Studies on Viruses in Water

A new procedure for the detection of viral antigens in fecal material was developed. The test is performed by first diluting a fecal sample with phosphate buffered saline to give a liquid consistency. The pH is then adjusted to 8.5-9.0 and the solids are allowed to settle for five minutes. Supernatant fluid from above the fecal sediment is placed on the upper surface of a well of an inverted Immulon microtiter plate and incubated for one hour at 37 degrees C to allow virus to adsorb to the plastic. The Immulon plate is then washed three times with a Tween 20 solution and dried. Adsorbed virus is stained with fluorescein labled antiviral antibody containing Evan\u27s Blue dye. The stained preparations are examined by epi-fluorescence microsopy for the presence of viral aggregates and virus-containing cellular membranes. The test is applied in a continuous water monitoring procedure that can be used to upplement methods in which infectious viruses are isolated from water. In another study a protamine sulfate procedure for concentrating and an immunofluorescent cell procedure for assaying infectious virus (IV, reovirus that is infectious without proteolytic enzyme treatment), and potentially infectious virus (PIV, enzyme enhanceable reovirus) from polluted waters have been developed. The presence of PIV inthe environment had not previously been investigated. In following these procedures, protamine sulfate concentratiosn of 0.005 percent for the first precipitation of the sample, and 0.0025 percent for the second were used. With these protamine concentrations and 0.25 percent fetal bovine serum, IV and PIV are concentrations over 500-fold from river water inoculated with virus. Virus recoveries are between 80 and 100 percent. The IV and PIV fractions are assayed respectively before and after treatment with 200 ug fo chymotrypsin per millileter. When PIV is precipitated from river water, and the precipitate is dissolved and stored at 20 degrees C as a protamine-virus concentrate, only 5 percent of the viral infectivity is lost after 14 days. Therefore, reovirus can be precipitated from water at the sampling site, and only the protamine concentrate needs to be taken to the laboratory to be examined for virus content. When reoviruses are treated with chlorine, PIV is more resistant to inactivation thatn IV, and PIV appears to be at least as resistant to chlorination as poliovirus and coxsackievirus A-2. Granular media filtration systems (i.e., sand, anthracite coal and sand; anthracite coal; sand and garnet) are ineffectual in the removal of the acteriophage MS 2 from water when used as in-line direct filters. Batch assays have indicated a 93 percent reduction of MS 2 can occur when polyelectrolytes are added to the water. In addition, alum concentrations of 20, 30, 40, and 50 mg/1 remove 80 to 98 percent of the virus by precipitation. No reduction of MS 2 was observed at alum concentrations from 1 to 10 mg/1

ERBB and P‐glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P‐glycoprotein

Chemotherapy resistance is a persistent clinical problem in relapsed high-risk neuroblastomas. We tested a panel of 15 drugs for sensitization of neuroblastoma cells to the conventional chemotherapeutic vincristine, identifying tariquidar, an inhibitor of the transmembrane pump P-glycoprotein (P-gp/ABCB1), and the ERBB family inhibitor afatinib as the top resistance breakers. Both compounds were efficient in sensitizing neuroblastoma cells to vincristine in trypan blue exclusion assays and in inducing apoptotic cell death. The evaluation of ERBB signaling revealed no functional inhibition, i.e., dephosphorylation of the downstream pathways upon afatinib treatment but direct off-target interference with P-gp function. Depletion of ABCB1, but not ERRB4, sensitized cells to vincristine treatment. P-gp inhibition substantially broke vincristine resistance in vitro and in vivo (zebrafish embryo xenograft). The analysis of gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a pivotal role of P-gp specifically in neuroblastoma resistance at relapse, while the ERBB family appears to play a minor part

Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.This work is part of the IMPROVE-PD project that has received funding from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement number 812699. M.B. is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—Projektnummer 419826430. R.H. was supported by a research fellowship of the European Renal Association and European Dialysis and Transplant Association (ERA-EDTA). E.L. was supported by the ÚNKP-18-2 New National Excellence Program of the Ministry of Human Capacities, Hungary. E.L. and H.J. were supported by Jellinek-Harry scholarship. S.G.Z. acknowledges the Alexander von Humboldt Stiftung/Foundation for an Experienced Researcher Fellowship (2019–2021) and the International Peritoneal Dialysis Society (ISPD) for an International Cooperation Research Grant (2019–2021). C.P.S. has obtained funding from European Nephrology and Dialysis Institute (ENDI).Peer reviewe

Association of the OPRM1 Variant rs1799971 (A118G) with Non-Specific Liability to Substance Dependence in a Collaborative de novo Meta-Analysis of European-Ancestry Cohorts

Peer reviewe

A large-scale genome-wide association study meta-analysis of cannabis use disorder

Summary Background Variation in liability to cannabis use disorder has a strong genetic component (estimated twin and family heritability about 50–70%) and is associated with negative outcomes, including increased risk of psychopathology. The aim of the study was to conduct a large genome-wide association study (GWAS) to identify novel genetic variants associated with cannabis use disorder. Methods To conduct this GWAS meta-analysis of cannabis use disorder and identify associations with genetic loci, we used samples from the Psychiatric Genomics Consortium Substance Use Disorders working group, iPSYCH, and deCODE (20 916 case samples, 363 116 control samples in total), contrasting cannabis use disorder cases with controls. To examine the genetic overlap between cannabis use disorder and 22 traits of interest (chosen because of previously published phenotypic correlations [eg, psychiatric disorders] or hypothesised associations [eg, chronotype] with cannabis use disorder), we used linkage disequilibrium score regression to calculate genetic correlations. Findings We identified two genome-wide significant loci: a novel chromosome 7 locus (FOXP2, lead single-nucleotide polymorphism [SNP] rs7783012; odds ratio [OR] 1·11, 95% CI 1·07–1·15, p=1·84 × 10−9) and the previously identified chromosome 8 locus (near CHRNA2 and EPHX2, lead SNP rs4732724; OR 0·89, 95% CI 0·86–0·93, p=6·46 × 10−9). Cannabis use disorder and cannabis use were genetically correlated (rg 0·50, p=1·50 × 10−21), but they showed significantly different genetic correlations with 12 of the 22 traits we tested, suggesting at least partially different genetic underpinnings of cannabis use and cannabis use disorder. Cannabis use disorder was positively genetically correlated with other psychopathology, including ADHD, major depression, and schizophrenia. Interpretation These findings support the theory that cannabis use disorder has shared genetic liability with other psychopathology, and there is a distinction between genetic liability to cannabis use and cannabis use disorder. Funding National Institute of Mental Health; National Institute on Alcohol Abuse and Alcoholism; National Institute on Drug Abuse; Center for Genomics and Personalized Medicine and the Centre for Integrative Sequencing; The European Commission, Horizon 2020; National Institute of Child Health and Human Development; Health Research Council of New Zealand; National Institute on Aging; Wellcome Trust Case Control Consortium; UK Research and Innovation Medical Research Council (UKRI MRC); The Brain & Behavior Research Foundation; National Institute on Deafness and Other Communication Disorders; Substance Abuse and Mental Health Services Administration (SAMHSA); National Institute of Biomedical Imaging and Bioengineering; National Health and Medical Research Council (NHMRC) Australia; Tobacco-Related Disease Research Program of the University of California; Families for Borderline Personality Disorder Research (Beth and Rob Elliott) 2018 NARSAD Young Investigator Grant; The National Child Health Research Foundation (Cure Kids); The Canterbury Medical Research Foundation; The New Zealand Lottery Grants Board; The University of Otago; The Carney Centre for Pharmacogenomics; The James Hume Bequest Fund; National Institutes of Health: Genes, Environment and Health Initiative; National Institutes of Health; National Cancer Institute; The William T Grant Foundation; Australian Research Council; The Virginia Tobacco Settlement Foundation; The VISN 1 and VISN 4 Mental Illness Research, Education, and Clinical Centers of the US Department of Veterans Affairs; The 5th Framework Programme (FP-5) GenomEUtwin Project; The Lundbeck Foundation; NIH-funded Shared Instrumentation Grant S10RR025141; Clinical Translational Sciences Award grants; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; National Institute of General Medical Sciences.Peer reviewe

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

Transancestral GWAS of alcohol dependence reveals common genetic underpinnings with psychiatric disorders

Liability to alcohol dependence (AD) is heritable, but little is known about its complex polygenic architecture or its genetic relationship with other disorders. To discover loci associated with AD and characterize the relationship between AD and other psychiatric and behavioral outcomes, we carried out the largest genome-wide association study to date of DSM-IV-diagnosed AD. Genome-wide data on 14,904 individuals with AD and 37,944 controls from 28 case-control and family-based studies were meta-analyzed, stratified by genetic ancestry (European, n = 46,568; African, n = 6,280). Independent, genome-wide significant effects of different ADH1B variants were identified in European (rs1229984; P = 9.8 x 10(-13)) and African ancestries (rs2066702; P = 2.2 x 10(-9)). Significant genetic correlations were observed with 17 phenotypes, including schizophrenia, attention deficit-hyperactivity disorder, depression, and use of cigarettes and cannabis. The genetic underpinnings of AD only partially overlap with those for alcohol consumption, underscoring the genetic distinction between pathological and nonpathological drinking behaviors.Peer reviewe

An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia

Identification of HDAC10 Inhibitors that Modulate Autophagy in Transformed Cells

Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells