There is a variation in the administration of antibiotics prophylaxis to reduce the perceived risk of SSI in patients undergoing non-emergency cholecystectomy. The aim of this study was to determine the effectiveness of antibiotic prophylaxis following non-emergency cholecystectomy to prevent 30-day superficial surgical site infections (SSIs) using non-selected, nationally collected, prospective data.Data were extracted from the CholeS study, which examined and independently validated the outcomes on consecutive patients following non-emergency cholecystectomy across 166 hospitals in the UK and Ireland. Patients who received antibiotic prophylaxis were exact matched to those who did not on variables associated with antibiotic prophylaxis. The primary outcome of interest was superficial SSI, and secondary outcomes included deep SSI, readmissions, complications and re-interventions within 30 days.p = 0.001) and all-cause complications , but similar rates of deep SSI , readmissions  and re-interventions . The number needed to treat to prevent one superficial SSI was 45 .Out of a total of 7327 patients included in the study, 4468 (61%) received antibiotic prophylaxis. These were matched to patients who did not receive antibiotic prophylaxis on a range of demographic and surgical factors, leaving 1269 pairs of patients for analysis. Within this cohort, patients receiving antibiotic prophylaxis had significantly lower rates of superficial SSI  contains supplementary material, which is available to authorized users. There are wide variations in management of patients undergoing non-emergency cholecystectomy. One example is antibiotics prophylaxis administered to reduce the perceived risk of surgical site infections (SSIs) , 2. A reCurrent guidelines from the USA and the UK do not recommend antibiotic prophylaxis in non-emergency cholecystectomy for low- or moderate-risk groups, due to the low risk of developing SSIs and cost to the health care system –7. DespiThe Clinical Variation in Practice of Cholecystectomy and Surgical Outcomes Study (CholeS study) was a multicentre prospective, population-based cohort study of variation in practice of cholecystectomy . It examhttp://www.hra-decisiontools.org.uk/research/) and further supported by written confirmation and advice from the Research and Development Director at University Hospitals Birmingham NHS Foundation Trust, UK.This was a secondary analysis of the CholeS study . The ChoThe study was registered as a ‘clinical audit’ or ‘service evaluation’ at each participating hospital under the supervision of a named senior investigator (consultant surgeon).The CholeS study-enrolled patients undergoing cholecystectomy for benign gallbladder diseases in acute UK and Irish hospitals participating in this study between 1 March and 1 May 2014 were included. This secondary analysis investigated the group of patients undergoing non-emergency cholecystectomy for benign gallbladder diseases. Patients undergoing non-emergency cholecystectomy as delayed operations  or elective operations  were included. Patients undergoing an emergency cholecystectomy were thus excluded. Open, laparoscopic and laparoscopic converted to open surgeries were included. Patients who had a cholecystectomy for known gallbladder cancer or as a part of another surgical procedure, e.g. pancreaticoduodenectomy, bariatric, anti-reflux or transplant operations or an emergency cholecystectomy (defined as a cholecystectomy during an acute admission) were excluded.Superficial SSI: (1) Purulent drainage from the incision; OR (2) At least two of: pain or tenderness; localised swelling; redness; heat; fever; AND. The incision is opened deliberately to manage infection or the clinician diagnoses a surgical site infection; OR (3) Wound organisms AND pus cells from aspirate/swab;Deep SSI: (1) A clinical diagnosis of wound infection with dehiscence of any layer below fat/Scarpa’s fascia; (2) A clinical diagnosis of intra-abdominal collection  with operative or radiological evidence of a collection;Re-interventions: a composite outcome of antibiotics, radiological drainage, re-laparoscopy, laparotomy.The primary outcome of interest was superficial SSI within 30 days. Secondary outcomes included a deep SSI, all-cause readmissions, all-cause complications, all re-interventions and post-operative administration of antibiotics within 30 days. These have been defined previously . BrieflyTo standardise data quality, a quality assurance programme was developed . This in2; <24.9, 25.0–29.9, 30.0–34.9, >35.0), indication . Surgical factors considered were grade of operating surgeon , operative method , operative difficulty  and intm 1 to 4 . Low-rist tests. Nominal variables were analysed using Chi-square tests, with Kendall’s tau used for ordinal variables, to account of the ordering of the categories.Initially, univariable analyses were performed to assess the relationships between antibiotic prophylaxis use, and a range of patient and surgical factors, as well as patient outcomes. Continuous variables were compared using independent samples To adjust for factors found to be associated with antibiotic prophylaxis, multivariable binary logistic regression models were produced for each outcome. Antibiotic prophylaxis was entered into the model as a factor, and all of the other potentially confounding factors were considered for inclusion, with a forward stepwise entry procedure used to select independent predictors of outcome.The data were also analysed using a matched approach. Patients who received antibiotic prophylaxis were exact matched to those who did not on all of the confounding factors considered, with age treated as categorical. The outcomes were then compared across the resulting pairs of patients using McNemar’s test, and odds ratios (OR) with 95% confidence interval (CI) were produced, in order to compare the results to those of the multivariable analysis for consistency.Finally, to test whether the effect of antibiotic prophylaxis differed by patient risk, an additional set of multivariable binary logistic regression models were produced. The patient risk group, antibiotic prophylaxis and the interaction term were entered into the model. One model included the whole cohort of patients, so confounding factors were added to the model using a forward stepwise approach, to give adjusted odds ratios. The analysis was also repeated for the subgroup of matched patients, which did not require this adjustment. The significance of the interaction terms was from these models which were interpreted as testing the difference in the effectiveness of antibiotic prophylaxis in the low- and high-risk groups.All analyses were performed using IBM SPSS 22 . Patients with missing data were excluded on a per-analysis basis, and p < 0.05 was deemed to be indicative of statistical significant throughout.n = 5482, 74.8%) and had a mean age of 51.1 years (SD = 16.3), and antibiotic prophylaxis was administered in 4468 (61.0%) cholecystectomies.Data were collected on 8914 patients undergoing a cholecystectomy between 1 March 2014 and 1 May 2014. Of these, 7400 (83.0%) patients underwent non-emergency cholecystectomy. Antibiotic prophylaxis data were unavailable in 73 cases, which were excluded, leaving 7327 cases for analysis. General patient demographics are presented in Table Patients receiving antibiotic prophylaxis were significantly older and more likely to be male, to have a delayed admission, be operated on by a consultant, have surgeries converted to open and to have greater ASA and operative difficulty Table . AntibioThe numbers of patients at 30 days with a superficial SSI was 147 2.0%), deep SSI 140 (1.9%), all-cause complications 714 (9.7%), post-operative antibiotics use 389 (5.3%) and re-interventions 416 (5.7%). Patients receiving antibiotic prophylaxis were observed to have significantly higher rates of all-cause complications, re-interventions and the requirement for post-operative antibiotics . No significant association was detected between antibiotic prophylaxis and all-cause readmission, complications, deep SSI or re-interventions.In order to account for the effect of selection bias, a set of multivariable analyses were performed to test the impact of antibiotic prophylaxis, after accounting for all of pre- and peri-operative factors in Table p = 0.001), with an odds ratio similar to that from the multivariable analysis . Of patients treated with antibiotic prophylaxis, 0.7% developed SSI, compared to 2.3% in the non-prophylaxis group, giving a number needed to treat (NNT) to prevent one SSI of 63 (95% CI 44–176). The paired analysis also found antibiotic prophylaxis to be associated with a significantly reduced rate of all-cause complications, with an odds ratio of 0.71 , giving a NNT of 45 (95% CI 24–662). The results of the two statistical approaches are reported graphically in Fig. A paired matched approach was also used to further understand the effect of antibiotic prophylaxis. A total of 1269 pairs of patients could be successfully matched on all of the factors in Table p = 0.666 and 0.481, respectively.An additional analysis was performed to test whether the effectiveness antibiotic prophylaxis differed by low-risk (46.7%) or high-risk (53.3%) patients for a SSI Fig. . The effThis study used validated, non-selected population-level data collected as part of the CholeS study –12. In tThe results of this study differ from the current body of evidence of antibiotic prophylaxis in non-emergency cholecystectomy. The majority of randomised studies have showed a small, but non-statistically significant decrease in infective complications when antibiotic prophylaxis is administered. These studies also failed to show a difference in overall SSI rates . The onlTwo recent meta-analyses have been published comparing the effectiveness of antibiotic prophylaxis in non-emergency cholecystectomy. SSIs were detected in 2.4% of patients given antibiotic prophylaxis and 3.2% who were not . This foPopulation-based series have also been analysed. The Swedish Register of Gallstone Surgery and ERCP  study analysed 10,927 patients and found a paradoxical increase in infective complications in the patients given prophylactic antibiotics. This effect was diminished when the results were adjusted for confounding factors, such as age, indication, conversion to open surgery, operative time and gallbladder perforation [The results derived from the CholeS study again show an increase in complications in non-adjusted outcomes. Outcomes were then adjusted for patient and surgical factors that influence the administration of antibiotic prophylaxis. In addition, a 1-to-1 paired analysis was performed, where patients were identically matched such that the only difference between the groups was the administration of antibiotic prophylaxis. Antibiotic prophylaxis reduced the rate of superficial SSI from 2.3 to 0.7%, an effect size that was consistent with the analysis that used multivariable adjustment on the whole cohort. This produced a NNT of 63 and 45 in the multivariable adjusted and matched analyses, respectively. This may reflect the inclusion of higher risk and unselected patients in the CholeS study compared to the randomised studies. Even in this analysis, the effectiveness of prophylactic antibiotics remains small.One explanation for the differences between our results and current evidence is the lack of studies assessing the effect of antibiotic prophylaxis in high-risk groups. The most recent guidelines from both the UK and the USA suggest antibiotic prophylaxis, but state that it should be considered in groups at high risk of SSI, such as having an intra-operative cholangiogram, bile spillage or conversion to open surgery . HoweverBenign gallbladder disease is a major global health burden with an estimated 115 patients for every 100,000 of the world’s population undergoing a cholecystectomy for benign gallbladder disease every year . There iThere are limitations to this study. The data represents a two-month ‘snap-shot’ of practice and may not have fully captured all complications requiring a family doctor attendance. However, it is likely that the proportion of these events is similar in both groups.In addition, this study prospectively collected and independently validated data obtained utilising trainee-led networks in the UK and Ireland. This methodology is powerful and accurate when studying surgical outcomes , 22.Data were not collected on antibiotic type, as this was left to the discretion of the operating surgeon and guided by local hospital policy. Also, it is unknown whether antibiotics were given pre-operatively, for instance in a difficult case, or intra-operatively, for instance when bile or stones are spilled. In the former case, the use of antibiotics is confounded by neither patients nor disease characteristics, while in the latter the administration of antibiotics is causative. This makes it difficult to interpret the association between antibiotics and infections, and suggests that further research is needed in high-risk patients.Despite the current evidence, 61% of the patients in the CholeS data set were administered antibiotic prophylaxis, which is consistent with other population data sets. When factors accounting for antibiotic prophylaxis use were accounted for, either using multivariable adjustment or matched analyses, antibiotic prophylaxis appeared to reduce superficial SSI at 30 days. There was no evidence antibiotic prophylaxis significantly reduced the rates of deep SSIs, readmissions, post-operative antibiotic use or re-interventions at 30 days. The results of this study suggest the need for a high-quality, pragmatic, randomised controlled trial looking at the potential benefit of antibiotic prophylaxis, particularly in high-risk patients.Supplementary material 1 (DOCX 33 kb)Supplementary material 2 (DOCX 33 kb)Below is the link to the electronic supplementary material.


World AIDS Day draws attention to the current status of the human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) epidemic worldwide. The theme for this year’s observance on December 1 is “Focus, Partner, Achieve: An AIDS-Free Generation.”MMWR. Today, an estimated 35 million persons are living with HIV infection , have resulted in approximately 11.7 million persons in low-income and middle-income countries receiving antiretroviral therapy for HIV infection in 2013 (In the United States, nearly 648,500 persons diagnosed with AIDS have died since the first cases were reported , and app


ALK was observed in GSC, but weak overexpression was detected by IHC in three of nine GSCs. Similarly, no MET amplification was found by FISH but three GSCs presented significant immunohistochemical staining. No ALK or MET mutation was found by Sanger's direct sequencing. In this study, we show no molecular rearrangement of ALK, ROS1, and MET that would lead us not to propose, as a valid strategy, the use of crizotinib to eradicate GSCs. However, MET was overexpressed in all GSCs with mesenchymal subtype and three GSCs presented an overexpression of ALK. Therefore, our study corroborates the idea that MET and ALK may assume a role in the tumorigenicity of GSC.Glioblastoma stem cells (GSCs) are believed to be involved in the mechanisms of tumor resistance, therapeutic failures, and recurrences after conventional glioblastoma therapy. Therefore, elimination of GSCs might be a prerequisite for the development of successful therapeutic strategies. ALK, ROS1, and MET are targeted by Crizotinib, a tyrosine kinase inhibitor which has been approved for treatment of ALK‐rearranged non–small‐cell lung cancer. In this study we investigated ALK, ROS1, and MET status in nine glioblastoma stem cell lines and tumors from which they arise. Fluorescent in situ hybridization (FISH), Sanger's direct sequencing, and immunohistochemistry were used to screen genomic rearrangements (or amplifications), genomic mutations, and protein expression, respectively. The immunohistochemical and FISH studies revealed no significant dysregulation of ROS1 in GSCs and associated tumors. Neither amplification nor polysomy of  Primary tumors of the central nervous system (CNS) account for about 2–3% of all cancers in humans and gliomas are the most frequent (70%). In the 2007 WHO classification, glioblastomas (GBM), the highest grade of gliomas (Grade IV) are classified among the group of astrocytic tumors. They represent about 49% of brain tumors in adults, almost 30000 new cases per year in Europe According to many studies, GBMs derive from malignant transformation of stem cells and/or glial precursor cells ALK gene. It was originally developed as an inhibitor of Mesenchymal‐epithelial transition MET but is also active against structurally related tyrosine kinases such as ALK and ROS proto‐oncogene 1 ROS1. It is an orally available ATP‐competitive selective inhibitor that prevents tyrosine phosphorylation on these receptors. Targeted alterations by crizotinib are found in more than 20 different cancers. Since June 2013, as part of a phase 2 clinical trial, the AcSé program of the French National Cancer Institute has been evaluating the efficiency of crizotinib in adult and pediatric cancers presenting an alteration of ALK, MET, or ROS1. This molecule is proposed to patients with cancer in situations of treatment failure and for whom genetic alterations have been identified in the tumor Crizotinib is a targeted therapy agent, which has been approved in adults since 2012 for treatment of non–small lung cancer with an activating translocation of MET gene is mapped to the chromosome region 7q31 and encodes a transmembrane tyrosine kinase receptor closely related in sequence to the insulin receptor. It is expressed by cells of epithelial or endothelial origin. The ligand is the hepatocyte growth factor (HGF) expressed by stroma mesenchymal cells and neutrophils. The HGF/MET activation pathway, essential in embryogenesis, plays a role in cell proliferation and cellular migration, particularly in cases of tissue aggression, in order to restore the integrity of injured tissues The ALK gene (Anaplastic Lymphoma Kinase) is mapped to the chromosome region 2p23.2 and encodes the ALK protein, a tyrosine kinase receptor (RTK) of the insulin receptor family. ALK native transcripts are essentially and transiently expressed during development in specific regions of the central and peripheral nervous systems, such as the thalamus, mid‐brain, olfactory bulb, and peripheral ganglia, and are localized mainly in neuronal cells. As ALK expression is maintained, albeit at a lower level, in the adult brain, it might play an important role in both the normal development and function of the nervous system ALK alterations have been described in tumors: first, intra, or interchromosomic rearrangements leading to formation of a fusion gene having an oncogene activity—the most common fusion partner being EML4 (Echinoderm microtubule‐associated protein‐like 4)—second, genomic amplifications, and, lastly, mutations especially in exons 20–25. Rearrangements and mutations of ALK gene result in ligand‐independent auto‐phosphorylation of the protein and activation of downstream signaling pathways that play a role in cell proliferation and survival. They have been described in anaplastic large‐cell lymphoma, non–small‐cell lung cancer (NSCLC), inflammatory myofibroblastic tumors, diffuse large B‐cell lymphoma, squamous cell carcinoma of the esophagus, and neuroblastoma. ALK amplification has been described in neuroblastoma, NSCLC, rhabdomyosarcoma, esophageal cancer, and endometrial carcinosarcomas. Misense mutations are present in neuroblastoma and anaplastic thyroid cancer. Tumors originating from various organs that harbor ALK abnormalities have been defined as “ALKoma” The ROS1 gene is mapped to the chromosome region 6q22.1 and encodes an orphan transmembrane tyrosine kinase receptor phylogenetically related to ALK and the insulin receptor family. It is expressed transiently in various tissues during development with little to no expression in adult tissues. Overexpression has been reported mainly in meningiomas (55%) and glioblastoma multiform (29%). Intra‐ and interchromosomic rearrangements involving ROS1 have been characterized in cholangiocarcinomas and NSCLC ROS1 gene and the 5′ region of the partner gene generate a gene fusion encoding a chimeric tyrosine kinase protein that initiates an intracellular signal resulting in an oncogenic activation cascade of MAP kinase pathways through phosphorylation of RAS ROS1 rearrangement could provide sensitivity to crizotinib and early clinical studies in ROS1‐positive NSCLC patients brought out promising responses MET, the translocation, amplification, and mutations of ALK, and the translocation of ROS1 have consequently been sought in GSC.The MGMT (O6‐methylguanine‐DNA‐methyltransferase) promoter methylation, EGFR copy number, IDH1, IDH2, EGFR‐variant III, p53, PTEN status, as well as LOH (Loss Of Heterozygosity) at loci 1p36, 19q13, 9p21, and 10q23 was performed and the results are presented in Table Nine cell lines of GSC have been investigated. These cell lines were established and characterized in our laboratory as we previously described in detail From the nine cell lines, Cell MicroArrays (CMA) were established. They were obtained after fixing stem cells in formalin  and paraffin embedding. A 1‐mm‐diameter biopsy core for each of the nine lines was transferred to the recipient block using a TMA workstation   were established using paraffin‐embedded tissue samples from tumor biopsies or surgical removal of the same patients. All specimens had >30% tumor content. To overcome tumor heterogeneity, three biopsy cores of 1 mm diameter were included in the same recipient paraffin block as the CMA Figs. .μm‐thickness section of the TMA/CMA‐embedded paraffin block were produced to achieve immunohistochemistry and Fluorescent in situ hybridization (FISH) experiments for each antibody or probe.Slides with a 3‐ALK immunohistochemistry (IHC) was performed using the prediluted D5F3 monoclonal antibody and the protocol developed by Ventana Rabbit Monoclonal Primary Antibody (ref: 792‐4794). ROS1 immunohistochemistry was performed using the prediluted D4D6 monoclonal antibody and the protocol developed by Genemed Rabbit Monoclonal Primary Antibody (ref: 60‐0180‐7). Cases were assessed as IHC score 0 (no staining), 1+ (faint cytoplasmic staining), 2+ , or 3+  by two expert pathologists. An IHC cytoplasmic staining with an intensity of 1+, 2+, or 3+ on more than 10% of tumor cells was defined as an ALK‐positive result. Any percentage of tumor cells with a cytoplasmic and membrane signal intensity of 1+, 2+, or 3+ was defined as a ROS1‐positive result.MET immunohistochemistry was performed using the prediluted SP44 monoclonal antibody and the protocol developed by Ventana Rabbit Monoclonal Primary Antibody (ref: 790‐4430). Only strong IHC staining with a 2+ or 3+ intensity present in more than 50% of tumor cells was defined as a MET‐positive result.ALK, ROS1, and MET by FISH was conducted using Vysis® LSI ALK Dual Color Break Apart FISH Probe, ZytoLight® ROS1 SPEC Dual Color Break Apart, and ZytoLight® MET/CEN7 Dual Color Probe, respectively. The Zeiss Axio Imager Z2 fluorescent microscope, ×100 objective, and Isis software were used for scoring each case and image acquisition.Analysis of ALK and ROS1 rearrangements were defined as more than 15% split or single signals. Concerning amplification, an average copy number of ALK or MET inferior to 6 was considered negative Reading was carried out in double filter or alternating red and green filters by two experienced molecular biologists trained in interpretation and applying the interpretative guidelines provided by the manufacturer and the published recommendations Genomic DNA from cultured cells and from paraffin‐embedded tissue samples was extracted using the DNAeasy Blood and Tissue DNA isolation kit and QIAamp DNA Mini Kit , respectively. The presence of at least 50% tumor cells in paraffin‐embedded tissue samples had previously been evaluated histologically.ALK gene and exons 14, 16, 17, 18, and 19 of MET gene because mutations in these exons, coding for the protein kinase domains, have been described as conferring sensitivity or resistance to Crizotinib Mutations were investigated by bidirectional sequencing using the standard Sanger method. The primer sequences are available on request. We chose to study exons 23, 24, and 25 of MET genes. There was no correlation between copy number in GSC and in associated tumor samples (P = 0.4 Spearman). Concerning Sanger's sequencing, no mutation was observed by direct sequencing for exons 14 and 16–19 of the MET gene, neither in tumor samples nor in GSC lines.The results of IHC were variable depending on the sample Table , Fig. 2.ALK  showed a weak signal 1+ without any staining of the associated tissue samples. In all cases, FISH showed neither rearrangement nor amplification of , and 9 sROS1 gene on any analyzed spots . Tissue MicroArray (TMA) was established from tumor tissue samples. CMA and TMA are very useful tools to compare samples in the same conditions  and they save valuable biological tissue material.MET gene, including amplification, overexpression, and mutations, have been described in numerous solid tumors and have been associated with a poor prognosis Various studies agree that HGF/MET signaling promotes biological activities, resulting in tumor growth, angiogenesis, and the development of invasive phenotypes, making this receptor an attractive target for potential anticancer treatment MET expression has been associated with cells of the stem compartment in several tissues and overexpression of HGF promotes the acquisition of stem‐like properties and malignant progression of glioma tumor cells et al., who showed overexpression in 29% of glioblastomas in a series comprising 62 cases. This overexpression was associated with poor prognosis with a survival of 11.7 months against 14.3 months for patients whose tumors did not express or had low expression of MET We found overexpression of MET by IHC in two cases of nine tumors, which is comparable to results of Kong MET in all GSC lines and all tissue samples attached to them with a MET/CEP7 ratio close to 1. The average copy number of MET was 5.3 copies/nucleus for tissue samples and was lower with 4.2/nucleus for GSC lines. There was no correlation between copy number in GSC and in associated tumor samples (P = 0.4). In our series, while increased expression in several cases could be explained by polysomy, it was not verified for all cases. However, the case with the largest copy number in terms of tumors and in GSCs had the highest MET overexpression.A polysomy was observed for MET, the target potential of MET alterations including polysomy, gene amplification, and gene mutation has not been well‐established. The clinical data suggest that MET amplification as strictly defined by a MET/CEP7 ratio of >2.2 has the potential to act as an oncogenic driver and thereby to render at least one subset of affected tumors responsive to MET‐TKIs such as crizotinib Although overexpression of MET potentially arises from genetic alteration of ALK rearrangement in GSC lines has never been reported in the literature. No translocations involving ALK have been observed in GSC or in tumors. We have found an expression of ALK in three GSCs and no expression in related tumors, supporting the idea that, consistent with previous reports, ALK plays a role in the maintenance of stemness ALK and its ligand, pleiotrophin, are highly expressed during embryonic brain development and in high‐grade brain tumors (glioblastoma and anaplastic oligodendrogliomas) compared to normal brain tissue and low‐grade tumors and contribute to the growth of glioblastoma ROS1 rearrangement had previously been observed in the glioblastoma U118MG cell line by Charest A et al., who first found a microdeletion on 6q21 region responsible for the fusion of FIG, a gene coding for a Golgi apparatus‐associated protein to the kinase domain of the proto‐oncogene ROS1. The fused protein product FIG‐ROS leads to constitutive kinase activation and results in oncogenic transformation FIG‐ROS1 rearrangement in 268 cases of gliomas. Our data suggested that this particular fusion is not present or has a relatively low occurrence in both high‐grade gliomas  and low‐grade gliomas (<1%) FIG‐ROS1 but all rearrangements involving ROS1. No tumors or GSCs were positive. Concerning the expression of ROS1 in gliomas, the results were contradictory. ROS1 was shown to be overexpressed in 30% of gliomas and dependent upon the methylation of its promoter ALK, ROS1, and MET was found in our study, several GSCs presented MET or ALK overexpression, which corroborates the important role MET and ALK may play in tumorigenicity of GSC.Cancer stem cells identified and isolated from solid tumors are interesting prospects in terms of targeted therapies, especially in glioblastoma; their involvement in the recurrence of the disease does not appear to require any additional demonstration. Even if no molecular rearrangement of None declared.Table S1. Initial pathological and molecular characterization of patients' original glioblastoma.Table S2. Molecular characteristics of glioblastoma stem cells.Click here for additional data file.


CCTOD) for the Two-Parameter Model, and the fracture energy (FG) for the Fictitious Crack Model were examined using the three-point bending notched beam test, according to the RILEM recommendations. The effects of polystyrene aggregate (PA) content and temperature levels on the fracture and mechanical properties of concrete were investigated. The results showed that the mechanical properties of PAC significantly decreased with increase in temperature level and the extent of which depended on the PA content in the mixture. However, at a very high temperature of 800 °C, all samples showed 80 percent reduction in modulus of elasticity compared to room temperature, regardless of the level of PA content. Fracture properties of control concrete (C) and PAC were influenced by temperature in a similar manner. Increasing temperature from 25 °C to 500 °C caused almost 50% reduction of the fracture energy for all samples while 30% increase in fracture energy was occurred when the temperature increased from 500 °C to 800 °C. It was found that adding more PA content in the mixture lead to a more ductile behaviour of concrete.This paper mainly reports an experimental investigation on the residual mechanical and fracture properties of polystyrene aggregate concrete (PAC) after exposure to high temperatures up to 800 degrees Celsius. The fracture properties namely, the critical stress intensity factor ( Light weight, good thermal properties, excellent sound insulation, improved durability and environmental friendliness  [3 densities with corresponding strengths of about 17 MPa minimum [The need for lightweight concretes is rapidly increasing worldwide due to their benefits  over Normal Concretes (NC). Among the various types of proposed lightweight concretes, Polystyrene Aggregate Concrete (PAC) is interesting because they can be tailored to suit specific needs by changing some of their constituents’ properties such as the bead size and volume fraction of polystyrene. In addition, this concrete can be fabricated in construction sites, which is a huge advantage against materials like autoclaved cellular concrete. PAC is a lightweight concrete with a wide range of densities from 1000 to 2000 kg/material) ,5,6,7. H minimum  was desi minimum ,10. The FG) is one of the most important material properties to assess the toughness and brittleness of concrete. The larger the value of FG, the less brittle or the more work is necessary to cause failure. Although a considerable amount of research work has been done on PAC, the information about its fracture properties, in particular the focus on fracture energy under high temperature is very limited. Trussoni et al. [FG) for PAC is considered to be higher than that for NC, as more energy is absorbed when creating more fracture surface in PAC. Sabaa [Fracture energy  and the fracture energy (FG). According to the RILEM Recommendation [A is the area under the load-deflection curve and δ0 is the deflection when the load capacity is zero. g, m, b, d and a0, are gravity factor, mass, width, height and notch depth of the beam. To describe the brittleness of a material, the characteristic length has been suggested as:In the Fictitious Crack Model (FCM), the main parameters are the tensile strength  are also determined from the three-point bending test on a notched beam in accordance with the RILEM recommendation. Firstly, the modulus of elasticity can be obtained from the initial compliance of the load-CMOD curve by usingiC, S, b, d and a0, are the initial compliance, span, thickness, height and initial notch depth respectively as shown in V1 is a geometric function expressed asα = a0/d, in which the thickness of holder of clip gauge was neglected. The critical stress intensity factor  by Jenq and Shah  the crite origin  as shownA material length is also introduced asIn this research, a control concrete (C) and two PAC mixes (PA25 and PA50) were studied. These two PAC mixes were proportioned by replacing 25 and 50 percent of the normal coarse aggregate from the control concrete with an equal bulk volume of polystyrene aggregate respectively. 3, respectively. The materials used in this study were Ordinary Portland cement (OPC) conforming to BS EN 197-1:2001 [The mean diameter and bulk density of the single-size polystyrene aggregate particles were approximately 4 mm and 24 kg/mThe residual properties of concrete specimens after exposure to four temperature levels  were investigated. Heating of specimens in a furnace was performed at a heating rate of 5 °C/min up to the desired temperatures. The hot specimens were then allowed to cool down naturally in room temperature and followed by the mechanical and fracture tests.cf, tensile strength, tf and elastic modulus, E) according to the BS standards [3 (see FG) according to RILEM recommendations [CCTOD) in the Two-Parameter Model were evaluated from the load-CMOD curves which were measured from the three-point bending tests on notched beams, in accordance with the RILEM Recommendation [Standard concrete specimens were cast to study the strength properties  and two PAC mixes PA25 and PA50) are presented against temperature and polystyrene aggregate content in  and PA50The residual compressive strengths of C and PAC mixes increased after heating up to 150 °C and then decreased rapidly at higher temperatures. Similar results were found in other structural lightweight concrete containing fly ash . CastillComparing the elastic modulus values obtained for C, PA25 and PA50 reveals that replacing each 25% of the normal aggregate with polystyrene aggregate lead to almost 30% to 50% reduction in modulus of elasticity depending on the test temperature. The lower stiffness of the polystyrene aggregate compared to the normal aggregate explains this manner. Moreover, a negative correlation is evident between temperature and the elastic modulus. Increasing temperature to 150 °C, caused about 2% decrease in modulus of elasticity for control concrete while elastic modulus of PA25 and PA50 showed respectively near 18% and 22% reduction as the result of the same temperature increase. Concurrent reduction of the modulus of elasticity and the compressive strength, pointed out in the preceding researches ,34, is sThe results presented in The fracture properties of C, PA25 and PA50 were investigated by three-point bending specimens with the same size at varying temperature and polystyrene aggregate content. The typical load-deflection curves at five different temperatures, for control concrete, PA25 and PA50, are presented in Detailed information regarding the fracture properties of control concrete, PA25 and PA50 are provided in The obtained critical effective crack length ratio exhibits erratic behaviour with temperature and polystyrene aggregate content. However, the highest value of The stress intensity factor is used to predict the stress state near the crack tip. The critical value of this factor is used to evaluate the toughness of the cracked specimen under load. The higher the −3/2 for control concrete, PA25 and PA50 respectively. However, the values of The obtained values for C are consistent with those published in literatures . It can perature . Compariu et al.  and Shahu et al.  on increAs it can be seen from 2 for PA25 and between 11.7 and 27 Nm/m2 for PA50. When temperature changes from 500 °C to 800 °C, this parameter increases dramatically for all samples reaching 65.0, 53.0 and 34.1 Nm/m2 for C, PA25 and PA50 respectively.The critical strain energy release rate is the required energy for crack to initiate. Similar to the trend in Q) of the two-parameter model were calculated to study the brittleness of materials. The characteristic length can be described as the length of fracture process zone, while the material length has no direct physical interpretation. The larger the value of these factors, the less brittle the material response to loading. According to The characteristic length (2 respectively at 500 °C. Increasing temperature to 800 °C, the fracture energy of samples begins to grow.Comparing the fracture energy at very high temperature, namely 800 °C, with that at room temperature for all three concretes, it is clear that PAC samples perform better in residual fracture energy so that at 800 °C fracture energy of the control concrete declines to near 60% of its original value at room temperature while both PAC samples show less than 20% reduction in fracture energy compared to the room temperature. The results, presented in Compressive strength, tensile strength and elastic modulus of both PAC samples and the control concrete also reduced under the effect of increasing temperature. However, mechanical properties of PAC samples showed marginally more temperature sensitivity than control concrete.The critical stress intensity factor, the fracture energy and the critical strain energy release rate decreased by increasing the polystyrene aggregate content. It means that less energy is required for crack initiation and crack propagation in PAC samples compared to control concrete. This could be explained by lower aggregate-cement paste bond in PAC in comparison to control concrete.It was observed that PAC samples have higher values of characteristic length. This means replacing more normal aggregate with polystyrene aggregate caused the material failure mode to be more ductile.Increasing temperature affects the fracture properties of all three concretes similarly. Elevating the temperature from 25 °C to 500 °C reduced the amount of In this study the effect of polystyrene aggregate and elevated temperatures on the mechanical and fracture properties of concrete were investigated experimentally. Two samples with 25% and 50% polystyrene aggregate content and a sample of control concrete were studied using three-point bending tests according to RILEM recommendations. Following are the main findings of the current research:


Its frequency varies greatly among ethnic groups and geographic regions ranging from less than 0.1% in Asia to 40% in North Africa. This mutation has three distinct haplotypes; haplotype 1 being the oldest and most common. Recent studies have dated haplotype 1 of the G2019S mutation to about 4000 years ago, but it remains controversial whether the mutation has a Near-Eastern or Moroccan-Berber ancestral origin. To decipher this evolutionary history, we genotyped 10 microsatellite markers spanning a region of 11.27 Mb in a total of 57 unrelated Moroccan PD patients carrying the G2019S mutation for which the Berber or Arab origin was established over 3 generations based on spoken language. We estimated the age of the most recent common ancestor for the 36 Arab-speaking and the 15 Berber-speaking G2019S carriers using the likelihood-based method with a mutation rate of 10−4. Data analysis suggests that the shortest haplotype originated in a patient of Berber ethnicity. The common founder was estimated to have lived 159 generations ago (95% CI 116–224) for Arab patients, and 200 generations ago (95% CI 123–348) for Berber patients. Then, 29 native North African males carrying the mutation were assessed for specific uniparental markers by sequencing the Y-chromosome  and mitochondrial DNA (mtDNA) hypervariable regions (HV1 and HV2) to examine paternal and maternal contributions, respectively. Results showed that the autochthonous genetic component reached 76% for mtDNA (Eurasian and north African haplogroups) and 59% for the Y-chromosome (E-M81 and E-M78), suggesting that the G2019S mutation may have arisen in an autochthonous DNA pool. Therefore, we conclude that LRRK2 G2019S mutation most likely originated in a Berber founder who lived at least 5000 years ago (95% CI 3075–8700).The most common cause of the monogenic form of Parkinson’s disease known so far is the G2019S mutation of the  Indeed, they have been found in about 1.8% of healthy controls, 3.6% of sporadic PD cases and 10% of autosomal dominant familial PD cases [LRRK2 gene so far [In the past two decades, 15 genes have been described for inherited forms of PD. Among them, there are at least 5 confirmed genes responsible for autosomal dominant forms of PD: CA PARK1/, LRRK2 . All patients agreed to participate and were recruited from October 2013 to June 2016 according to criteria described earlier . Age at −4 at each marker per generation was assumed [Genomic DNA was extracted from peripheral blood leukocytes using Isolate II Genomic DNA kit from Bioline. Haplotypes were constructed using the following microsatellite markers: D12S1648, D12S2080, D12S2194, D12S2514, D12S2516, D12S2518, D12S2519, D12S2520, D12S1048 and D12S1301, covering an interval of 11.27Mb. The genotyping of microsatellite markers was performed using fluorescently labeled primers, and the products were pooled and analyzed on a 310 Genetic Analyzer using Genotyper Software version 3.7 (Applied Biosystems). The CEPH DNA sample 1331–01 was used as external standard to control for consistency between runs. We used Phase v2.1.1 software [Since the G2019S mutation can be transmitted by either parent, and the uniparental markers can only be studied simultaneously in males, the 29 males carrying the G2019S mutation  were studied for their Y-chromosome and mtDNA markers. These markers are known to be inherited unchanged from one male or female generation to the next unless mutations occur.The biallelic markers E-M81 and E-M78, reported as being the specific male lineage of autochthonous Berbers of North Africa ,22 and JLRRK2 G2019S mutation, 29 were males and 28 were females. Of these patients, 52 were heterozygous and 5 were homozygous for the mutation. Their ethnic origin was investigated over three generations on the basis of their spoken language (Berber or Arab). Surprisingly, we found that 63% (36/57) of patients with the G2019S mutation were from Arab-speaking families and only 26% (15/57) were from Berber-speaking families for at least three generations. In the six remaining families (11%), the patients’ grandparents were found to speak both languages, and were therefore considered to be of mixed descent.Among the 57 unrelated PD patients carrying the The 36 Arab-speaking and 15 Berber-speaking patients were genotyped for 10 microsatellites around the G2019S mutation; patients of mixed origin were excluded because it is not possible to determine precisely the parent from whom the mutation was inherited, since their DNA was not available for analysis. The shared haplotype was inferred from patients homozygous for the G2019S mutation . This haInterestingly, 75% (38/51) of G2019S carriers showed the shared haplotype of 575 kb in length, which corresponds with the already reported MENA haplotype and is referred to as haplotype 1, indicating that they shared a common ancestor. The shortest haplotype was found in a patient of Berber origin . We estiIn The maternal lineage was evaluated by sequencing the two mtDNA hypervariable regions, HV1 and HV2. Haplotypes and their haplogroup assignment for the 29 G2019S male carriers are presented in Furthermore, analysis of uniparental markers revealed that most males (13/29) carrying the G2019S mutation have both parents of Berber origin. The 4 male patients with the Arab J1-M267 marker have both of the native maternal lineage (mtDNA) haplogroups, H and K.In this study, to determine whether the G2019S mutation has a Berber or an Arab origin in Morocco, the ethnic background of 57 unrelated Moroccan patients carrying the mutation was investigated. Surprisingly, the results suggest that the majority of patients with the G2019S mutation were from Arab-speaking families 63% (36/57) and only 26% (15/57) were from Berber-speaking families for the last three generations. Based on these relative frequencies, the G2019S mutation seems to be of Arab origin. If this is the case, however, one would expect to find the mutation at high frequencies in other Middle-Eastern Arab countries from where the Islamic conquest descended to Morocco and North Africa. The only studies on the G2019S mutation in Arab countries have come recently from Egypt, which reported a frequency among sporadic PD patients of 9.7% (11/113) in the north  and onlyTo address this issue, we estimated the age of the most recent common ancestor in the 36 G2019S Arab-speaking patients and the 15 G2019S Berber-speaking patients separately by genotyping 10 flanking microsatellites. Using best-call haplotypes, we estimated the age of the common ancestor of the 36 Arab-speaking carriers with haplotype 1 to 159 generations ago (95% CI 97–161), and considering an intergenerational time interval of 25 years, this corresponds to about 3975 years. The common founder in the 15 G2019S Berber-speaking patients was estimated to have lived 200 generations ago (95% CI 123–348) corresponding to 5000 years (95% CI 3075–8700). These results, along with the fact that the shortest haplotype was observed in a Berber-speaking patient, indicate that the G2019S mutation might have arisen in Morocco for the first time in a Berber ancestor about 5000 years ago.Our age estimation of the G2019S mutation in Berbers is older than what is reported in recent studies using the same study design. Also considering an intergenerational time interval of 25 years and using the ESTIAGE program, Lesage and collaborators reported an age estimate of about 3000 years using data collected from 67 North African patients and 8 microsatellites (D12S2080 to D12S1301) . Howeverwww.yfull.com/tree/E-M81). This haplogroup, referred to as a genetic Berber marker, reaches a mean frequency of 42% in North Africa, with a decreasing gradient ranging from 98.1% in the southern and Berber regions of Morocco to 11.7% in the north of Egypt [The estimated age of the G2019S mutation for Arab-speaking patients 3975 years) is older than the date of the Islamic conquest, which occurred 1300 years ago. This suggests that current Arab-speaking populations in Morocco are the result of interbreeding between Berbers and Arabs over many generations, and that the majority of people who declare themselves as Arabs are in fact Arabized Berbers ,21,33. Iof Egypt ,35. In tof Egypt , 5% amonof Egypt , but it of Egypt ,38. In E75 years of Egypt .In our sample of G2019S male carriers, the specific autochthonous Y-chromosome haplogroups, E-M81 and E-M78, which coalescence time has been estimated in North Africa to 15000 years , are theHomosapien dating back to 300000 years supports [Since antiquity, the Berber people of the Maghreb have been invaded by many civilizations, all of which have been assimilated to various degrees: Phoenicians, Romans, Vandals, Byzantines and Arabs . Moreovesupports .The G2019S mutation is mainly found in Mediterranean populations with the highest frequencies seen in the Maghreb countries . Among tS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableA: Arab-speaking; B: Berber-speaking; M: Mixed.(XLSX)Click here for additional data file.


IgG4-related disease is a newly recognized fibroinflammatory disorder, characterized by tumefactive lesions, storiform fibrosis and IgG4-positive plasma cells infiltration. IgG4-related sclerosing cholangitis (IgG4-SC) is the most common extrapancreatic manifestation of IgG4-related disease, but it is frequently associated with autoimmune pancreatitis(AIP). Only few case was reported to be diagnosed with IgG4-SC in the absence of AIP, with a striking male preponderance. Here we report a female case of isolated IgG4 related sclerosing cholangitis mimicking cholangiocarcinoma.A 58-year-old woman complaint of one-month history of jaundice and right upper quadrant discomfort, and the biliary reconstruction showed full-length wall thickening and segmental stenosis.Cholangiocarcinoma was then diagnosed.Choledochoplasty was performed, followed by Roux-en-Y anastomosis.However, pathological examination revealed IgG4-related sclerosing cholangitis (IgG4-SC) and the retrospective measurement of serum IgG4 was 346 mg/dL post-operatively. The patient was followed for another nine monthswithout recurrence.The differential diagnosis between cholangiocarcinoma and IgG4-SC is challenging due to significant overlap of clinical manifestations, lab tests and imaging characteristics. However, as an afterthought of this case, typical cholangiocarcinoma rarely presents full-length wall thickening. What the case taught us was pre-operative IgG4 measurement for patients with long bile duct involvement was highly recommended in order to rule out IgG4-SC. The clinical and cholangiographic abnormalities observed in IgG4-SC may resemble those observed in cholangiocarcinoma. Notably, IgG4-SC frequently keeps accompany with concurrent autoimmune pancreatitis (AIP). Only few cases were reported to be diagnosed with IgG4-SC in the absence of AIP, with a striking male preponderance.IgG4-related disease is a newly recognized fibroinflammatory disorder, which is characterized by tumefactive lesions, storiform fibrosis, IgG4-positive plasma cells infiltration as well as frequent but not always elevated serum IgG4 level.2Written informed consent was obtained, and institutional Ethics Committee of the Peking Union Medical College Hospital approved this case report.A 58-year-old female was admitted to hospital, presented with a 1-month history of jaundice, right upper quadrant discomfort, pruritus, pale stools, and dark urine. Laboratory data revealed: total bilirubin 4.6 mg/dL, direct bilirubin 3.3 mg/dL, alanine aminotransferase 314 U/L, alkaline phosphatase 387 U/L, gamma-glutamyl transpeptidase 1190 U/L, and carbohydrate antigen 19-9 71.7 U/mL. Contrast-enhanced computed tomography with biliary reconstruction showed wall thickening of the entire bile duct and segmental stenosis between 2 dilational parts  and involvement of extra-biliary organs also strongly suggest IgG4-SC. Due to the clinical rarity and poor recognition of this entity, it is not surprising that preoperative measurement of serum IgG4 could be missed, especially in those without explicit clinical manifestations in extra-biliary organs. Alternatively, surgical resection was indicated in patients with suspected malignancy, proved to be effective for symptom relief. Notably, relapse occurs in 53% of patients after steroid withdrawal and 44% after surgical intervention.Without therapeutic guidelines, glucocorticoids remain the first-line agents for symptoms improvement and organ functions restoration.Given the age, gender, clinical, and imaging characteristics of this patient, hilar cholangiocarcinoma was the primary suspect. Endoscopic retrograde cholangiopancreatography might fail to examine the whole bile duct due to the full-length stenosis, so diagnostic laparotomy was performed. However, as an afterthought, typical cholangiocarcinoma rarely presents full-length wall thickening and stenosis of bile duct. Thus we propose preoperative IgG4 measurement for patients presented with long bile duct involvement, and IgG4-SC should be considered if elevated IgG4 exists.


MAOA) is associated with alcoholism and smoking behavior. However, the association between rs1137070 and heroin addiction remains unclear. In this study, we examined the allelic distribution of rs1137070 in 1,035 heroin abusers and 2,553 healthy controls and investigated the interactive effects of rs1137070 and heroin addiction on gray matter volume (GMV) based on 78 heroin abusers and 79 healthy controls. The C allele frequency of rs1137070 was significantly higher in heroin abusers. Heroin addiction and the rs1137070 variant interactively altered measures of GMV in the anterior cingulate cortex, orbital frontal cortex, temporal pole, and insula, which were correlated with cognitive function. Heroin abusers with the C allele had lower measures of GMV in these regions than the healthy controls with the same allele, whereas those with the T allele displayed a different trend. The altered brain regions were connected with white matter tracts, yielding a structural network that partially overlapped with the salience network. These findings suggest that the low activity-related C allele of MAOA rs1137070 is associated with an increase in the sensitivity to heroin addiction and the damaging effects of heroin abuse on cognition and the salience network.The rs1137070 polymorphism of monoamine oxidase A ( Drug addiction is characterized by uncontrollable drug intake and drug-seeking behavior. It is a chronic psychiatric disorder with serious consequences, such as loss of productivity, social marginalization, and criminalityMAOA) gene is an X-linked gene that has been associated with various psychiatric disorders6MOAO vary widely among individualsMAOA is crucial for early brain development and has long-term effects on human behavior that may play an important role in the pathogenesis of addictionMAOA knockout mice exhibit impairments in nicotine preference but normal responses to novel stimuliMAOA is an important candidate gene that is responsible for interindividual differences in the susceptibility to substance addiction.Genetic factors contribute 40–60% to the development of addiction and operate at all stages of addictionMAOA is a restriction endonuclease site that is located at position 1460 in the MAOA gene. The C allele of rs1137070 lacks a restriction endonuclease site and is associated with lower MAOA activityThe rs1137070 polymorphism of MAOA rs1137070 and heroin addiction. We first measured the genetic distribution of the rs1137070 polymorphism based on a large sample of heroin abusers and normal controls and then evaluated its effects on gray matter volume (GMV) and neurocognitive performance in a subgroup of subjects with imaging data.We examined the association between The demographic and addictive characteristics of the genetic sample are shown in p = 0.102; heroin: p = 0.188). Significant differences in the allele frequencies of MAOA rs1137070 were found between heroin abusers and healthy controls in males, females, and the entire cohort. The C allele frequency of rs1137070 was significantly higher in heroin abusers than in healthy controls , bilateral temporal pole (TPO), bilateral anterior cingulate cortex (ACC), and right insula INS.R; , Table 2A structural connectivity analysis further revealed that the heroin abusers and healthy controls had different structural connections among these brain regions, measured by mean fractional anisotropy (FA) of white matter fiber tracts. Heroin abusers had stronger ACC-OFC.R, ACC-TPO.R, ACC-INS.R, ACC-OFC (TPO).L, OFC (TPO).L-TPO.R, and OFC (TPO).L-OFC.R connections and a weaker INS.R-TPO.R connection compared with healthy controls. Interestingly, the ACC was a hub node that was significantly connected with all of the other regions , Fig. 3.MAOA rs1137070 polymorphism and heroin addiction and related brain morphological and neurocognitive abnormalities. The results showed that the MAOA rs1137070 had an allelic association with heroin addiction. Furthermore, rs1137070 and heroin addiction interactively contributed to GMV alterations in the OFC, TPO, ACC, and INS.R. These affected brain areas are related to cognition and partially overlap with the salience network13The present study investigated the association between the MAOA rs1137070 was associated an increased susceptibility to heroin addiction. The rs1137070 polymorphism is a synonymous variant, and allelic differences at this position do not alter the amino acid sequence but rather affect the presence or absence of restriction sites and consequent levels of MAOA activityMAOA activity levels were correlated with the number of active enzyme moleculesMAOA rs1137070 were similar to the low-expression variant of the upstream variable number tandem repeat region in the MAOA promoter, which was shown to increase the risk for alcohol addictionThe low-activity C allele of MAOA rs1137070 had neural system-level effects on GM. In the present study, a subset of male subjects were scanned to obtain imaging data because the allelic effect can be assessed more clearly in male subjects who carry a single X chromosome. We found that the decrease in MAOA activity interacted with addictive drug use to decrease GMV, which is consistent with a previous studyMAOA is predominantly localized in catecholaminergic neurons in the primate brainMAOA activity increases the actions of dopamine and other monoamine neurotransmitters that are responsible for drug reinforcement and motivationMAOA genotype on the brain begins in early brain development and may be augmented by drug addiction. We postulated that epigenetic modifications might contribute to the impact of MAOA on GM. Notably, the brain areas that were altered, including the OFC, TPO, INS, and ACC, are predominantly involved in cognitive functionMAOA rs1137070 was associated with both the risk of onset of heroin addiction and also its progression.The present study also found that the brain regions that were interactively affected by rs1137070 and heroin use were connected by white matter tracts, thus yielding a structural network with the ACC as the hub node. The function of the anterior insula and ACC is to segregate the most relevant stimuli among internal and external stimuli to guide behavior, and this function is altered in addiction13The present study was limited by its cross-sectional design; therefore, we could not differentiate the effects of genetic factors during different stages of addiction. The further follow-up study about the rs1137070 effects on drug relapse is desirable. We also did not collect functional magnetic resonance imaging (MRI) data and thus were unable to investigate functional brain networks, although we identified abnormal brain regions that partially overlapped with the salience network.MAOA rs1137070 was significantly associated with the susceptibility to heroin addiction and changes in GM. The low activity-related C allele of rs1137070 was associated with an increased risk of heroin addiction and the deleterious effects of heroin abuse on GM. The brain areas that were found to be altered are involved in neurocognitive performance, and MAOA rs1137070 may play an important role in impaired cognition in heroin abusers. The function of the ACC-centered network may be critical for mediating the genetic effect of MAOA rs1137070 on heroin addiction.In conclusion, we found that the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria for opiate dependence and had no history of poly-substance abuse , with the exception of nicotine dependence. The subjects had been abstinent from heroin for less than 2 years at the time of the study. Healthy subjects were excluded if they had any history of substance abuse or dependence other than nicotine. All of the subjects had no past or current major medical conditions and no major psychiatric disorders other than substance addiction (and no family history of these). Alcohol abusers were also excluded based on the Michigan Alcoholism Screening Test (≤4 points).We recruited 1,035 heroin abusers  from multiple drug addiction treatment centers in China’s Guangdong and Hubei provinces and 2,553 healthy controls  from local communities. Only Han Chinese whose parents were native to southern China were included in the study. Inclusion criteria for the heroin group were the following: met the Magnetic resonance imaging scans and neurocognitive performance were examined in a subset of the initial sample that consisted of 78 male heroin abusers and 79 male healthy controls  who had an ability to understand Mandarin and a level of education that was higher than primary school. Additional exclusion criteria that were applied for subjects who participated in the imaging study included left-handedness and contraindications for the MRI environment .This study was approved by the Peking University Institutional Review Board, and the experiment was performed in accordance with relevant guidelines and regulations. The subjects signed a written informed consent form and were paid for their participation.Genomic DNA was extracted from approximately 200 μl peripheral blood samples using the QIAamp DNA Mini Kit . The single-nucleotide polymorphism was genotyped using the Sequenom Mass Array system (SequenomiPLEX assay). Locus-specific polymerase chain reaction was performed on the DNA samples, and the detection primers were designed using MassARRAY Assay Design 3.0 software . The resulting products were then desalted and transferred to a 384-element SpectroCHIP array, and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy was used for allele detection. The mass spectrograms were analyzed using MassARRAY TYPER software (Sequenom).2, number of averages = 2). T2-weighted images were also acquired to exclude subjects with abnormal brain images, which were checked by two experienced neuroradiologists. Diffusion-weighted images were acquired using a single-shot twice-refocused echo planar imaging sequence .The MRI data were acquired using a GE Signa Twin speed MRI 1.5 T scanner at Zhongshan Traditional Chinese Medicine Hospital. The subjects laid supine with their heads snugly fixed by straps and foam pads to minimize head movements. T1-weighted, sagittal three-dimensional images were acquired with a spoiled gradient recalled echo sequence that covered the entire brain . All T1 scans were segmented into GM, white matter, and cerebrospinal fluid using the New Segment toolboxStructural MRI data were preprocessed using SPM8  using the following steps: eddy-currents and head motion correction, estimation of the diffusion tensors, and calculation of FA. Structural MRI scans were co-registered with their corresponding DTI b0 image so that ROIs that were defined in the structural MRI space could be transformed to DTI space.Diffusion tensor imaging (DTI) data preprocessing was performed using the FMRIB Software Library 5.0.7 , BIS-11 (higher scores indicated greater impulsiveness), and IGT (higher scores indicated better decision-making ability). Additional details are provided in the MAOA is an X-linked gene, we separately analyzed the genetic association in each sex group, and only female subjects were used to perform the test of Hardy-Weinberg equilibrium. Deviation of the genotype counts from Hardy-Weinberg equilibrium was evaluated by the χ2 goodness-of-fit test. The level of significance was set at a two-tailed p < 0.05.Because http://afni.nimh.nih.gov/pub/dist/doc/manual/AlphaSim.pdf; accessed February 1, 2017) with a corrected p < 0.05 and voxelwise threshold of p < 0.025 was used to correct for multiple comparisons, resulting in a cluster size >117 mm. Brain regions with statistically significant morphometric alterations that were derived from the ANOVA were used as ROIs in the structural connectivity analysis. Independent two-sample t-tests were then used to compare the structural connectivity measures between heroin abusers and healthy controls. Post hoc analyses were performed using SAS 9.0 software . Correlation analyses were performed using SPSS 13.0 software.A voxelwise two-way analysis of variance (ANOVA) model was used to explore the interactive effects of genetic variants and addiction status on brain regions in which differences in GMV were found between heroin abusers and healthy controls, with age and cigarette use as regressors. AlphaSim .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death. In patients with advanced or unresectable HCC, there are few treatment options. Conventional chemotherapy has limited benefits. Sorafenib, a multi-kinase inhibitor, improves survival, but options for patients intolerant of or progressing on sorafenib are limited. There has been much interest in recent years in molecular therapeutic targets and drug development for HCC. One of the more promising molecular targets in HCC is the cellular-mesenchymal-epithelial transition (c-MET) factor receptor. Encouraging phase II data on two c-MET inhibitors, tivantinib and cabozantinib, has led to phase III trials. This review describes the c-MET/hepatocyte growth factor (HGF) signalling pathway and its relevance to HCC, and discusses the preclinical and clinical trial data for inhibitors of this pathway in HCC. Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide . TreatmeAt least three recent phase III trials of molecularly targeted agents as second-line treatment in HCC failed to meet their primary endpoints. These trials studied brivanib (BRISK-PS) , everoliThe tyrosine kinase receptor, cellular-mesenchymal-epithelial transition (c-MET) factor receptor, has been studied as a potential therapeutic target, and phase II data with the c-MET inhibitors, tivantinib and cabozantinib, have been encouraging, prompting ongoing phase III trials.In this review, we discuss the c-MET/HGF pathway, its relevance to HCC, and summarise the preclinical and clinical data to date regarding c-MET inhibitors in HCC.The MET proto-oncogene was first identified in an osteosarcoma cell line . The genBinding of HGF to c-MET’s Sema domain leads to receptor homodimerisation, autophosphorylation of tyrosine residues in the tyrosine kinase domain, and downstream activation of the Ras/MAPK, PI3K/Akt, and Ras/Rac/Pho pathways . These pAbnormal activation of HGF/c-MET signalling can occur in several ways ,12:(1) Overexpression of HGFElevation of HGF protein levels, both intratumoural and systemic, has been noted in many tumour types, such as lung cancer (50%), breast cancer (91%), stomach cancer (87%), colon cancer (95%), cancer of the head and neck (45%) and liver cancer (33%) . ElevatePlasma HGF levels have been consistently shown to be higher in patients with HCC compared to controls. Biomarker analyses using samples from the SHARP trial  and phas(2) Overexpression of c-METOverexpression of c-MET in tumour tissue has been noted in many cancers, such as lung cancer, stomach cancer, breast cancer, kidney cancer, colon cancer, and HCC.In HCC, high c-MET expression has also been found to be a poor prognostic marker, correlating with poorly differentiated tumours and lower survival rates ,16. Tumo(3) MET amplificationHigh MET gene copy number can be due to general ploidy status, or true focal gene amplification. MET amplification is less common than overexpression of the protein receptor tyrosine kinase , but has(4) Activating mutationsActivating mutations in c-MET’s tyrosine kinase domain have been reported in hereditary and sporadic papillary renal cell carcinomas , paediatThe interaction between c-MET and HER2 family members is well-documented. MET amplification has been reported to lead to EGFR tyrosine kinase inhibitor (TKI) resistance by HER3-mediated activation of PI3K/AKT signalling in NSCLC ,24. MET c-MET/HGF signalling promotes angiogenesis through increasing vascular endothelial growth factor (VEGF)-A expression and interaction with the VEGF receptor (VEGFR) pathway . It has Small molecule c-MET inhibitors can be classified as selective inhibitors, which specifically target c-MET tyrosine kinase in an ATP-competitive or non-competitive manner, or non-selective inhibitors, which target other kinases in addition to c-MET.Alternatively, blockade of the HGF/c-MET pathway can also be effected through anti-HGF neutralising antibodies, which block only HGF-dependent c-MET activation, or anti-MET antibodies . Anti-HGStudies have shown the role of HGF in enhancing liver regeneration, hepatocyte survival and tissue remodelling after acute injury , and in In human HCC cell lines, c-MET positive cells were noted to have cancer stem cell-like characteristics. Treated with c-MET inhibition however, c-MET positive cells had increased apoptosis, decreased proliferation and suppressed tumour growth, while c-MET reduced cells survived the inhibition treatment. This suggests that c-MET inhibition may be an effective therapy only for selected patients with strong c-MET expression .c-MET pathway activation is also postulated to promote cancer metastasis by inducing epithelial-to-mesenchymal transition (EMT) , which cc-MET inhibitors are sequenced in this section by how extensively they have been investigated in HCC, in descending order. Selected active clinical trials involving these agents are summarised in Tivantinib is an oral non-ATP-competitive selective small molecular inhibitor of c-MET. Binding of tivantinib to c-MET stabilises the receptor in its inactive conformation, hence blocking both ligand-dependent and ligand-independent receptor phosphorylation and thus reducing downstream signalling ,34. Tivaet al. showed that c-MET decreases in tumours treated with tivantinib [Phase I studies for tivantinib, both as monotherapy ,36,37 anet al. [In the phase Ib trial by Santoro et al. , tivantiet al. [The combination of tivantinib and sorafenib was examined in a phase I study of 20 HCC patients by Martell et al. , followiet al. . Of noteet al. performed a pooled analysis to summarise the outcomes of 53 patients with HCC or biliary tract cancer receiving tivantinib in phase I trials [Chai I trials . These ivs. 1.4 month; hazard ratio, HR 0.64). Progression-free survival (PFS) and overall survival (OS) however were not statistically different.Based on the phase I data, tivantinib was studied as a second-line therapy for advanced HCC compared against placebo in a randomised multi-centre phase II trial in 107 patients with Child-Pugh A cirrhosis . All of vs. 1.4 month; HR 0.43; 95% CI 0.18–0.81), median PFS  and median OS . The survival advantage for patients on the lower dose of 240 mg twice daily of tivantinib was at least equivalent to that observed among patients on the higher dose of 360 mg twice daily. Among MET-low patients, however, there was no difference in response rates or survival outcomes between tivantinib and placebo, suggesting that c-MET expression is predictive of response to tivantinib.Pre-specified subgroup analysis according to c-MET expression status was performed, with MET-high defined as more than 50% of HCC cells with 2+ or stronger staining intensity on immunohistochemistry (IHC) . Only thp = 0.0385) [The same authors subsequently showed that the interaction test between treatment and tumour c-MET levels in OS was positive ( 0.0385) . They alSelected phase I and II studies of tivantinib monotherapy are summarised in Extending from the phase II data, there are currently two phase III double-blind, randomised controlled trials that are recruiting patients with advanced HCC and high c-MET-expression to compare tivantinib as second-line treatment against placebo, namely the METIV-HCC trial in the West  and the JET-HCC trial in Japan .In the METIV-HCC trial, the original dose of 240 mg twice daily was reduced to 120 mg twice daily, after a higher than expected rate of neutropaenia and higher than expected drug exposure levels were observed. These were attributed to the switch in formulation from capsules used in the phase II study to tablets ,46. The Cabozantinib is a non-selective oral multi-kinase inhibitor targeting c-MET, VEGFR2, KIT, RET, FLT3 and TIE-2. Cabozantinib has been shown to prolong survival in a c-MET-driven transgenic mouse model of HCC and to show efficacy against human HCC xenografts grown in mice .A phase I dose-escalation study of cabozantinib in 85 patients with advanced solid tumours established the maximum tolerated dose (MTD) at 175 mg daily . Dose-liN = 41) [A phase II randomised discontinuation study evaluated cabozantinib in advanced solid tumours of nine different tumour types, including HCC (N = 41) ,50. The Of note, this study did not evaluate for c-MET expression as a predictor of response to cabozantinib, and given the broad spectrum of targets of cabozantinib, it is unclear how much of the activity is attributable to c-MET inhibition alone. In fact, the combined inhibitory effects of c-MET and VEGF may be particularly effective, which is postulated to be due to upregulated c-MET signalling from VEGF inhibition, either from resultant hypoxia or direct interactions between VEGFR2 and MET .Selected phase I and II studies of cabozantinib monotherapy are summarised in Given the encouraging data from the phase II study, a phase III randomised double-blind study is currently recruiting to compare cabozantinib against placebo as second-line treatment for advanced HCC patients who have previously received sorafenib . EnrolmeCapmatinib is a highly selective c-MET inhibitor. It has demonstrated strong dose-dependent anti-tumour activity and dose-dependent reduction of phosphorylated MET (pMET) levels in c-MET-dependent murine tumour models .In a phase I dose-escalation study, capmatinib was tested in 33 patients with confirmed c-MET-dysregulated advanced solid tumours, with HCC representing the commonest tumour type (45%), which were refractory to current therapy or for which effective therapy was lacking . Stable A phase II trial is currently ongoing testing the efficacy and safety of capmatinib as first-line treatment for patients with c-MET-dysregulated advanced HCC . The trial is actively recruiting patients.There was also a phase II randomised trial for capmatinib as second-line treatment for patients with advanced HCC after sorafenib, but the trial was suspended without any patient recruitment .Tepotinib is a specific, reversible, ATP-competitive c-MET inhibitor.Following encouraging preclinical data in liver cancer models , a phaseThere are currently two phase Ib/II trials ongoing for tepotinib. The first is a single-arm trial evaluating tepotinib as second-line treatment for MET-positive advanced HCC . The other trial is a randomised open-label trial comparing tepotinib against sorafenib as upfront treatment in Asian patients with MET-positive advanced HCC . MET positivity in the trial is defined as moderate or strong protein overexpression on IHC .Foretinib is an ATP-competitive TKI with activity against c-MET, AXL, RON, VEGFR2, TIE-2 and PDGFR. It has been shown to inhibit tumour growth and prolong mouse survival in patient-derived HCC xenograft models .A phase I study of foretinib showed DLT of transaminitis and elevated lipase levels, and common adverse events of hypertension, fatigue, diarrhoea and vomiting, proteinuria, and haematuria . The RP2Foretinib has been studied as first-line therapy in a phase I/II study in Asian patients with advanced HCC. The phase I portion of the study showed ORR of 24%, disease stabilisation rate of 79% and median time to progression of 4.2 months, with no DLT observed at 30 mg once daily .Golvatinib is a non-specific c-MET inhibitor, with activity also against VEGFR2, c-KIT and RON. It has been shown to promote tumour regression and prolong survival in mouse xenograft models .Two dose-finding phase I studies were conducted in patients with advanced solid tumours. The Japanese study found that the MTD was 200 mg twice a day , while tA phase Ib/II clinical trial is currently recruiting, in which the phase II cohort will compare golvatinib plus sorafenib against sorafenib alone as first-line use in patients with advanced HCC . The phase I portion of the trial suggested that the golvatinib and sorafenib combination had manageable toxicity and showed an encouraging 17% of patients with partial responses and durable stable disease in another 31% .Onartuzumab is a monovalent, humanised monoclonal antibody specific for an epitope in the HGF-binding domain of the c-MET receptor. It was developed to overcome the limitation of bivalent antibodies which was thought might cause receptor dimerisation . OnartuzA phase I dose-escalation study of onartuzumab as a single agent and in combination with bevacizumab was carried out in patients with advanced solid malignancies. The maximum tolerated dose was not reached, while the most common drug-related adverse events included fatigue, peripheral oedema, nausea and hypoalbuminaemia . A seconA phase Ib open-label study evaluating onartuzumab as a single agent and in combination with sorafenib in patients with advanced HCC has completed recruitment .Emibetuzumab is a bivalent c-MET-specific monoclonal antibody that blocks HGF binding to c-MET, and neutralises and accelerates internalisation and degradation of the c-MET receptor upon binding, decreasing its level of cell surface expression .A phase I dose-escalation study of emibetuzumab was performed in advanced solid tumours, establishing a RP2D of 750 mg every 2 weeks . No DLT A phase Ib/2 study examining emibetuzumab in combination with ramucirumab in advanced solid tumours including HCC is actively recruiting .versus-host disease, and after surgical procedures.It is unclear whether systemic levels of HGF predict response to anti-HGF/c-MET therapies. HGF levels are also known to be elevated in many other clinical settings including infections, graft-Use of IHC for determination of c-MET protein overexpression has been extensively reviewed ,72, but in vitro, but has not been validated in larger studied in histological specimens.Determination of pMET as a biomarker has been used In the onartuzumab trial , additioAnother possible strategy is the assessment of MET sequence status, including MET mutations, MET amplification, and chromosome 7 polysome .Preclinical studies of different c-MET inhibitors revealed variable efficacy based on the specific MET mutation, e.g., PF-2341066/4217903, has greater activity against certain c-MET ATP-binding site mutations compared to c-MET kinase domain activations . One sucTherapeutic options for patients with advanced HCC intolerant to or progressive on sorafenib are scant. Data from c-MET inhibitors is promising, with phase III trials in progress for tivantinib and cabozantinib.Considering that preclinical and clinical data suggest that the benefit of c-MET inhibition may be restricted to a patient subpopulation with high c-MET expression, future trials may need to be enriched by prospectively incorporating biomarker analyses to validate this hypothesis, so as to better select patients who would benefit from these therapies. Already, the METIV-HCC trial has adopted such a strategy of enrolling only patients with MET-high HCC, and its results are eagerly awaited.


Prevention is a major factor in fighting HIV epidemics in the world. However, a main obstacle for prevention is the lack of knowledge on HIV and its transmission ways, so reinventing ways to spread more awareness about HIV/AIDS is of utmost importance. A novel way to do this is by making use of mass media in order to influence policies and public opinions, to create a supportive environment and finally to enable the prevention of the disease. TV is the main source of information for the general Iranian population, although other sources such as radio and the internet are also used frequently. Unfortunately, information on HIV transmission methods was largely overlooked previously, due to taboos, which resulted in a drastic increase of AIDS infection statistics in Iran , 2. BroaHowever recently, a TV series called” Paria” aired on national media and has effectively changed the taboo surrounding HIV/AIDS. This, coupled with the fact that national media has not paid attention to this disease until now, has made, “Paria” a bold and controversial TV series with a much greater impact on the audience than dialogue-driven programs . In factThis seems to be a positive step for Iran towards achieving the WHO`s goal to eliminate AIDS-related deaths by 2030 around the globe. However, this is only the beginning for Iran and in order to target seriously this goal the Iranian Deputy Minister of Health and the national media broadcaster IRIB must increase cooperation for decreasing infections and finally eliminating “AIDS”. Iran still has a long way to go, but incorporating media in the fight against HIV/AIDS seems to be an unlikely, yet effective, way by which to achieve this.


Salmonella enterica sv. Typhimurium and Escherichia coli were exposed to common co-formulants of formulations, and S. enterica sv. Typhimurium was exposed to active ingredients dicamba, 2,4-D and glyphosate to determine what ingredients of the commercial formulations caused this effect. Co-formulants Tween80 and carboxymethyl cellulose induced changes in response, but the pattern of the responses differed from the active ingredients, and effect sizes were smaller. A commercial wetting agent did not affect antibiotic responses. Active ingredients induced changes in antibiotic responses similar to those caused by complete formulations. This occurred at or below recommended application concentrations. Targeted deletion of efflux pump genes largely neutralized the adaptive response in the cases of increased survival in antibiotics, indicating that the biochemistry of induced resistance was the same for formulations and specific ingredients. We found that glyphosate, dicamba, and 2,4-D, as well as co-formulants in commercial herbicides, induced a change in susceptibility of the potentially pathogenic bacteria E. coli and S. enterica to multiple antibiotics. This was measured using the efficiency of plating (EOP), the relative survival of the bacteria when exposed to herbicide and antibiotic, or just antibiotic, compared to survival on permissive media. This work will help to inform the use of non-medicinal chemical agents that induce changes in antibiotic responses.Herbicides are frequently released into both rural and urban environments. Commercial herbicide formulations induce adaptive changes in the way bacteria respond to antibiotics.  The widespread use of antibiotics has led to antibiotic-resistant human pathogens. Multi-drug-resistant pathogens are now a serious but common occurrence complicating treatment, increasing morbidity and mortality, and resulting in increased costs to health systems . While m‘Adaptive’ responses to antibiotics that temporarily increase the MIC can be caused by changes in gene expression . These oin vitro under laboratory conditions in the absence of such triggers   . Changesml−1)LB] . In brieP values reported in R was used for all statistical analyses of change in EOP (antibiotic effectiveness) or inducing concentration (dose) of ingredients . For the0 scores among herbicide concentrations  experiments, many data points were near or below the detection limit, and consequently the residuals from a standard ANOVA were not always normally distributed. In these cases, a Kruskal–Wallis one-way ANOVA, the equivalent nonparametric test, was used to test for differences in log-transformed EOP/EOPS. enterica exposed to herbicide active ingredients were measured [−1) of a culture on treatment plates to the titre (c.f.u. ml−1) on LB. The limit of detection was determined by the number of bacteria in, and practical volumes of, culture that could be transferred to a Petri plate. This provided a range of 9 orders of magnitude (EOP ≈1-10−8).Responses to the antibiotics ampicillin (Amp), chloramphenicol (Cam), ciprofloxacin (Cip), kanamycin (Kan) and tetracycline (Tet) by measured . BacteriThe series of EOP measurements from high to low reveals the concentrations of antibiotic that affect survival and thus is indicative of antibiotic effectiveness. The active ingredient alone at the concentrations used did not reduce EOP. At low antibiotic concentrations the EOP was high, and it dropped below the detection limit as concentrations approached the MIC. In most cases the addition of active ingredient led to an increase in survival shown by higher EOP measurements at higher concentrations of antibiotic. However, decreases in survival and no change in response for some combinations of antibiotic and ingredient were also observed. The response to a single antibiotic could be different depending on the herbicide to which the bacteria were exposed. For example, glyphosate increased and 2,4-D decreased EOP on medium with Kan. Furthermore, the same herbicide could induce different responses depending on the antibiotic to which the bacteria were exposed. For example, 2,4-D increased EOP on Cam, but decreased it on Kan.3 with or without herbicide exposure  or increases  in survival in all tested antibiotics, with EOP ratios reaching 104–105.At the antibiotic concentration that caused the biggest change in EOP , the min107-fold . The ratWe restricted our comparisons to cases where there is a difference of at least 100-fold between the herbicide and non-herbicide treatments . This crCommercial herbicide formulations contain a range of inert ingredients in addition to the herbicidal compounds . These iS. enterica. However, the low cost of surfactants allowed us to measure the responses of both S. enterica and E. coli.The change in EOP was measured at increasing concentrations of the different antibiotics on medium supplemented or not with co-formulants .For cost3-fold change in EOP (E. coli exposed to CMC*Amp and Tween80*Kan) and the other nine increased the necessary antibiotic concentrations. To quantify the effect, we identified the change in antibiotic concentration when the EOP changed by a factor of ≥103  and Tween80*Cam (<0.05 %), and E. coli exposure to Tween80*Kan (0.5 %) and CMC*Amp (0.25 %).The concentration (or dose) of co-formulant needed to induce a response is presented again as a log-transformed ratio of EOP measurements: (1) EOP on antibiotic at a given co-formulant concentration to (2) EOP on antibiotic without co-formulant. The extremes . Most ofextremes . ExceptiS. enterica antibiotic susceptibility for any of the five antibiotics when it was added at 2 %, 10 times the recommended application level. Any observed differences in EOP were <100-fold and only observed at one antibiotic concentration (data not shown).We also tested the commercial wetting agent Pulse Penetrant which, according to the manufacturer, is used to ‘improve penetration and rainfastness of Roundup herbicide’, and contains organo-modified polydimethyl siloxane as the active ingredient. Pulse Penetrant did not induce significant changes to acrA, acrB, acrD, tolC) and one associated with influx (ompF) were tested in a common genetic background called the Keio collection [E. coli BW25113 (‘wild-type’).Adaptive resistance can be caused by increases in efflux or decreases in influx . This isllection . The KeiTwo antibiotics from different classes, Cip and Tet, and the commercial herbicide formulations Kamba and Roundup, were chosen to represent the response patterns observed previously. The change in EOP was measured at increasing concentrations of the different antibiotics on medium supplemented or not with herbicide.∆ompF and ∆acrD strains compared to the wild-type. A decrease in MIC was observed for strains with a ∆acrA, ∆acrB, or ∆tolC genotype. This effect was much more pronounced for Roundup than for Kamba. For these strains, the Roundup concentration used to measure changes in the EOP at different antibiotic concentrations was reduced from 1240 to 25 ppm ae, a concentration that caused no decrease in EOP by itself (The MIC of each herbicide and antibiotic was measured for each strain (Table S2). No change or an increase in the MIC of the herbicides was observed for the y itself , Fig. 3.P-values for the antibiotic*herbicide interaction term were calculated for each genotype to determine whether the effects of the herbicides on antibiotic response could still be observed when the gene was deleted (2). We express this measure as a percentage, with 0 % indicating that none of the variation in EOP is explained by the interaction term and 100 % indicating that all variability in the dataset is explained by the interaction term. Antibiotics and herbicide ingredients on their own explain the majority of variability in the dataset. The pattern observed for the wild-type resembled that seen for E. coli JB578  and S. marRAB operon increased when E. coli were exposed to the Tet*Kamba combination (Table S3). We chose this combination because resistance to Tet is mediated by the efflux pump TetA [Consistent with the observed role of efflux pumps, transcription of the ump TetA , and Kamump TetA .We show that bacteria exposed to the ingredients of herbicides respond differently to clinically relevant antibiotics. This is important because bacteria can be exposed to herbicides and antibiotics in environments as diverse as the human body, farms and in urban environments such as the lawns and gardens surrounding hospitals and homes. Exposure to herbicide active ingredients in food is regulated. Maximum residue levels (MRLs) of herbicides on food commodities are set by national and international bodies such as the Codex Alimentarius Commission (CAC) of the UN FAO. However, many other potential exposures in both urban and rural environments are not monitored. Thus, both the frequency of herbicide-induced changes in antibiotic resistance in any approved use of herbicides and antibiotics, and the effects of the change escape regulatory oversight.Toxicity testing of herbicides before registration is usually restricted to the active ingredient tested in isolation, and in any case never extends to sub-lethal effects on potential human, companion animal or livestock pathogens. The concentrations of active ingredients at which change to antibiotic response was observed were within application levels, as would be seen in the field or urban environments . They weIt is tempting to directly compare the results obtained for active ingredients with those for the respective commercial formulations. However, the active ingredients have different MICs and solubility compared to the commercial formulations, and hence the concentrations used often differed. For example, the glyphosate concentration in the commercial formulation used was 1240 ppm ae , while iIn commercial use, active ingredients are mixed with co-formulants to improve spreading, absorbing or penetrating properties. Toxicity data for these co-formulants are not usually required by regulators, and residues of co-formulants left by herbicides are not monitored. The concentrations of Tween80 and CMC at which change to antibiotic response was observed were within levels permitted in food. The United States Food and Drug Administration guidelines allow concentrations of Tween80 up to 1 % in some processed foods, where it is used as an emulsifier. Herbicide formulations before dilution are exempt from this concentration limit . HerbiciStreptococcus agalactiae to platensimycin and triclosan was increased by as little as 0.2 % Tween80, while susceptibility to triclosan increased and to platensimycin decreased for MRSA when Tween80 was added [It is noteworthy that the tested co-formulants did not induce the same pattern of antibiotic response observed for any of the herbicide formulations. While we observed mainly decreases in antibiotic susceptibility or no effect induced by the two co-formulants and a commercial wetting agent, increases in susceptibility to some of these co-formulants have been reported elsewhere. For example, susceptibility of as added .E. coli and S. enterica in higher concentrations of the fluoroquinolone antibiotic Cip at exposures of only 2 % Tween80. It is worth investigating whether the choice of co-formulants will influence the frequency of resistance or the efficacy of treatments using these drugs. In addition to use in processed foods, polysorbates are widely used as solubilising agents in products intended for applications that result in chronic human exposures. These products include cosmetics, mouthwash, and medical preparations. Thus, the patchwork of non-antibiotic resistance-inducing chemicals of human origin that microbes routinely encounter may conflict with antibiotic stewardship strategies, undermining goals to substantially reduce antibiotic resistance, or sustain susceptibility, even if antibiotic use were decreased.Adaptive responses leading to antibiotic cross-response have been reported in many bacterial species upon sub-lethal exposure to a variety of substances, including bile salts , weak acacrA (Roundup*Tet), ∆acrB (Kamba*Cip), or ∆tolC (Roundup*Cip). For these combinations, the deleted gene explained most if not all of the adaptive response. No other statistically robust interactions were observed. It is noteworthy that the herbicide concentrations tolerated by these strains were much lower than those tolerated by the wild-type, confirming that adaptive resistance contributes to the intrinsic herbicide and antibiotic response level. This is consistent with an increase in acrB (efflux) and a decrease in ompF (influx) transcripts observed for Tet*Kamba interactions. In contrast, AcrD did not contribute to the response to either antibiotic or Kamba. Because deleting some components of the AcrAB-TolC system did not uniformly impact responses to herbicide/antibiotic combinations, the response of E. coli to the compounds tested probably involves additional genes [mar-dependent and an undescribed mar-independent pathway as suggested by others [The phenotype and transcription assays confirmed that multiple genes respond to herbicide exposures, and some also contributed to adaptive resistance. Several genes known to participate in adaptive resistance were tested in phenotype assays and by transcriptomics. Three strains with gene deletions had a different phenotypic pattern of response compared to the wild-type. These had deletions in ∆al genes . This suy others . The datMultiple strategies are needed to address the antibiotic resistance crisis. While new drugs will certainly be required, invention alone will fail unless we can maintain the effectiveness of antimicrobial agents, both old and new . SustainAntibiotic resistance is influenced by more factors than just exposure of bacteria to antibiotics. These other factors include manufactured products released into the environment. We demonstrated that the pure forms of dicamba, 2,4-D, and glyphosate, as well as common surfactants, can change the susceptibility of bacteria to a diverse range of antibiotics upon concurrent exposure. The inducing concentrations were well within the working concentrations of herbicides to which people, pets and farm animals may be exposed.