear-positive, and the remaining 97 (10.3%) were not recorded. A breakdown of results is shown in Massive contamination rates were higher in March (13.7%) and in November (8.5%). They decreased to 0.1% in January and did not occur (0%) in February, April, June, and August; Single tube contamination rates were higher in January (28.2%), November (25.2%), and in March (24.5%) .Out of 1,125 specimens included in this study, 180 (16%) samples were excluded from the statistical analysis. The remaining 945 samples (84%) had complete data regarding independent variables and are eligible to be included in the statistical analysis; details were shown in The WHO laboratory quality management system LQMS document recommends management reviews of all information gathered in the laboratory records regularly ; the typThe accepted culture contamination rate in L\u00f6wenstein\u2013Jensen (LJ) solid medium is 3\u20135% . In thisMajor limitations of this study were due to the missing, not recorded, or not transcribed data; this could increase the uncertainty of generated information rather than the appropriate interpretation of the study findings , as in tThe study concludes that high culture contamination is the greatest risk to the quality of laboratory service and can end in either the loss of specimens or delay in the decisions of initiating patients\u2019 treatment. In addition, the low quality of data increases the uncertainty and undermines the measurement of key performance indicators. Furthermore, the team conducting the experiment was found to be associated with high culture contamination rate. Therefore, the study recommends that all culture laboratory staff should be on the same level of knowledge and proficiency according to their role and should be under regular restricted supervision; in addition, laboratory management should have to use regular assessment tools for internal audits.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MM wrote the preliminary draft. RE contributed to data entry and data cleaning. SB critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.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": "Prosthetic service development and delivery rely on data describing population needs. These needs are context-specific, but most existing data come from high-income countries or small geographic areas, which are often not comparable. This study analysed routinely collected digital patient record data at multiple time points to provide insights into characteristics of people accessing Cambodian prosthetic services.We investigated trends in birth year, sex, year and reason for limb absence, and prosthesis type, over three decades. Then, we observed data from 2005 and 2019 indicating how the population actively accessing prosthetics services has changed.Temporal trends in prosthetics service user demographics corresponded with events in Cambodia\u2019s socio-political history. The predominant historical reason for limb absence prior to 2000 was weapon trauma during and following conflict. Since 2000, this was replaced by non-communicable disease and road accidents. Transtibial remained the most prevalent amputation level but transfemoral amputation had higher incidence for people with limb loss from road accidents, and people with limb loss due to disease were older. These observations are important as both transfemoral and older-aged groups experience particular rehabilitation challenges compared to the young, transtibial group.The study shows how standardised, routinely collected data across multiple clinics within a country can be used to characterise prosthetics service user populations and shows significant changes over time. This indicates the need to track client characteristics and provides evidence for adapting services according to population dynamics and changes in patient need. Fifteen percent of the world\u2019s population has a disability, over a billion people, and 80% live in lower and middle income countries (LMICs) . The GloIn many LMICs, the high prevalence of amputation has resulted from conflict. In Cambodia, from 1979 to 2021, 19\u2009779 people were killed and 45\u2009144 people were injured by landmines, cluster munitions and other explosive remnants of war (ERW) following the Vietnam war, and subsequent civil conflict. This included 9067 people with amputations [While long-term economic growth and technological development may improve prosthetics service provision the specific needs may change, and ability to deliver services may be disrupted. This may arise from gradual growth in non-communicable disease (NCD), or situations of conflict and natural disaster, where a sudden increase in need can occur alongside an interrupted ability to provide care. Such problems are often felt most acutely in LMICs. The demography of people accessing prosthetics services is expected to change considerably as landmines and ERW are cleared and urban environments grow rapidly, with an associated high incidence of road traffic accident injuries, especially involving young people using motorcycles ,8. Type Prosthetics service need, access and delivery data are varied. Much of the existing data come from small geographic areas. The majority of available evidence on need has been generated through registry and health insurance data, which are concentrated in high income country (HIC) settings  established a standardised digital patient management system (PMS) which offers an opportunity to identify and compare context-specific needs for prosthetics services at repeated time points, with homogeneous client demographics, standardised clinical data and outcome information. This data set has been used to study the demographics of people accessing ICRC physical rehabilitation services across 14 countries , and theProsthetics service delivery has systems level challenges; service providers must ensure and evidence quality and provide continuity between health and rehabilitation services. In the context of growing NCD, the aim is early detection, intervention and prevention . HoweverThis study aimed to provide novel insight into the demographics of those accessing prosthetic services in Cambodia and indicate their context-specific prosthetics service needs. Further, the study aims to show how Cambodian prosthetics service needs have changed over time and identify future service requirements.An observational analysis of routinely collected longitudinal data over multiple time points was undertaken, to investigate the demographics of people accessing prosthetics services between 1992 and 2019 at three clinics run by charity Exceed Worldwide, in Phnom Penh, Kampong Som and Kampong Chhnang, Cambodia. These prosthetics services were provided alongside orthotics services by dual-qualified staff. Approval was granted by national (230&311NECHR) and institutional ethics review boards (ERGO45577&51898) to analyse episodal statistics extracted from Exceed\u2019s digital clinical records. Records were collected in a standardised manner and stored in the \u201cPMS-5\u201d database  for all individuals or \u201cclients\u201d accessing Exceed\u2019s clinics for prosthetics services from 1998 until December 31, 2019. The earliest clinical contact recorded within this data set was the December 2, 1992 in Phnom Penh, February 14, 1993 in Kampong Som and September 14, 1995 in Kampong Chhnang. Records from before 1998 were input by the clinics from paper records for specific individuals whose care was ongoing. Each line of data described a single clinical contact such as an assessment, a prosthetic device provision, a replacement, or a repair. An individual could have multiple contacts for provision of prosthetic devices, and an individual device could have multiple repairs. Individuals were considered \u201cactive\u201d if they had at least one appointment in the last 7 years, on the Exceed Country Director\u2019s recommendation (SK).2 tests were performed to assess the null hypotheses i) that each demographic category was the same for \u201call\u201d and \u201cactive\u201d groups, and ii) that each demographic category did not change over time. Hypothesis tests of independent sample proportions were conducted to assess demographic differences between particular groups and the rest of the study population. Finally, two exemplar cross-sectional analyses were reconstructed from the data set, representing the active clients at the end of 2005  and the end of 2019 . \u03c72 tests were performed to assess the null hypotheses that demographic categories were the same for the reconstructed cross sections.Data analysis was performed in Stata , to describe the demographics of clients accessing the prosthetics service and the devices they were prescribed, including the client\u2019s year of birth, gender, year of limb absence or amputation, reason/cause of limb absence/amputation, and type of prosthesis as a proxy for limb absence level. Descriptive statistics were extracted at 6 time points to assess the temporal trends in these demographics, and \u03c72 tests revealed differences in the distribution of all demographics between \u201call\u201d and \u201cactive\u201d client groups  except for side .After removing duplicate appointments, the data set contained 50\u2009144 entries representing clinical contacts for 7117 individual clients. Of these clients, 2820 were classified as active. Most clients had at least partial data recorded. 351 individuals (4.9%) did not have any data apart from the date of the initial assessment appointment. Person characteristics for all and active clients, including reason for limb loss and type of prosthetic supplied, are shown in , Table S1 in the P\u2009<\u20090.001). Upper limb devices were considerably more common for people with limb loss following accident at work (30%) vs other reasons for absence (P\u2009<\u20090.001). Partial foot absence represented 16% of people with congenital limb absence and 11% of people with amputation following animal bite, which was more common than for other reasons for limb absence (both P\u2009<\u20090.001).Weapon injury was the most common reason for limb absence at all levels .The most common band of age at limb absence or amputation due to road traffic accident, weapon injury and accident at work was 20-29 years , and a proportional rise in transfemoral absences over the full period . All other levels of limb absence remained below 12.5%.P\u2009<\u20090.001).2\u2009=\u20092109, P\u2009<\u20090.001). While the overall distribution of absence levels has changed little between 2005 and 2019 , inspecting subgroups shows significant differences with a proportional decrease in the transtibial level in the 10-49-year-old age groups . The distribution of absence levels in the 50+ year age group was unchanged in 2019 compared to 2005.Using routinely collected data this study provides insight into the clinical and demographic characteristics of people who have experienced limb absence or loss in Cambodia over more than six decades. By presenting temporal trends in patient characteristics and prosthetic device types, this study indicates how the Cambodian prosthetics service needs have changed over time, based on reasons for limb absence and the patients\u2019 demographics. In Cambodia the prevalence of landmine and ERW injuries is understood anecdotally, but researchers report a mismatch between in-country data and published literature . The des, Table S1-S2 in the ,, Table S3 in the , Table S4 in the The present study facilitated an overall description of the people accessing prosthetics services in Cambodia , congenital defects predominated, believed to be linked to the defoliant Agent Orange used during the Vietnam War (1961-71) . The dat, Table S3 in the Online Supplemetary Document) but has declined since the 1980-89 decade, and there is a gradual increase in the proportion of transfemoral absences. Possible explanations may be found in the changing reasons for limb absence, most notably the increasing incidence of amputation following road traffic accidents. Traumatic injury in road traffic accidents has a historic burden and is a growing cause of lower extremity loss in many LMICs [The transtibial absence level clearly had the highest incidence in all decades  and at the end of 2019, to allow comparison of the demands upon service providers at particular time points (Table S5 in the This study is limited by reliance on secondary analysis of clinical data sets, which represent only partial national coverage for three of eleven clinics. Its accuracy was dependent on reporting practices and is influenced by any differences in data input regimes between the three centres, and some data were missing. These clinics serve clients across a wide range of the population in Cambodia both in urban and rural regions, free of charge, so while the study\u2019s generality cannot be confirmed, the findings are not restricted by socioeconomic status and therefore broadly reflect the wider population. There may also be cohort effects associated for example with the changing survivorship of trauma, development and treatment of non-communicable disease, and the proportion of clients characterised as inactive. The study provides no information on people in need who do not access physical rehabilitation services, and no reason for why people stop appearing in the data set. This may be due to death, people moving and accessing a different prosthetics service, or ceasing to use their prosthesis. Some people may have accessed more than one centre, which could only be accounted for if the data set noted a national identification record. Future work might investigate the declining number of people accessing prosthetics services in the last decade, and whether this is due to the existing population reaching their life expectancy, standardised prosthetic devices lasting longer or no longer meeting the needs of an emerging middle class who then choose to access services elsewhere, or people experiencing more difficulty in accessing services due to reducing financial support. Further consideration of dimensions including sex should be made, noting that female clients are in a clear minority in this and similar data sets ,20,22. FRoutinely collected clinical data are regularly used in high income countries, but less in LMICs, partly because data are not so easily available, and partly due to the dearth of professionals to analyse them, and necessary focus on service delivery above all . This fi"}
+{"text": "Escherichia coli weighs \u223c20\u00a0MDa, without considering its filament portion, which is by itself a \u223c1.6\u00a0GDa structure arranged as a multimer of \u223c30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way.Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from  In Gram-positive bacteria, most of the flagellar appendage is extracellular, with only part of the basal body including cytoplasmic and transmembrane structures Salmonellructures . A similructures .Figure\u00a01Vibrio, Helicobacter, Brucella, and related genera, protrude extracellularly, yet are fully surrounded by the uninterrupted sheath of the outer membrane  from the flagellar ancestor . The T3Smembrane .A more extreme modification is observed in the entire Spirochete phylum, where flagella, known in this group as endoflagella, are entirely confined within the periplasm, with the filament helically wrapped around the cell body . AlthougThe ongoing revolutionary progress of three-dimensional cryo-EM and cryo\u2013electron tomography (cryo-ET) approaches , 28 has The flagellar basal body harbors the motor device that drives flagellar rotation, while also including additional protein components that play key roles : (i) a mIn a general sense, motors are engines that perform mechanical work by using a two-component arrangement: a static part (stator) is fixed to a reference framework, whereas a moving part (rotor) rotates with respect to the stator. Some form of chemical and/or electrical input energy is needed to drive rotation unidirectionally. Brownian (random) back and forth small rotations because of thermal energy in the system would not produce effective work, as for the purpose of cell movement. For any form of energy to be useful at the nanometric scale of protein parts, such energy input must produce allosteric changes in transmission proteins, otherwise fuel consumption  would be largely dissipated in the form of heat. For example, if ATP is hydrolyzed at a particular nucleotide-binding pocket within a motor, energy liberation from covalent bond breaking through phosphoryltransfer to water would dissipate as heat in a matter of picoseconds  and withvia allosteric conformational/dynamic rearrangements of key motor proteins  and Gram-positive (Clostridium sporogenes and Bacillus subtilis), as well as across the entire family of MotA/MotB orthologs, such as PomA/PomB, the Ton transport ExbB/ExbD systems, and even the nonhomologous GldL/GldM gliding machines (A). The 5:2 ratio constrains each MotB monomer to sit into nonequivalent environments at any given time: each MotB helix is forced to interact with a different constellation of MotA residues. It is precisely this helix that includes the key cation transporter amino acid within MotB, namely aspartate 22 (according to the C.\u00a0jejuni numbering scheme), a strictly conserved residue in bacterial MotB orthologs across phyla (A). The dissimilar environments play a central role in allowing for a \u201csee-saw\u201d alternating motion, of the carboxylate-bearing Asp22 side chains.In the bacterial flagellar motor, ATP hydrolysis does not drive rotation. Instead, it is the regulated transport of protons or sodium cations along their electrochemical gradient, from the outside to the inside of the cell. Such proton-motive potential is transduced to mechanical work proteins . Recent proteins , 36, uncproteins . For manproteins , 38. Howetations . Furthermachines A. The 5:ss phyla A. The diB), particularly as a means of powering a rotating motor device that produces effective work, received statistical thermodynamics support in the famous lecture \u201cratchet and pawl\u201d by Richard Feynman . The energy instilled onto the pawl counteracts the random motion, and the asymmetric interaction with the ratchet is maintained, obstructing reversed rotation . The recent cryo-EM data : (i) the force injected to the aspartic acid/aspartates (the pawls) on MotB is due to the binding of protons on one side, and their dissociation on the other, along the electrochemical gradient; the Asp carboxylate\u2013bearing side chains rearrange spatially, according to them being charged or neutral, alternating such shifts in a see-saw fashion between both MotB monomers; and (ii) the molecular surface of the MotA pentamer (the ratchet) is irregular and offers an asymmetric junction to the moving aspartic acid/aspartates. It is thus clear that MotA and MotB constitute a fully functional motor on their own. MotA acts as the moving rotor, whereas MotB is the stator, fixed to a static reference such as the PG through its C-terminal PG-binding OmpA domain.The conformational change linked to the transport of each proton (a hydronium cation in water) is not very large, approximately 100-fold smaller than the estimated arc length traversed by the rotor (\u223c20\u201338\u00a0\u00c5) per cation passage . This feFig.\u00a02 Feynman . Essenti-EM data , 36 indeIn situ images of entire motors embedded within the inner membrane of Borrelia burgdorferi and Vibrio alginolyticus were recently obtained by cryo-ET at \u223c20\u00a0\u00c5 resolution Caulobact00 pN\u00b7nm ,\u00a050 . The hook functions as a universal joint . In other organisms, such as \u03b5-proteobacteria , two additional domains are found, D3 and D4, significantly extending the length of more external portion of FlgE . This notable observation raises the possibility that similar continuously flexible configurations could also be at play in the flagellar filament, for which more studies of native naturally curved filaments will be relevant. The change of paradigm, from a simpler two-state model to a continuous array of protomer conformations, is the result of technical improvements over the last several years in the field of cryo-EM image analyses. The initial hypothesis received support from observations using mutated variants of flagellin. Such mutants resulted in conformationally homogenous filament preparations that were better suited for high-resolution cryo-EM studies (B). However, the molecular basis of the defined asymmetric bending is still a puzzling issue. Each protofilament is composed of similarly configured FlgE protomers such that, as the assembly rotates, each protofilament will rapidly traverse the other 10 different conformations: what constrains each position among the different longitudinal directions of the assembly to adopt a unique conformation? That each protofilament position of the curved rotating hook only lodges a particular FlgE conformation, appears to imply that external forces\u2014perhaps the connection to the rotating basal body\u2019s rod\u2014impose some degree of asymmetry, subsequently fixed and amplified via inter-FlgE cooperativity. Indeed, recent observations of temporary \u201chook locking\u201d during CCW to CW motor reversal in Salmonella and E.\u00a0coli . The crosslinking site sits at the D1\u2013D2 domain interface, whereby D1 residue Lys165 reacts with Cys178 on the D2 domain of an adjacent FlgE protomer, both residues being close to each other in the assembled hook (B). Such covalent bonding eventually results in the crosslinking of all FlgE protomers in the hook  protofilaments. Such straight filaments dramatically improved cryo-EM resolution, a breakthrough for bacterial flagellar structural biology. Since then, many other filament structures have been solved, confirming a well-conserved organization with 11 flagellin protofilaments positioned around a central \u223c2 nm-wide channel. Flagellin protomers are secreted by the flagellar secretion apparatus and reach the distal growing tip of the filament through the central channel S.\u00a0enteri channel , 78. Whi channel , it has  channel , 81.A). The N- and C-terminal regions of FliC comprise \u223c250 amino acids each, folding together into two all-helical domains named D0 and D1, each incorporating portions from both terminal sequences . Often, in between the highly conserved D0 and D1 sequence segments, sits a stretch of sequence of variable length, which encodes surface-exposed domains, radiating as spikes from the central D0\u2013D1 core . Such additional domains are frequently two, globular and \u03b2-sheet\u2013containing (named D2 and D3), which typically include more variable sequences that provide interspecies diversity (S.\u00a0enterica FliC). On the other hand, it is also well known that many species only possess the conserved core D0\u2013D1 structure within their flagellins, such as in most Firmicutes, including B.\u00a0subtilis . Moreover, while B.\u00a0burgdorferi possesses a homopolymeric FlaB core . Flagellin protomers are arranged according to a simple helical symmetry, that is, a defined helical radius, with a precise rise (\u00c5) and twist (\u00ba) per consecutive FliC protomer. The FliC protomer shares a very similar structure to the hook FlgE and to the cap protein FliD A. The N-equences . D0 and equences , 83. Theiversity , and caniversity . Even moiversity , all theiversity , 88, somsubtilis , or in SlaB core , most SplaB core , 91, wit of them . ConcernB, rightmost panel). The universal component of the filament sheath in Spirochetes is FlaA, which has no homology to FlaB and can be present in one or more isoforms. FlaA proteins include a signal peptide, strongly suggesting that they are secreted into the periplasm via the classic Sec-dependent pathway, and thereafter recruited onto the filament . The cryo-EM data of Leptospira filaments combined with X-ray crystallographic analyses of individual flagellar proteins , composed of two FlaA isoforms (FlaA1 and FlaA2) (Leptospira genus: (i) flagellar coiling protein A (FcpA), one of the most abundant components of Leptospira endoflagella and essential for motility and pathogenicity . The convex, or outer side of the sheath, is composed of a two-tiered polymer, with FcpA protomers organized as an inner layer directly contacting the FlaB core, and a surface-exposed layer of FcpB moieties largely covering the FcpA sheet. The intricate network of intermolecular contacts, exerting asymmetric forces onto the FlaB core, elucidate the molecular basis of filament stiffness and supercoiled curvature  compared with \u0394fcpB genicity . The shegenicity , 99, in urvature , 99 thatth \u0394fcpB . The conth \u0394fcpB , with deTaking all the evidence together, phylogenetic variations of bacterial flagella, most likely related to environmental adaptations, can lead to filament structures that lose their characteristic symmetry, adding a richer complexity to the system.In situ images of entire motors embedded within the inner membrane of bacterial cells have been obtained by cryo-ET (B). The two-cogwheel gear allows the sense of rotation of one of the cogwheels (the C ring of the rotor on the base of the flagellum) to switch by simple inversion of the direction of coupling between the two. More precisely in the case of the motor, this switching device corresponds to the interface between MotA  and FliG on the C ring (known to turn either CW or CCW). Flipping the FliG\u2013MotA interface is enacted by a conformational rearrangement of the C-terminal domain of FliG , there is no equivalent bearing to envelop the inner-membrane motor components . The strong asymmetry in the organization of the filament sheath, observed for the first time in Leptospira (Flagellar diversity during evolution makes the Spirochetes a most fascinating model. Several recent accomplishments studying their unique endoflagella show how the same structural elements can design a radically different locomotion mechanism in bacteria. Endoflagella share the same general organization as exoflagella, yet the former possess no extracellular portions, so that the long filaments cannot work as free beating propellers to produce thrust and push the cell body. In a way that is not yet fully understood, Spirochetes use the flagellar machinery to drag their own cell body. Taking advantage of the sinusoidal, or even bluntly spiraled shape of the cell itself, the effected rotation produces a screw motion of the cell body and consequent forward thrust . The perFig.\u00a06ptospira , 99, is The impressive technological progress in the field of structural biology, which is now merging with advanced bioimaging techniques, provides us with powerful tools to uncover the detailed mechanisms of bacterial locomotion. The relevant questions that remain unanswered and the captivating ways that different bacteria employ to swim in different environments guarantee an exciting research journey ahead.The authors declare that they have no conflicts of interest with the contents of this article."}
+{"text": "APC, ARID1A, AXIN1, BAP1, EGFR, FGFRs, IDH1/2, RAS, SMAD4, and TP53. Actionable targets include alterations of FGFRs, IDH1/2, BRAF, NTRK, and HER2. \u201cPrecision oncology\u201d is emerging as a promising approach for CCA, and it is possible to inhibit the altered function of these genes with molecularly oriented drugs . In this review, we provide an overview of new biologic drugs  to treat metastatic CCA, providing readers with panoramic information on the trajectory from \u201cold\u201d chemotherapies to \u201cnew\u201d target-oriented drugs.Cholangiocarcinoma (CCA) is a malignant neoplasm arising in the epithelium of the biliary tract. It represents the second most common primary liver cancer in the world, after hepatocellular carcinoma, and it constitutes 10\u201315% of hepatobiliary neoplasms and 3% of all gastrointestinal tumors. As in other types of cancers, recent studies have revealed genetic alterations underlying the establishment and progression of CCA. The most frequently involved genes are  ARID1A, AXIN1, BAP1, EGFR, FGFRs, IDH1/2, KRAS, SMAD4, and TP53 (BRAF (p.V600E), NTRK fusions, and HER2 amplifications. \u201cPrecision oncology\u201d has recently emerged as a promising approach for CCA, and it is possible to inhibit the altered function of these genes with molecularly oriented drugs. In this review, we present an overview of the current drugs under investigation to treat metastatic CCA, providing readers with panoramic information from \u201cold\u201d chemotherapies to \u201cnew\u201d target-oriented drugs.Cholangiocarcinoma (CCA) is a malignant neoplasm arising in the epithelium of the biliary tract. It represents the second most common primary liver cancer in the world, after hepatocellular carcinoma, and it constitutes 10\u201315% of hepatobiliary neoplasms and 3% of all gastrointestinal tumors . CCAs arand TP53 . Among tDuring the last three decades, the incidence rates of intrahepatic forms of CCA, although less common than extrahepatic ones (approximately 20% of CCAs), have increased in Western Europe and Japan compared to eastern countries ,4.2 followed by gemcitabine 1000 mg/m2 on days 1 and 8 every three weeks) became the standard treatment for the first line therapy of metastatic disease. The association of these drugs achieves a response rate of 26.1% (versus 15.5%) and a median overall survival rate of 11.7 months (versus 8.1) in patients treated with gemcitabine alone  [2, folinic acid 350 mg, fluorouracil 400 mg/m2 bolus, and fluorouracil 2400 mg/m2 as a 46 h continuous intravenous infusion) as a second line over ASC in 162 patients progressing to a standard first-line therapy [p = 0.031). The overall survival rate in the ASC group was 35.5% (95% CI: 25.2\u201346.0) at 6 months and 11.4% (5.6\u201319.5) at 12 months compared to 50.6% (39.3\u201360.9) at 6 months and 25.9% (17.0\u201335.8) at 12 months in the ASC plus mFOLFOX group. The response rate was 5% in 81 patients in the ASC plus mFOLFOX group .Specific palliative treatment options for the late stages of CCA include chemotherapy and radiotherapy. Based on the results of the randomized phase three ABC-02 study, the combination of cisplatin and gemcitabine  and their associated receptors (FGFRs) have been studied to exploit the therapeutic potential of inhibiting their signaling. In fact, recent studies have shown that FGFRs have important roles in the pathogenesis and biology of CCA. Indeed, in vitro data has revealed the expression of FGFRs in human CCA specimens by immunohistochemistry  . The binFGFR2 is the most common gene fusion in CCAs [FGFR2. The approval for infigratinib was based on a phase two study that showed a surprising response rate (RR) of 23.1% and a median progression-free survival (PFS) rate of 7.3 months. Pemigatinib showed a robust RR of 35.5% and a median PFS rate of 6.9 months [Fusion proteins arise from chromosomal translocations that merge two genes and their consequent protein products.  in CCAs . To date in CCAs . In part in CCAs , FGFR2-A in CCAs , FGFR2-P in CCAs , FGFR2-K in CCAs . Many st in CCAs ,19,20. F in CCAs  and pemi in CCAs  have bee9 months . This st9 months . Further9 months  emerged IDH1 are detected in approximately 13% of iCCA and 1% of eCCA tumors [Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are cytosolic and mitochondrial enzymes that catalyse the conversion of isocitrate to \u03b1-ketoglutarate (\u03b1KG) while reducing NADP to NADPH (nicotinamide adenine dinucleotide phosphate hydrogen). NADPH is a crucial cellular reducing agent in detoxification processes involved in protection against the toxicity of reactive oxygen species and oxidative DNA damage . In factA tumors . NumerouA tumors ,31,32. IA tumors ,34. The IDH1mut may represent an early event in CCA carcinogenesis, as observed in glioblastoma and acute myeloid leukemia [Interestingly, high D-2HG levels cause the inhibition of hepatocellular differentiation and the uncontrolled proliferation of liver progenitor cells, suggesting that leukemia . Howeverleukemia .IDH1 mutations are more common than those of IDH2 [IDH1mut. Inhibitors of IDH1mut (IDH305 and FT-2102), IDH2mut (AG221), and pan-IDH1/2mut (AG881) are under investigation for CCA patients [IDH1-mutated CCA patients\u2019 refractories to previous systemic therapies, ivosidenib (AG120) showed a significant\u2014albeit small\u2014improvement in the median PFS versus the placebo  and a DCR of 53.2% (3 RP and 63 SD out of 124 patients). Based on the results of this randomized phase three study, the FDA approved the use of ivosidenib in this clinical setting [ of IDH2 . After tpatients ,39,40,41DH2mut AG1, and paThe mitogen-activated protein kinase (MAPK) pathway is involved in the crucial cellular processes of proliferation and survival . BRAF isBRAF mutations have been found in several malignancies including melanoma, non-small cell lung cancer, CCA, and colorectal cancer [BRAF mutations have been identified in CCA, but the most common is the p.V600E variant [BRAF p.V600E mutation detected by immune-histochemistry in CCAs was associated with (i) a higher TNM stage, (ii) resistance to systemic chemotherapy, and (iii) an aggressive clinical course with worse survival rates  [BRAF mutations in metastatic biliary tract cancers was a phase two basket trial [BRAF mutations, patients treated with BRAF inhibitors develop disease progression within a few months from the start of treatment. It has been demonstrated that resistance is predominantly mediated by downstream MAPK pathway alterations, including MEK activating mutations. For this reason, the concomitant BRAF and MEK inhibition overcomes the acquired resistance to BRAF inhibitors and potentiates the anti-tumor effects [BRAF p.V600E-mutated biliary tract cancers, with an ORR of 51% and a median PFS and median OS of 9.0 months and 14.0 months, respectively [BRAFmut CCAs [BRAF p.V600E mutation in CCAs, genetic testing should always be performed in these neoplasms.l cancer ,46,47,48 variant . In an iatients) . The firet trial . In thiset trial ,53. In m effects ,55. Of nectively . To datemut CCAs ,57. The NTRK-1, -2, -3) genes encode for TRK-A, -B, and -C (Tropomyosin receptor kinase-A). These are the high affinity receptors for the \u201cneurotrophin\u201d  that is crucially involved in neural development. NTRK is a member of the tyrosine kinases family associated with the MAPK signaling pathway [NTRK gene fusions.The neurotrophic tyrosine receptor kinase , in CCAs only NTRK-1 has been detected so far [NTRK gene, fusion partner, and chromosomal localization involved in CCAs [However, the estimated prevalence of NTRK fusions in CCAs is very low, and they are considered rare events (0.75%) . Furtherd so far . The NTR in CCAs ,65,66,67NTRK fusions have been intensively study as potential antitumor targets focusing on the development of a large number of TRK small molecule inhibitors. There are only two NTRK inhibitors that have been approved by both the FDA and the EMA: larotrectinib and entrectinib. These inhibitors have achieved high response rates and durable responses in metastatic solid tumors (including those of patients with CCA). Larotrectinib and entrectinib were associated with ORRs of 75% and 57%, respectively. The median response duration was 10 months with larotrectinib, though none was reached with entrectinib [NTRK-fusion-positive CCAs.Over the past decade, rectinib ,69. ThesHER2 aberrations in CCAs.The human epidermal growth factor receptor 2 (EGFR2 or HER2 or ERBB2) belongs to a family of tyrosine kinase receptors with four distinct domains that, following ligand binding, undergo to homo- or hetero-dimerization . ConsequHER2 amplification was found in 1.4% of more than 400 surgically resected and histologically confirmed CCAs. More variable results have been reported in South American or Asian populations [HER2 in CCA is trastuzumab; it is a humanized monoclonal antibody able to bind the extracellular binding domain of HER2. It suppresses the signaling pathways and induces HER2 degradation [In an interesting and large study , HER2 amulations ,77,78,79ulations ,81,82. Iradation . Currentradation . Trastuzradation . Neratinradation .An overview of the pharmacodynamics and molecular characteristics of the target-oriented drugs used in the treatment of CCA is provided in CCA incidence is increasing and, unfortunately, its prognosis remains very poor. The reasons are large genetic heterogeneity (from a molecular point of view) and scarce chemotherapy responsiveness . These characteristics render the CCA a perfect model to study and apply innovative and molecularly oriented drugs. A new class of drugs is now emerging, and its application is necessarily preceded by the genetic profiling of CCA towards a \u201cprecision oncology\u201d . Every patient will deserve a \u201cfrom bench to bad\u201d approach and commitment. In this expanding context, future clinical research paradigms  must be pursued that revolutionize our dogmatic approaches to sequential treatments, molecular testing, and response monitoring."}
+{"text": "Alsophila spinulosa) and unique plants (cool-adapted plants) are present in Tiankeng. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, the species diversity of Cordyceps-like fungi in Monkey-Ear Tiankeng was investigated. Seven species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were identified based on internal transcribed spacer sequences and morphological characteristics. Eight new species in the genera Akanthomyces, Cordyceps, and Samsoniella were established and described according to a multilocus phylogenetic analysis and morphological characteristics. Our results revealed that Cordyceps-like fungi were abundant in Monkey-Ear Tiankeng, providing new insights into the diversity of Ascomycota in this special eco-environment.Tiankeng acts as a refugium for biodiversity amid a changing global climate, and a previous study has shown that some ancient (IMPORTANCE Karst Tiankeng has a special eco-environment and acts as a refugium for biodiversity. However, there are few reports on Ascomycota from the Tiankeng karst region. In this research, seven known species and eight new species in the genera Akanthomyces, Beauveria, Cordyceps, and Samsoniella were reported. The results showed that Cordyceps-like fungi are abundant in Monkey-Ear Tiankeng. Interestingly, the month of the sampling was November, which is not an active period of growth and reproduction for Cordyceps-like fungi. These results revealed that unconventional time sampling should not be ignored, especially for a special eco-environment, and provided new insights into the diversity of Ascomycota in this special eco-environment. Tiankeng is a kind of negative karst terrain, which was first named by Zhu in 2001 \u20135.\u2013Research on the Tiankeng karst region has been conducted for many years and has focused on geology  \u201310, anim\u201313\u201316\u2013Trichoderma sp. was able to degrade feathers efficiently. Lan et al.  Luangsa-ard, Houbraken, and Hywel-Jones & Samson (CBS 431.87) were used as the outgroup in analysis 1, whereas P. lilacinum (CBS 284.36 and CBS 431.87) was used as the outgroup in analysis 2. The concatenated sequences of analyses 1 and 2 included 32 and 74 taxa, respectively, and consisted of 516  and 2,480  characters with gaps, respectively.In the phylogenetic tree of analysis 1 (establishing the genus placement of the new strains) and analysis 2 (determining the establishment of the new species)  Vuill. with a high bootstrap value (99/1). Strains KY11071, KY11211, KY11111, KY11771, and KY11101 were clustered into the subclade of B. pseudobassiana S.A. Rehner & Humber with a high bootstrap value (92/0.97). Strains KY11231, KY11031, KY11221, KY11661, and KY11731 were clustered into the subclade of Cordyceps tenuipes (Peck) Kepler, B. Shrestha & Spatafora with a high bootstrap value (92/0.99). Strains KY11511 and KY11361 were clustered into the subclade of C. fumosorosea (Wize) Kepler, B. Shrestha & Spatafora with a high bootstrap value (99/1). Strains KY11431 and KY11671 were clustered into the subclade of C. cateniannulata (Z.Q. Liang) Kepler, B. Shrestha & Spatafora with a high bootstrap value (95/1). Strains KY11021, KY11061, KY11171, KY11331, KY11391, KY11371, KY11711, KY11551, and KY11051 were clustered with C. amoene-rosea (Henn.) Kepler, B. Shrestha & Spatafora and C. cateniobliqua (Z.Q. Liang) Kepler, B. Shrestha & Spatafora. Strain KY11701 was clustered into the subclade of C. javanica  Kepler, B. Shrestha & Spatafora with a high bootstrap value (86/1). Strains KY11121, KY11122, KY11161, KY11162, KY11741, KY11742, KY11041, KY11042, KY11321 ,and KY11322 were clustered into the clade of the genus Samsoniella Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard. Strains KY11341, KY11342, KY11571, and KY11572 were all clustered with the Akanthomyces species. Strains KY11141 and KY11142 were clustered into the clade of the genus Cordyceps.Analysis 1: The final value of the highest scoring tree was \u20133,525.3844, which was obtained from a maximum likelihood (ML) analysis of ITS sequence. The parameters of the general time-reversible (GTR) model used to analyze the data set were estimated using the following frequencies: A\u2009=\u20090.2280, C\u2009=\u20090.3330, G\u2009=\u20090.2552, and T\u2009=\u20090.1838 with the substitution rates AC\u2009=\u20091.9959, AG\u2009=\u20092.6768, AT\u2009=\u20091.0000, CG\u2009=\u20091.9959, CT\u2009=\u20096.7579 and GT\u2009=\u20091.0000. The gamma distribution shape parameter was \u03b1 = 0.4764. The selected model for the Bayesian inference (BI) analysis was GTR+F+G4 (ITS). The phylogenetic trees  construcBeauveria bassiana. Strains KY11071, KY11211, KY11111, KY11771, and KY11101 were identified as B. pseudobassiana. Strains KY11231, KY11031, KY11221, KY11661, and KY11731 were identified as Cordyceps tenuipes. Strains KY11511 and KY11361 were identified as C. fumosorosea. Strains KY11431 and KY11671 were identified as C. cateniannulata. Strains KY11021, KY11061, KY11171, KY11331, KY11391, KY11371, KY11711, KY11551, and KY11051 were identified as C. cateniobliqua. Strain KY11701 was identified as C. javanica. Multilocus phylogenetic analysis was required for the further identification of the strains KY11121, KY11122, KY11161, KY11162, KY11741, KY11742, KY11041, KY11042, KY11321, KY11322, KY11341, KY11342, KY11571, KY11572, KY11141, and KY11142.Comparing their typical morphological characteristics, strains KY11411, KY11721, KY11081, KY11451, KY11281, KY11191, KY11151, KY11261, KY11601, KY11251, and KY11181 were identified as Cordyceps tenuipes (Peck) Kepler, B. Shrestha & Spatafora, C. coleopterorum (Samson & H.C. Evans) Kepler, B. Shrestha & Spatafora, and C. bifusispora O.E. Erikss. in a subclade. Strains KY11571 and KY11572 were clustered with Akanthomyces coccidioperitheciata (Kobayasi & Shimizu) Spatafora, Kepler & B. Shrestha, A. kanyawimiae Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard, and A. thailandicus Mongkols., Spatafora & Luangsa-ard in a subclade. Strains KY11341 and KY11342 were clustered with A. pissodis  W.H. Chen, Y.F. Han & Z.Q. Liang in a subclade. Strains KY11321 and KY11322 were clustered with Samsoniella alboaurantia (G. Sm.) Mongkols., Noisrip., Thanakitp., Spatafora & Luangsa-ard and S. cardinalis H. Yu, Y.B. Wang, Y. Wang, Q. Fan & Zhu L. Yang in a subclade. Strains KY11041, KY11042, KY11161, and KY11162 were clustered with S. pupicola W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang in a subclade. Strains KY11741, KY11742, KY11121, and KY11122 were clustered into two independent clades.Analysis 2: The final value of the highest scoring tree was \u221228,566.0385, which was obtained from the ML analysis of the data set (ITS+LSU+RPB2+TEF). The parameters of the GTR model used to analyze the data set were estimated based on the following frequencies: A\u2009=\u20090.2351, C\u2009=\u20090.2835, G\u2009=\u20090.2710, and T\u2009=\u20090.2104 with the substitution rates AC\u2009=\u20091.0000, AG\u2009=\u20092.2438, AT\u2009=\u20091.0000, CG\u2009=\u20091.0000, CT\u2009=\u20095.0707, and GT\u2009=\u20091.0000. The gamma distribution shape parameter was \u03b1 = 0.5592. The selected models for the BI analysis were GTR+F+I+G4 , GTR+F+G4 (LSU), and SYM+G4 (RPB2). The phylogenetic trees  construcAkanthomyces, Cordyceps and Samsoniella, were established based on a multi-locus phylogeny, their morphological and ecological characteristics. The morphological and ecological characteristics of the new species were as follows. Akanthomyces araneosus W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov (MycoBank: 844985) (Eight new species distributed in the three genera  844985) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a dead spider (Araneae), 16 November 2020, Wanhao Chen, GZAC KY1134 (holotype), ex-type living cultures, KY11341.Description: Spider host was completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on potato dextrose agar (PDA) were 2.4 to 2.6\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish. Prostrate hyphae were smooth, septate, hyaline, and 1.0 to 2.5\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of two. Phialides were 16.9 to 18.1\u2009\u00d7\u20091.3 to 1.9\u2009\u03bcm with a cylindrical basal portion and tapered into a short, distinct neck. Conidia were hyaline, fusiform, one-celled, and 3.1 to 5.0\u2009\u00d7\u20091.0 to 1.8\u2009\u03bcm. The sexual state was not observed.Host: Spider (Araneae).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to the mycelium covering the spider like a spider web.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a dead spider (Araneae), 16 November 2020, Wanhao Chen, KY11342.Akanthomyces araneosus was easily identified as Akanthomyces, according to the phylogenetic analysis of the combined data sets   and its spider host.Remarks: B2, TEF)  and 2, aAkanthomyces tiankengensis W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844986) ( 844986) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a dead spider (Araneae), 16 November 2020, Wanhao Chen, GZAC KY1157 (holotype), ex-type living cultures, KY11571.Description: Spider host was completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on PDA were 2.8 to 3.6\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish. Prostrate hyphae were smooth, septate, hyaline, and 1.2 to 2.9\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of two. Phialides were 13.9 to 17.1\u2009\u00d7\u20091.1 to 1.6\u2009\u03bcm with a cylindrical basal portion and tapered into a short, distinct neck. Conidia were hyaline, fusiform, one-celled, and 2.3 to 3.0\u2009\u00d7\u20091.5 to 2.3\u2009\u03bcm. The sexual state was not observed.Host: Spider (Araneae).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its location in Tiankeng.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a dead spider (Araneae), 16 November 2020, Wanhao Chen, KY11572.Akanthomyces tiankengensis was easily identified as Akanthomyces, according to the phylogenetic analysis of the combined data sets   and fusiform conidia. It was distinguished from A. thailandicus by its smaller conidia (2.3 to 3.0\u2009\u00d7\u20091.5 to 2.3\u2009\u03bcm) and cylindrical phialides.Remarks: B2, TEF)  and 2, aCordyceps tiankengensis W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844987) ( 844987) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, GZAC KY1114 (holotype), ex-type living cultures, KY11141.Description: Insect pupa was completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on PDA were 2.2 to 3.2\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish. Prostrate hyphae were smooth, septate, hyaline, and 1.3 to 1.9\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of two. Phialides were 7.1 to 12.2\u2009\u00d7\u20091.9 to 2.5\u2009\u03bcm with a cylindrical basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, fusiform, one-celled, and 4.1 to 5.1\u2009\u00d7\u20091.8 to 2.7\u2009\u03bcm. The sexual state was not observed.Host: Pupa (Lepidoptera).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its location in Tiankeng.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, KY11142.Cordyceps tiankengensis was easily identified as Cordyceps, according to the phylogenetic analysis of combined data sets  , and it was easily distinguished from C. coleopterorum by its smaller conidia (4.1 to 5.1\u2009\u00d7\u20091.8 to 2.7\u2009\u03bcm).Remarks: B2, TEF)  and 2, aSamsoniella formicae W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844988) ( 844988) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On an ant (Formicidae), 16 November 2020, Wanhao Chen, GZAC KY1104 (holotype), ex-type living cultures, KY11041.Description: Ant host was completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on PDA were 5.0 to 5.1\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish or pale orange. Prostrate hyphae were smooth, septate, hyaline, and 1.3 to 1.9\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of two. Phialides were 8.3 to 13.5\u2009\u00d7\u20091.2 to 1.8\u2009\u03bcm with a cylindrical basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, fusiform, one-celled, and 2.5 to 3.2\u2009\u00d7\u20091.6 to 2.2\u2009\u03bcm. The sexual state was not observed.Host: Ant (Formicidae).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its ant host.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On an ant (Formicidae), 16 November 2020, Wanhao Chen, KY11042.Samsoniella formicae was easily identified as Samsoniella, according to the phylogenetic analysis of the combined data sets   and its ant host. It was distinguished from S. guzhouensis by its longer phialides (8.3 to 13.5\u2009\u00d7\u20091.2 to 1.8\u2009\u03bcm) and its bigger conidia (2.5 to 3.2\u2009\u00d7\u20091.6 to 2.2\u2009\u03bcm).Remarks: B2, TEF)  and 2, aSamsoniella erucae W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844989) ( 844989) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a caterpillar (Lepidoptera), 16 November 2020, Wanhao Chen, GZAC KY1112 (holotype), ex-type living cultures, KY11121.Description: Synnemata arose from different parts of the insect host. Conidiophores were synnematous and arose from the lateral hyphae of the synnemata. Colonies on PDA were 4.6 to 4.8\u2009cm in diameter after 14 d at 25\u00b0C, white, comprised of a basal felt hyphal overgrowth, and powdery in the middle during mass sporulation with the reverse light yellowish. Prostrate hyphae were smooth, septate, hyaline, and 1.4 to 1.9\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of three. Phialides were 6.8 to 13.7\u2009\u00d7\u20091.1 to 1.5\u2009\u03bcm with a cylindrical or ellipsoidal basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, fusiform to ellipsoidal, one-celled, and 2.3 to 2.9\u2009\u00d7\u20091.1 to 1.5\u2009\u03bcm. The sexual state was not observed.Host: Caterpillar (Lepidoptera).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its caterpillar host in the order Lepidoptera.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a caterpillar (Lepidoptera), 16 November 2020, Wanhao Chen, KY11122.Samsoniella erucae was easily identified as Samsoniella according to the phylogenetic analysis of the combined data sets   and its bigger conidia (2.3 to 2.9\u2009\u00d7\u20091.1 to 1.5\u2009\u03bcm). S. erucae clustered into an independent subclade  . When cosubclade  and was Samsoniella guizhouensis W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844990) ( 844990) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, GZAC KY1116 (holotype), ex-type living cultures, KY11161.Description: Insect pupa was covered completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on PDA were 4.4 to 4.5\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish to pale brown or green. Prostrate hyphae were smooth, septate, hyaline, and 1.3 to 2.5\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of two. Phialides were 4.9 to 7.9\u2009\u00d7\u20091.7 to 2.1\u2009\u03bcm with an ellipsoidal basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, fusiform, one-celled, and 2.2 to 2.5\u2009\u00d7\u20091.5 to 1.9\u2009\u03bcm. The sexual state was not observed.Host: Pupa (Lepidoptera).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its location in Guizhou Province.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, KY11162.Samsoniella guizhouensis was easily identified as Samsoniella, according to the phylogenetic analysis of the combined data sets   and its shorter phialides (4.9 to 7.9\u2009\u00d7\u20091.7 to 2.1\u2009\u03bcm).Remarks: B2, TEF) , and hasSamsoniella neopupicola W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844991) ( 844991) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, GZAC KY1132 (holotype), ex-type living cultures, KY11321.Description: Insect host was completely covered by white mycelium. Conidiophores were mononematous and arose from the lateral hyphae. Colonies on PDA were 5.5 to 5.7\u2009cm in diameter after 14 d at 25\u00b0C, white, and comprised of a basal felt and a floccose hyphal overgrowth with the reverse yellowish or pale orange. Prostrate hyphae were smooth, septate, hyaline, and 1.2 to 2.4\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of five. Phialides were 8.2 to 11.7\u2009\u00d7\u20091.5 to 2.3\u2009\u03bcm with a cylindrical basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, fusiform, one-celled, and 2.5 to 3.0\u2009\u00d7\u20091.6 to 2.3\u2009\u03bcm. The sexual state was not observed.Host: Pupa (Lepidoptera).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its pupa host in the order Lepidoptera.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, KY11322.Samsoniella neopupicola was easily identified as Samsoniella, according to the phylogenetic analysis of the combined data sets   and its Lepidoptera pupa host. It was distinguished from S. cardinalis by its smaller phialides (8.2 to 11.7\u2009\u00d7\u20091.5 to 2.3\u2009\u03bcm).Remarks: B2, TEF) , and hasSamsoniella tiankengensis W.H. Chen, Y.F. Han, J.D. Liang & Z.Q. Liang, sp. nov. (MycoBank: 844992) ( 844992) .Type: China, Guizhou, Guiyang, Kaiyang County, Monkey-Ear Tiankeng . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, GZAC KY1174 (holotype), ex-type living cultures, KY11741.Description: Synnemata arose from different parts of the insect pupa. Conidiophores were synnematous and arose from the lateral hyphae of the synnemata. Colonies on PDA were 5.3 to 5.6\u2009cm in diameter after 14 d at 25\u00b0C, white to light pink, and comprised of a basal felt and a cottony hyphal overgrowth with the reverse light yellowish. Prostrate hyphae were smooth, septate, hyaline, and 1.5 to 2.5\u2009\u03bcm in diameter. Erect conidiophores usually arose from the aerial hyphae. Phialides were solitary or in groups of four. Phialides were 5.4 to 10.4\u2009\u00d7\u20091.3 to 2.2\u2009\u03bcm with a cylindrical or subellipsoidal basal portion and tapered into a short, distinct neck. Conidia were in chains, hyaline, ellipsoidal, one-celled, and 2.3 to 2.8\u2009\u00d7\u20091.6 to 1.8\u2009\u03bcm. The sexual state was not observed.Host: Pupa (Lepidoptera).Locality: Kaiyang County , Guiyang, Guizhou Province, China.Etymology: Referring to its location in Tiankeng.Additional strain examined: China, Guizhou, Guiyang, Kaiyang County . On a pupa (Lepidoptera), 16 November 2020, Wanhao Chen, KY11742.Samsoniella tiankengensis was easily identified as Samsoniella according to the phylogenetic analysis of the combined data sets  , its bigger conidia (2.3 to 2.8\u2009\u00d7\u20091.6 to 1.8\u2009\u03bcm), and its pupa host. S. tiankengensis clustered into an independent subclade  . When cosubclade  and was Monkey-Ear Tiankeng is an important modern refugium. It contains cliffs, caves, and an underground river. Following a vertical distribution pattern, the bottom of Tiankeng is the center of the biodiversity, and the species distribution gradually increases from the pit mouth to the pit bottom, such that it can be used as a natural refuge for organisms in rocky karst, desert, and mountainous areas , 27. PreCordyceps-like fungi are important Ascomycota that originated in remote mountains and dense forests and have become closely intertwined with people\u2019s lives , B. pseudobassiana (5 strains), Cordyceps tenuipes (5 strains), C. fumosorosea (2 strains), C. cateniannulata (2 strains), C. cateniobliqua (9 strains), and C. javanica (1 strain). A total of 16 strains were clustered into the following genera: Akanthomyces (4 strains), Cordyceps (2 strains), and Samsoniellla (10 strains). More information is needed for further identification. Eight new species: A. araneosus, A. tiankengensis, C. tiankengensis, S. formicae, S. erucae, S. guizhouensis, S. neopupicola, and S. tiankengensis, were established and described according to a multilocus phylogenetic analysis and their morphological characteristics. Our results showed that Cordyceps-like fungi are abundant in Monkey-Ear Tiankeng. Interestingly, the month of the sampling was November, which is not an active period of growth and reproduction for Cordyceps-like fungi, revealing that unconventional time sampling should not be ignored, especially for a special eco-environment, such as Tiankeng. However, further research is needed to confirm whether the diversity of the Cordyceps-like fungi in Tiankeng, especially for the new species, is related to the sampling time and the environment.In the present study, the species diversity of a Cordyceps-like fungi are all-rounders in their nutrition intake. The nutritional model of Cordyceps-like fungi was found to range from plants (including living plants and plant residues) to animals  and even to fungi  and unique plants (cool-adapted plants) are present in Tiankeng  were collected from Monkey-Ear Tiankeng , Kaiyang County, Guiyang, Guizhou Province, on 16 November 2020. The area belongs to a subtropical monsoon humid climate zone that receives an annual precipitation of 1,141 to 1,547\u2009mm and has an annual average temperature of 10.6 to 15.3\u00b0C . The isoThe macroscopic and microscopic morphological characteristics of the fungi were examined, especially for the arrangement, shape, and measurement of phialides and conidia. The growth rates were also determined from potato dextrose agar cultures incubated at 25\u00b0C for 14\u2009days. The hyphae and conidiogenous structures were mounted in lactophenol cotton blue or a 20% lactate solution and observed with an optical microscope .DNA extraction was carried out using the Fungal Genomic DNA Extraction Kit  in accordance with Liang et al. . The extLasergene software  was applied for the assembly and editing of the DNA sequences in this study. ITS, LSU, RPB2, and TEF sequences were downloaded from GenBank, based on Sung et al. , Chen etThe combined loci were analyzed using Bayesian inference (BI) and maximum likelihood (ML) methods. For the BI, a Markov chain Monte Carlo (MCMC) algorithm was used to generate phylogenetic trees with Bayesian probabilities for the combined sequence data sets using MrBayes v.3.2 . The Bay"}
+{"text": "The analysis of \u201cbig data\u201d identified superoxide dismutase-1 (SOD1) as a novel target of CHI3L1 and interacted with CHI3L1. The depletion of CHI3L1 increased SOD1 expression, resulting in ER stress. Furthermore, the depletion of SOD1 reduced the expression of ER chaperones and ER-mediated apoptotic marker proteins, as well as apoptotic cell death induced by the depletion of CHI3L1 in in vivo and in vitro models. These results suggest that the depletion of CHI3L1 increases ER stress-mediated apoptotic cell death through SOD1 expression, and subsequently inhibits lung metastasis.Chitinase-3-like protein 1 (CHI3L1), which is secreted by immune and inflammatory cells, is associated with several inflammatory diseases. However, the basic cellular pathophysiological functions of CHI3L1 are not well characterized. To investigate the novel pathophysiological function of CHI3L1, we performed LC-MS/MS analysis of cells transfected with Myc-vector and Myc-CHI3L1. We analyzed the changes in the protein distribution in Myc-CHI3L1 transfected-cells, and identified 451 differentially expressed proteins (DEPs) compared with Myc-vector-transfected-cells. The biological function of the 451 DEPs was analyzed and it was found that the proteins with endoplasmic reticulum (ER)-associated function were much more highly expressed in CHI3L1-overexpressing cells. We then compared and analyzed the effect of CHI3L1 on the ER chaperon levels in normal lung cells and cancer cells. We identified that CHI3L1 is localized in the ER. In normal cells, the depletion of CHI3L1 did not induce ER stress. However, the depletion of CHI3L1 induces ER stress and eventually activates the unfolded protein response, especially the activation of Protein kinase R-like endoplasmic reticulum kinase (PERK), which regulates protein synthesis in cancer cells. CHI3L1 may not affect ER stress owing to the lack of misfolded proteins in normal cells, but instead activate ER stress as a defense mechanism only in cancer cells. Under ER stress conditions induced by the application of thapsigargin, the depletion of CHI3L1 induces ER stress through the upregulation of PERK and PERK downstream factors (eIF2\u03b1 and ATF4) in both normal and cancer cells. However, these signaling activations occur more often in cancer cells than in normal cells. The expression of Grp78 and PERK in the tissues of patients with lung cancer was higher compared with healthy tissues. It is well known that ER stress-mediated PERK-eIF2\u03b1-ATF4 signaling activation causes apoptotic cell death. ER stress-mediated apoptosis induced by the depletion of CHI3L1 occurs in cancer cells, but rarely occurs in normal cells. Consistent with results from the  The endoplasmic reticulum (ER) is an important cellular organelle responsible for the synthesis of proteins, protein modification through glycosylation or disulfide bond formation, protein folding into a normal structure, calcium storage, and signaling transduction The activation of UPR by ER stress is mediated by IRE-1 (inositol-requiring 1), PERK (Protein kinase R-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor) Chitinase-3-llke protein 1  was first discovered in mouse breast cancer cells and was named breast regression protein 39 (BRP-39). CHI3L1 binds to chitin; however, unlike chitotriosidase (CHIT1) and acid mammalian chitinase (AMCase), it has an intrinsic function independent of chitin. In humans, CHI3L1 plays an important role in maintaining homeostasis in many organs Mus musculus skin melanoma cells were obtained from the American Type Culture Collection . U-2 OS cells were cultured in McCoy's 5a medium supplemented with 10% fetal bovine serum (FBS), 100 \u03bcg/mL penicillin, and 100 \u03bcg/mL streptomycin. A549, MRC-5, and B16F10 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 \u03bcg/mL penicillin, and 100 \u03bcg/mL streptomycin. A172 and Hep3B cells were cultured in DMEM medium supplemented with 10% FBS, 100 \u03bcg/mL penicillin, and 100 \u03bcg/mL streptomycin. Cell cultures were maintained in an incubator with a humidified atmosphere of 5% CO2 at 37 \u00b0C. Reduced serum medium and X-tream GENE HP DNA transfection reagent were used for transfecting plasmid and siRNA oligonucleotides following the manufacturer's instructions.A549 lung carcinoma, MRC-5 normal lung cell, U-2 OS bone osteosarcoma, A172 glioblastoma, Hep3B hepatocellular carcinoma, and B16-F10 Animal experiments were performed as described previously All protocols involving mice in this study were reviewed and approved by the Chungbuk National University Institutional Animal Care and Use Committee (IACUC) and complied with the Korean National Institute of Health Guide for the Care and Use of Laboratory Animals (CBNUA-1073-17-01).g for 20 min at 4 \u00b0C. Cells were harvested and lysed using lysis buffer and centrifuged. The proteins were loaded to SDS-PAGE. The specific primary antibodies and HRP-conjugated secondary antibodies were incubated and visualized by using a FUSION Solo S chemiluminescence detection system .The tissues were homogenized with lysis buffer  and centrifuged at 13,000 X For immunoprecipitation, the cell lysates were incubated with specific primary antibodies for 4 h at 4 \u00b0C. Then protein A/G PLUS-Agrose (Santa cruz) were added to antibody containing cell lysates and incubated for 2 h at 4 \u00b0C. Immunoprecipitated proteins were loaded to SDS-PAGE.Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells then were permeabilized with 100% cold-Methanol for 5 min and blocked by 4% BSA in PBS with 0.1% Triton X-100 (PBST) for 1 h. Primary antibodies were incubated for overnight at 4 \u00b0C and Alexa Fluor 488  or Texas Red  conjugated secondary antibodies were incubated for 1h at room temperature. Fixed cells were incubated with 1 \u03bcg/mL of DAPI  for 5 min at room temperature and then covered with Fluoromount-G Mounting Medium . Cells were visualized using Ziess AxioObserver  fluorescence microscope system. Digital images were analyzed using the ZEN 2.1 (Carl Zeiss) software.The tumor and lung tissue sections were blocked with 3% normal goat serum diluted in PBS, for 30 min; the sections were then incubated with antibodies for CHI3L1, Grp78, PERK, CHOP, caspase 12, and SOD1, at the appropriate dilution in blocking serum, for overnight at 4 \u00baC. The slides were washed in PBS, followed by the avidin-biotin-peroxidase complex . The slides were washed, and the peroxidase reaction was developed with diaminobenzidine and peroxide , mounted in Aqua-Mount, and evaluated under a light microscope (Olympus).A549 cells were transfected with either control siRNA or CHI3L1-siRNA for 1 day with thapsigargin for 18 h. Then cells were fixed with 4% paraformaldehyde in DPBS for 30 min at room temperature, permiabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 10 min at room temperature. The cleavage DNA fragments were stained for 1 h in the dark at 37 \u00b0C using the TUNEL reaction mixture. Cells were observed on an Axio Observer Z1 inverted fluorescence microscope.For trans-well assay, A549 cells were seeded on upper chamber inserts . After incubation for 18 h, the cells were fixed with 4% formaldehyde for 5 min, permeated with 100% methanol for 15 min, and stained with 0.1% crystal violet for 20 min. In the upper chamber, non-migrated cells were removed with a cotton swab. The migrated cells were visualized using a light microscope (Olympus) and analyzed using ImageJ software (NIH).Cell pellets were stored at -80 \u00b0C until cell lysis was performed. Lysis of cell pellets was done at room temperature. Biological replicates (one cell pellet from one cell line) were processed simultaneously to minimize the effect of error. Pellets were resuspended in 100 \u03bcL 8 M Urea  were added. Protein concentration was estimated with the bicinchoninic acid assay . Proteins were reduced with 10 mM dithiothreitol (Sigma) and alkylated 25 mM iodoacetamide (Sigma). Samples were diluted in 50 mM AmBic and trypsinized  overnight at 37 \u00b0C at a trypsin/protein ratio of 1:50, w/w). The resulting peptide mixture was lyophilized overnight and digested peptides were cleaned by flowing through an Oasis HLB 1cc (10 mg) solid phase extraction (SPE) catridges . Samples were dried using a Speed-Vac  and stored at -80 \u00b0C until time for analysis. The peptides digestion protocols were performed modifying the method described previously 6, and a maximum injection time of 80 ms. Fragmentation was performed with a normalized collision energy of 25.LC-MS/MS was performed as described previously Human tissue samples from lung cancer patients and normal controls were obtained from Chungbuk National University Hospital Biobank, Keimyung University Dongsan Hospital Biobank and the Biobank of Ajou University Hospital, members of Korea Biobank Network. All studies using human samples were conducted in accordance with the Declaration of Helsinki and were approved by the Ethics Committee of Chungbuk National University Medical Center (IRB No. CBNU-201910-BR-941-01).t-test. Multiple comparisons were using one-way analysis of variance followed by Tukey's tests. Differences between groups were considered significant at P-values of <0.05.Statistical analyses were performed using the GraphPad Prism 5 software. All error bars reported are the standard deviation (SD) unless otherwise indicated. Pairwise comparisons were performed using Student's To evaluate the global changes in protein abundance in CHI3L1-expressing cells, we performed nano-high-performance liquid chromatography (nano-LC). In total, we identified 1146 proteins and 1120 proteins in CHI3L1-expressing cells and 1053 proteins in control vector-expressing cells. There were 1027 proteins differentially expressed in CHI3L1-expressing cell and vector-expressing cell samples  treatment, an ER stress inhibitor. The western blotting results showed that 4-PBA treatment had no obvious effect on ER chaperone protein levels in cells transfected with control siRNA. However, ER chaperone protein levels were lower in CHI3L1 depletion in the presence of 4-PBA, indicating that the degree of ER stress is reduced . Compared with DMSO-treated control cells, thapsigargin treatment did not alter cell proliferation (75.34\u00b14.07%). The proliferation of cancer cells transfected with CHI3L1 siRNA and treated with thapsigargin was significantly decreased (49.88\u00b17.80%)  Figure A. In sevin vivo metastasis in CHI3L1 WT and KO mice, A549 cells were injected into tail veins and control siRNA and SOD1 siRNA were intravenously injected into CHI3L1 WT and KO mice a three times per week for 7 weeks. The CHI3L1 KO group had significantly fewer metastatic nodes in lung tissues compared with the CHI3L1 WT group  and PERK signaling proteins compared with the depletion of CHI3L1 in lung cancer cells and metastatic lung tumor tissues. In addition, ER stress-mediated apoptotic marker protein levels also much greatly decreased in the dual depletion of CHI3L1 and SOD1 in in vitro and in vivo. Here, we showed that the depletion of CHI3L1 induces ER stress-mediated lung cancer cell death through SOD1 upregulation.However, the exact mechanism of how CHI3L1 causes ER stress and thus induces apoptosis and inhibits tumorigenesis is unknown. From the GENEMANIA database, we found that the SOD1 protein forms an interaction network between CHI3L1 and ER stress. In addition, we confirmed that CHI3L1 physically interacts with SOD1. Several papers reported that SOD1 accumulation activates the ER stress-mediated UPR pathway The present study provides important insights into the role of CHI3L1 in the regulation of ER stress associated UPR function and apoptosis, as well as the SOD1 dependent regulation of tumor growth.Supplementary figures and table.Click here for additional data file."}
+{"text": "Employment as a critical domain of functioning is an important target of recovery-oriented programs for people with psychiatric disabilities. Evidence shows that persons participating in competitive employment which meets their vocational needs are more likely than people in sheltered work programs to feel included in their communities, to report satisfaction with work and a high quality of life.The Vineyard Project is a program engaging young people with different forms of mental ill-health in local practices of hand-harvesting grape. The program stems from a pilot carried out in September 2022 with a group of people aged 16-25, who worked with professional vine growers in the renowned area of Langhe . Aims were manifold: i) for the group: involvement in a culturally meaningful activity that is part of the transformative process of winemaking, as a way to overcome social anxiety symptoms and poor self-efficacy; ii) for the community: to attempt overcoming structural stigma that may undermine an employer\u2019s willingness to hire a person with a psychiatric diagnosis; iii) to develop an evidence-based rehabilitation program aimed at competitive employment.To foster community integration, the program was hosted in a real-world setting. The program is multidisciplinary, involving psychiatrists, psychologists, rehabilitation specialists and sociologists. Clinical assessment and semi-structured interviews with participants are performed.Preliminary research findings provide evidence to develop the program according to the following principles: on-site practical training provided in a 1:1 relationship with professional workers , supplemented by extra educational resources available in the community ; covering of all the vineyard activities throughout the year ; growth of expertise on an individual as well as on a group level, to foster the building of a cohesive team that can compete on the labour market and that provides participants with a sense of membership and identity; opportunity for new participants to join the team on an annual basis, acknowledging their peers as experts who can in turn pass on their knowledge; continual assessment of the ever-changing needs of participants and qualitative inquiry of their perspective, to provide time-unlimited support and ongoing adjustment of the program.The Vineyard Project aims to eventually establish a rehabilitation tool, resulting from the combination of multi-disciplinary approaches, that can be tested and applied to work settings different from the viticultural environment where it had its origin.None Declared"}
+{"text": "DTI methods of white matter analysis using standardised methods of ROI extraction can help in differentiation of iNPH patients not only from healthy patients but also from patients with other causes of gait disturbances with cognitive decline and ventriculomegaly.The aim of this study was to investigate whether white matter changes as measured by diffusion tensor imaging (DTI) can help differentiate shunt-responsive idiopathic normal pressure hydrocephalus (iNPH) patients from patients with other causes of gait disturbances and/or cognitive decline with ventriculomegaly whose clinical symptoms do not improve significantly after cerebrospinal fluid derivation (non-iNPH). Between 2017 and 2022, 85 patients with probable iNPH underwent prospective preoperative magnetic resonance imaging (MRI) and comprehensive clinical workup. Patients with clinical symptoms of iNPH, positive result on lumbar infusion test, and gait improvement after 120-h lumbar drainage were diagnosed with iNPH and underwent shunt-placement surgery. Fractional anisotropy (FA) and mean diffusivity (MD) values for individual regions of interest were extracted from preoperative MRI, using the TBSS pipeline of FSL toolkit. These FA and MD values were then compared to results of clinical workup and established diagnosis of iNPH. An identical MRI protocol was performed on 13 age- and sex-matched healthy volunteers. Statistically significant differences in FA values of several white matter structures were found not only between iNPH patients and healthy controls but also between iNPH and non-iNPH patients. ROI that showed best diagnostic ability when differentiating iNPH among probable iNPH cohort was uncinate fasciculus, with AUC of 0.74 ( Idiopathic normal pressure hydrocephalus (iNPH) is a clinical syndrome classically characterized by a triad of progressive dementia, gait disturbances, and urinary incontinence, with imaging findings of ventriculomegaly despite normal opening pressures on lumbar puncture . The rep2. iNPH is associated with certain imaging findings [However, clinical features of iNPH are grossly non-specific as individual components of Hakim\u2019s triad are ubiquitous in the elderly population and often coincide without an underlying iNPHfindings , 10 and findings . Howeverfindings \u201314. Therfindings , a magnefindings .Several studies , 18 inveIn order to further investigate the clinical utility of DTI in diagnosis of iNPH, this study focused on differentiation of shunt-responsive iNPH patients from non-responders among a cohort of probable iNPH patients. Intending to achieve better reproducibility, data was co-registered to a standard MNI152 space, regions of interest were extracted using JHU white matter atlas, and FSL\u2019s TBSS pipeline was used for analysis of white matter skeleton.This study was approved by the ethics board of the Military University Hospital Prague. Informed consent was obtained from all patients prior to the inclusion into the database and following procedures.Patients referred to the Military University Hospital Prague between 2017 and 2022 with a working diagnosis of probable iNPH were prospectively included in the study. Criteria for inclusion in the study included (1) age above 40\u00a0years old, (2) insidious onset of symptoms over period of at least 3\u00a0months, (3) ventriculomegaly defined as Evans\u2019 index\u2009>\u20090.3, (4) gait disturbance and at least one of the remaining Hakim\u2019s triad symptoms, i.e., urinary incontinence and/or cognitive deficit, and (5) no other known underlying condition that would account for the symptoms (1). Gait disturbance was evaluated using the Dutch Gait Scale.2O) and/or abnormal laboratory findings in CSF were excluded from the study.After admission, patients underwent a complex diagnostic protocol consisting of clinical, psychological, imaging, and CSF studies. Toward the end of these tests, all patients underwent a lumbar infusion test (LIT). Once the test was completed, lumbar drainage was performed using the same needle, CSF was drained for 120\u00a0h, and clinical evaluation was repeated. Ventriculoperitoneal shunt (VPS) implantation was indicated for all patients with (1) positive result on LIT (resistance to CSF outflow\u2009>\u20099\u00a0mmHg/ml/min) and (2) a minimum of 15% clinical improvement in the Dutch Gait Scale following the 120-h lumbar drainage (LD). If these criteria were not fulfilled, the patient was determined as non-iNPH. Patients with abnormal CSF opening pressure  stationed at the Department of Radiology of The Military University Hospital Prague. Standardized imaging protocol consisted of 3D T1, 3D T2, and DTI sequences, accompanied by a phase-contrast CSF-flow study and fMRI acquisition. The DTI sequence covering the whole brain had the following parameters: FOV 256\u00a0mm, acquisition matrix 128\u2009\u00d7\u2009128, slice thickness 4\u00a0mm, TR 8000\u00a0s, TE 77\u00a0s, and bandwidth 1930\u00a0Hz. Monopolar diffusion scheme with 27 diffusion directions at DTI data were processed using the FSL toolkit  , 20. Pret-tests for independent samples. Multiple intergroup comparisons were performed using ANOVA with subsequent Fisher LSD post hoc testing. Significant predictors were further implemented for their diagnostic accuracy evaluation by calculating sensitivity, specificity, receiver operating characteristic (ROC) curve, and the area under the curve (AUC). All calculations and graphical interpretations were performed in STATISTICA (TIBCO Software) and OriginPro Software (OriginLab Corporation).Differences of categorical variables were standardly evaluated using the chi-square test. Comparisons of two continuous variables were calculated using During the 6-year period, a total of 173 patients with suspected iNPH were prospectively enrolled in the study. Patients with other discernible causes of their symptoms  were excluded. Three patients refused shunt insertion. The final study cohort consisted of 85 participants with adequate data from DTI and CSF functional testing. Out of these 85, 47 patients were diagnosed with shunt-responsive iNPH and indicated for VPS surgery. The remaining 38 patients did not exhibit any significant improvement on LIT and/or LD tests and were considered to have either shunt non-responsive iNPH or another neurodegenerative disorder (collectively referred to as non-iNPH group in the following text for the sake of conciseness). Remaining 13 participants consisted of age- and sex-related healthy controls. Demographic data related to included patients are presented in Table According to the predefined binary groups, i.e., (1) iNPH and healthy controls and 2) iNPH and non-NPH, individual diagnostic ability for each group was calculated. Overview of detailed ANOVA analysis regarding FA values of individual anatomical structures among NPH, non-NPH, and healthy controls is presented in Table  iNPH andBy studying differences between iNPH and healthy controls, statistically significant differences in FA as well as MD were found in the majority of investigated white matter structures. ROI with best discriminating power were the uncinate fasciculus  and cingulate gyrus (AUC 0.83 and 0.84). Figure\u00a0In a clinical setting, iNPH is often a challenging condition to diagnose due to relatively non-specific imaging and clinical features, and currently there is a lack of non-invasive outpatient procedures that can reliably confirm the diagnosis or at least trim the cohort of probable iNPH patients to numbers that would be feasible for thorough inpatient evaluation.This is one of the possible reasons for the focus on MRI in the iNPH-related scientific literature, as a variety of complex evaluations of brain morphology and function are achievable in the outpatient setting. Unfortunately, standard brain MR protocols lack sufficient sensitivity and/or specificity to be a confirmatory or screening test on its own, and therefore search for novel approaches to imaging diagnosis of iNPH is ongoing.A common challenge for application of DTI in the clinical setting is the lack of standardized approaches to extraction of DTI measures. Results reported in this study were achieved using standardized approaches widely employed in the literature (TBSS pipeline), with alignment of FA and MD data into standard MNI152 space and ROI placement according to JHU white-matter atlas. Both MNI152 template and JHU white-matter atlas are accessible open-source references that allow for easy reproduction of the methodology.There is a decent amount of studies  showing UF and CG are considered to be a part of the limbic pathway, and while the former is reported to play a role in certain types of learning  and memory retrieval  , the forThis study suggests that DTI can be a useful tool in iNPH workup, as it has the ability to discriminate not only between iNPH patients and healthy controls but also between iNPH patients and other causes of gait disturbances and cognitive decline. DTI measures of several structures showed high diagnostic ability in differentiating iNPH and non-iNPH, like, e.g., FA values of the UF, which showed sensitivity of 92% and specificity of 67% (AUC of 0.735), which is more than what is usually achievable by MRI evaluation solely based on morphological features \u201314. OtheA common limitation in the research of neurodegenerative disorders is the decreased compliance of these patients, older age, and presence of other conditions often precluding  or confounding  certain diagnostic or therapeutic approaches. In the case of our study, this meant excluding patients with insufficient quality of the imaging data or another pathology found on MRI exam . In this context, however, DTI is a technique relatively resilient to motion artifacts, with possible motion-correction during post-processing, which makes it a suitable method for low-compliance patients.An important thing to consider is also how the diagnosis of iNPH was reached. At our institute, the diagnosis of iNPH was made when (1) the clinical criteria for probable iNPH were met along with (2) clinical improvement after 120-h lumbar drainage  and when (3) no other underlying cause explaining patient symptoms was found. As these criteria were set as relatively strict, it is possible that some patients harboring iNPH in its initial phase were tested as false negatives. A 120-h lumbar drainage was used to maintain diagnostic uniformity and at the same time to compare DTI with the gold standard of iNPH functional testing. Some patients may also improve clinically during cerebrospinal fluid diversion with delay . AdditioiNPH is a challenging condition to correctly diagnose and manage, with novel iNPH-specific biomarkers being in high demand. This study focused on the application of DTI in differentiation of shunt-responsive iNPH patients from other causes of gait disturbances and cognitive decline, refractory to CSF derivation. When using ROIs extracted from standardized JHU white-matter atlas, DTI measures of several white matter regions showed promising results, reaching 92% sensitivity and 67% specificity when differentiating shunt-responsive iNPH patients from patients who fulfilled criteria of probable iNPH but did not improve on CSF derivation. We find these results encouraging and warranting further search for iNPH-specific diffusion MRI biomarkers."}
+{"text": "Violence against healthcare workers is a global health problem threatening healthcare workforce retention and health system resilience in a fragile post-COVID \u2018normalisation\u2019 period. In this perspective article, we argue that violence against healthcare workers must be made a greater priority. Our novel contribution to the debate is a comparative health system and policy approach.We have chosen a most different systems comparative approach concerning the epidemiological, political, and geographic contexts. Brazil (under the Bolsonaro government) and the United Kingdom (under the Johnson government) serve as examples of countries that were strongly hit by the pandemic in epidemiological terms while also displaying policy failures. New Zealand and Germany represent the opposite. A rapid assessment was undertaken based on secondary sources and country expertise.We found similar problems across countries. A global crisis makes healthcare workers vulnerable to violence. Furthermore, insufficient data and monitoring hamper effective prevention, and lack of attention may threaten women, the nursing profession, and migrant/minority groups the most. There were also relevant differences. No clear health system pattern can be identified. At the same time, professional associations and partly the media are strong policy actors against violence.In all countries, muchmore involvement from political leadership is needed. In addition, attention to the political dimension and all forms of violence are essential. Violence against healthcare workers (HCWs) is a persistent and pressing concern, and the COVID-19 pandemic has added new threats. Systematic data and monitoring are still lacking, yet international organisations and mounting individual cases call to action, highlighting sharp increases and qualitatively new dimensions of hate, harassment, and severe violent attacks against HCWs . An incrIncreased violence against the HCWs comes at a critical point in the global health crisis when countries worldwide struggle to meet population health demands due to severe HCWF shortages . Given tThis perspective article brings policy and politics into the debate on violence against HCWs. Available evidence shows that violence was heightened during the pandemic, even in countries with formal democratic institutions and upper-middle to high-resourced healthcare systems. This raises questions as to whether and how institutional/systemic, epidemiological, and pandemic policy conditions shape the debate on violence. Applying a comparative lens and exploring the problem within various countries may help identify policy gaps and develop new policy solutions.We have chosen a most different systems comparative approach concerning the epidemiological, political, and geographic contexts. In our research design , Brazil We rapidly assessed available data, policy responses and actors, and material on the discourse surrounding violence and actions taken against it in the four selected countries. A topic guide served as a framework for the comparative assessment, drawing on country expertise and secondary sources .Our comparative assessment  highlighAvailable data is scattered, and access is generally limited in all countries. Evidence is mainly based on either criminal (police) statistics or surveys, both of which are limited in their ability to tell a holistic story. While pre-COVID survey data exists in New Zealand and the UK, suggesting that violence was a relevant health system problem before the pandemic, a lack of systematic data and monitoring systems makes it difficult to explore to what extent and why violence actually increased during the pandemic. Insufficient empirical evidence hampers a critical debate and the development of effective policy solutions and also opens the door for various forms of interest-driven politics.Strong political leadership and effective policies play a critical role in aiding HCWs. Unfortunately, political leadership in the examined countries has remained sparse; however, health professional associations  have proven to be important and valuable supporters. The nurses\u2019 associations appear to play the biggest supportive role in Brazil, while doctors\u2019 associations take the lead in Germany. The associations in New Zealand and the UK also matter, including hospital organisations and paramedics associations.The policy initiatives among the cases reflect country-specific governance arrangements, particularly centralised vs. decentralised governance structures. The most centralised efforts can be seen in the UK, where the NHS is working to improve data collection and analysis across NHS trusts, propelled by the #WorkWithoutFear campaign. The associations and some regional (L\u00e4nder) governments called for a centralised register system to monitor attacks in Germany. In addition, legal action was taken to improve policy statistics; here, we can observe more decisive action taken on the organisational and operational levels of governance . The other two countries showed limited initiative. Overall, sensitivity to the problem seems to be increasing, yet change is incremental, action is limited to piecemeal work, and actor collaboration is poorly developed . Much more involvement from political leadership is necessary to set the agenda throughout government and society, thereby increasing the likelihood of action and, hopefully, changing the status quo on violence against the HCW.If violence is addressed, this mainly relates to doctors and nurses as the most significant groups, with some country-specific variation. However, health workforce policy primarily focuses on health labour markets and system needs rather than on HCWs as human beings with specific conditions and needs related to age, sex, gender, ethnicity/race, and other social positions. Ignoring the human behind every HCW seriously obstructs the opportunity to protect HCWs better and improve prevention. This creates additional policy gaps exacerbating existing social inequalities in the HCWF, especially in professional groups with more women and migrant HCWs.The connection to the COVID-19 pandemic was substantial, especially in Germany and Brazil, where increased violence against HCWs was most prominent. New Zealand and especially the United Kingdom have faced the challenge of violence well before the pandemic; however, only the latter country has developed the beginnings of a strategy to combat it. We generally observed an overall lack of attention to the political dimension of violence against HCWs. However, there were also some examples of explicit connections to the populist radical right movement in Brazil.Violence against HCWs is and will remain a problem long after the pandemic subsides. If political action is not taken, HCWs will have an additional reason to leave their profession and workplace, and potential candidates will be made to consider the increasing risks of HCWs and pursue a different line of work. In a time when countries across the globe are struggling with HCW retention and recruitment protecting the health and care workforce is essential. Getting support and protection right enhances the retention of the existing workforce and will attract new generations of HCWs. Improved working conditions, mental health, and physical safety of HCWs are an obligation not only of organisations and employers but also of governments and policymakers. This will require governments to prioritise developing feasible and effective policy responses that tackle the many individual risk factors the HCWs face on a daily basis, as well as the health workforce and system-related risks.The original contributions presented in the study are included in the article/EK and MF had the idea, developed the framework, supervised the analysis, and prepared a draft. EK, MF, GL, TT, and AD-J collected the country cases, contributed to the analysis, commented on the draft, and have read and approved the final version. All authors contributed to the article and approved the submitted version.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": "Prostate cancer affects one out of every eight men over the course of their lifetimes, and recurrence of the disease after initial cancer treatment occurs in almost one-third of men. The aim of our review was to assess the role of salvage radical prostatectomy in patients with prostate cancer recurrence after initial treatment with radiotherapy. Outcome data demonstrate that radical prostatectomy has oncologic benefits, although it incurs several intraoperative and postoperative functional risks including rectal injury, erectile dysfunction, and incontinence. Given that there is no clinical consensus on which management approach is superior for patients with localized prostate cancer recurrence, this review provides evidence that radical prostatectomy offers meaningful cancer control and should be considered for select patients with radiation-resistant disease.There are multiple treatment strategies for patients with localized prostate adenocarcinoma. In intermediate- and high-risk patients, external beam radiation therapy demonstrates effective long-term cancer control rates comparable to radical prostatectomy. In patients who opt for initial radiotherapy but have a local recurrence of their cancer, there is no unanimity on the optimal salvage approach. The lack of randomized trials comparing surgery to other local salvage therapy or observation makes it difficult to ascertain the ideal management. A narrative review of existing prospective and retrospective data related to salvage radical prostatectomy after radiation therapy was undertaken. Based on retrospective and prospective data, post-radiation salvage radical prostatectomy confers oncologic benefits, with overall survival ranging from 84 to 95% at 5 years and from 52 to 77% at 10 years. Functional morbidity after salvage prostatectomy remains high, with rates of post-surgical incontinence and erectile dysfunction ranging from 21 to 93% and 28 to 100%, respectively. Factors associated with poor outcomes after post-radiation salvage prostatectomy include preoperative PSA, the Gleason score, post-prostatectomy staging, and nodal involvement. Salvage radical prostatectomy represents an effective treatment option for patients with biochemical recurrence after radiotherapy, although careful patient selection is important to optimize oncologic and functional outcomes. Prostate cancer remains the most common non-dermatologic cancer among men in the United States . With anThe serial evaluation of serum PSA is the primary surveillance method in males who have undergone initial treatment for localized prostate cancer. Despite undergoing definitive treatment for localized prostate cancer, an estimated 20\u201350% of men will experience rising PSA levels with no clinical evidence of disease relapse \u2014also knoAmong men with biopsy-proven post-radiation recurrence, there is currently no consensus on the optimal approach for salvage therapy . CurrentSystematic literature searches in MEDLINE (via Pubmed), Google Scholar, and clinicaltrials.gov databases were undertaken to identify published materials on oncologic and functional outcomes of salvage RP after primary RT for prostate cancer. All articles available in English and published after the year 1993 were considered. Letters, editorials, meeting abstracts, replies from authors, and case reports with fewer than 10 patients were excluded. Given that there is no strong evidence or clinical consensus on which of the several therapeutic options for patients with localized post-radiation recurrence is superior, this review will highlight the advantages and discuss the disadvantages of surgical intervention in previously irradiated patients based on surgical approach and primary radiotherapy type.Our search identified 1331 studies for consideration, including 182 registered clinical trials. After exclusion and screening, 24 total studies were included in our review, including three clinical trials identified via the clinicaltrials.gov register. Flow diagram for study selection according to PRISMA 2020 guidelines is shown in Although no randomized clinical trial data for salvage radical prostatectomy after radiotherapy exist, several multi- and single-center studies have reported both retrospective and prospective oncologic outcomes in this population . The stup = 0.65). Instead, pathologically organ-confined disease was a statistically significant predictor of BFS; seminal vesicle invasion (SVI) and positive lymph nodes were the worst prognosticators [p = 0.0002). Serum PSA > 10 ng/mL also correlated with lower PFS , but the interval between radiotherapy and salvage prostatectomy did not significantly affect PFS [In one of the earliest studies on salvage RP after radiotherapy, Rogers et al. examined 40 patients with clinically localized (T1-T3N0) disease. The authors reported 55% PFS at 5 years, and the only factor significantly predictive of progression was a pre-RP serum PSA level greater than 10 ng/mL . Tefilliticators . In the fect PFS . Taken tp < 0.05), and preoperative PSA > 10 ng/mL was again associated with worse survival (p = 0.007). The interval from RT to surgery did not correlate with PFS [p = 0.014) and metastasis (p < 0.001), suggesting that the ideal salvage RP patients have pre-RP PSA < 4 ng/mL and post-radiation biopsy Gleason scores of 7 or less. Notably, freedom from clinical metastasis was observed in 77% of patients 10 years after salvage prostatectomy [As concerns around the surgical morbidity associated with salvage radical prostatectomy faded, and urologists became less reluctant to perform salvage RP, results of earlier studies were built upon by more contemporary outcomes in the 21st century. Ward et al. retrospectively analyzed outcomes from 199 patients at a single center and reported an overall PFS of 48% at 10 years. Ploidy demonstrated significant predictive power for PFS . The authors found no difference in positive surgical margin rates (p = 0.13) and the pathologic stage after surgery (p = 0.55) when comparing robotic-assisted surgery to open surgery [p < 0.001), 5-year BFS , and 5-year OS  [Studies published within the last five years provide the most comprehensive data on salvage radical prostatectomy outcomes and underscore improvements in surgical technique as well as novel robotic-assisted approaches. In 2019, Mohler et al. published the results of CALGB 9687 (Alliance), the first prospective, multi-institutional, single-arm salvage RP trial. The authors found that the 10-year BFS was 33% and that the 10-year OS was 52% . These l surgery . Catarin surgery . Most re surgery . Of the  = 0.01) . Overallp < 0.001) and potency (p = 0.043) [The high rates of post-surgical incontinence and erectile dysfunction have been major impediments to the acceptance of salvage radical prostatectomy. Functional morbidity associated with salvage radical prostatectomy is significant but has decreased over time with improvement in surgical techniques . For exa= 0.043) .p = 0.02), including the rectal injury rate  [p < 0.001) [Stephenson et al. were the first to illuminate that the functional morbidity associated with salvage RP has declined significantly over time. In a review of 100 cases, the authors found that grade II or greater complication rates had decreased significantly over the twenty-year time period from 1984 to 2003  . Contine = 0.01) . Althoug< 0.001) . This suThe advent of Retzius-sparing robotic-assisted prostatectomy\u2014which historically improves continence rates\u2014 led Mason et al. to examine functional outcomes between 45 traditional robotic-assisted cases and 81 Retzius-sparing salvage prostatectomies. Thirty-day complication rates were lower for the Retzius-sparing group (10% vs. 26%), with 0 rectal injuries. Continence outcomes were also significantly improved in the Retzius-sparing group (59% vs. 38%), although erectile impotency was similar across groups [p = 0.785) or severity  of complications between the robotic and open groups at 90 days [p = 0.709). In a larger review comparing open and robotic-assisted approaches for 395 salvage radical prostatectomies, Gontero et al. reported no differences in overall and major complications  [p = 0.0014) and rectal injuries  were more common for the open RP group while urine leakage was more common for the robotic RP group . Again, the authors found that the robotic group had a lower average blood loss . Ultimately, these studies highlight that, regardless of the operative approach, incontinence remains common and is the greatest disadvantage of undergoing salvage RP. When considering the approach for post-radiation salvage RP, robotic RP seems to incur a lower risk of rectal injury and lower blood loss compared to open, although overall complication rates are statistically similar.In a small but early study of 18 patients who underwent salvage robotic-assisted RP, Eandi et al. reported a relatively low continence rate of 33% but demonstrated that functional outcomes were otherwise comparable to those of open salvage RP. Moreover, there were 0 rectal injuries when using the robotic approach . Other r 90 days . Notably 90 days . From a ctively) . Howeverp = 0.002), while no difference was noted in five-year BFS  [p < 0.001) and a lower complete continence rate (49% vs. 83% pad-free), whereas potency and 3-year BFS were statistically similar between groups [As mentioned previously, primary treatment options for localized prostate include radical prostatectomy, radiation therapy, and focal therapy. As such, salvage RP may be offered in multiple scenarios, namely, post-radiation and post-focal therapy, and there is growing evidence that outcomes after salvage RP may differ based on which primary therapy a patient receives. Onol et al. retrospectively evaluated a group of 126 patients who underwent salvage RP following radiation or focal therapy. Postoperatively, those who received primary focal therapy were noted to have significantly higher continence rates compared to those who received primary RT  . Ribeiron groups . TogetheWhile the presence of nodal involvement has been correlated with negative outcomes, the practice of pelvic lymph node dissection (PLND) at time of salvage RP is not universal. In a retrospective study of the Surveillance, Epidemiology, and End Results (SEER) database, PLND was performed in fewer than 30% of patients undergoing salvage RP . Furtherp < 0.001), and 5-year OS was also higher in men without SVI  [The presence of seminal vesicle invasion may also be a relevant prognosticator for patients undergoing salvage radical prostatectomy after radiation therapy. The rate of SVI is approximately 7% in high-risk prostate cancer, and SVI has been associated with an increased risk of BCR and worse survival ,54. In m= 0.003) . This su= 0.003) . Thus, a= 0.003) .p = 0.004) and were an independent predictor of OS , although they did not predict BFS [One recent study explored the role of a novel biomarker commonly ordered during anesthesia workup, serum cholinesterase (ChE), as a prognostic factor for patients with radiation-recurrent prostate cancer undergoing salvage radical prostatectomy. ChE modulates cellular proliferation and differentiation, and ChE levels are decreased in patients with cancer ,60. Whildict BFS . This hiCompared to salvage focal therapy, salvage radical prostatectomy treats the entire gland, which may improve the prognostic value of post-salvage RP PSA screening. Salvage RP also affords the ability to treat cancer recurrence within the seminal vesicles, which may not be possible using other salvage modalities. Whereas brachytherapy seeds or EBRT fiducial markers may complicate salvage HIFU, salvage RP can also be performed in patients who have previously undergone either EBRT or brachytherapy. Additionally, salvage surgery offers the possibility of performing lymph node dissection, which may aid in restaging. Still, the procedure is extremely technically demanding, and intraoperative and postoperative complications should not be understated. Furthermore, compared to other salvage modalities such as HIFU or cryoablation, salvage RP may increase the risk of incontinence and impotence .The results of the abovementioned studies underscore the clinical utility of salvage radical prostatectomy in patients with radiation-resistant prostate cancer. Nevertheless, there is still a need to improve outcomes and optimize the therapeutic approach in patients with radio-recurrent disease. Despite the demonstrable safety and efficacy of post-radiation salvage RP, important questions remain unanswered. Perhaps most importantly, there are no prospective, randomized control trials evaluating the efficacy or toxicity of salvage RP after radiation compared to other modalities such as HIFU or cryoablation. Furthermore, it is unknown whether the type of primary radiotherapy  impacts functional or oncologic outcomes following salvage RP.While there is a plethora of ongoing clinical trials in the setting of biochemically recurrent prostate cancer, most are focused on improving the detection of recurrent disease or on novel treatment modalities, and there is a relative paucity of current trials specifically examining post-radiation salvage RP. Still, there are several current clinical trials that include patients undergoing post-radiation salvage RP, which will hopefully add to the understanding of salvage surgery after radiation. The Trace-II trial (NCT05555017) is a phase II trial that seeks to assess the feasibility of PSMA-bound radiotracers to guide salvage surgery in patients with BCR. The trial is not yet recruiting but will include patients with local recurrence who have previously undergone primary treatment with either RT or RP, and therefore may represent an alternative to salvage RP. Functional and oncologic outcomes from trials such as this will be key in determining the utility and role of PSMA-radioguided salvage surgery . The VA Salvage radical prostatectomy remains an effective but underutilized therapeutic option for men with localized prostate cancer recurrence after radiotherapy. While it offers long-term oncologic and survival benefits, salvage RP after RT is a technically difficult procedure due to obliterated tissue planes and fibrotic reaction. Although functional outcomes have improved over time, post-radiation salvage RP still poses an elevated risk of erectile dysfunction and urinary incontinence. Given these risks, patient selection is paramount in determining when to offer salvage RP, a recommendation that has found its way into international society guidelines. Patients who are likely to benefit most from salvage RP include those with long life expectancy who have lower preoperative PSA, a lower Gleason score, and lower post-prostatectomy staging and who do not have nodal involvement. As the detection of radio-recurrent disease continues to improve with novel imaging techniques, decision making surrounding salvage therapy will continue to become more individualized, and oncologic outcomes will similarly become more promising."
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