Molecular Biology

Introduction : Le Covid-19 est la première pandémie du 21ème siècle. Causée par un virus émergent, le SARS-CoV-2, son diagnostic de confirmation actuel est basé sur la RT-PCR. La présente revue systématique avait comme objectif de décrire la performance des différents tests antigéniques et sérologiques dans le diagnostic du COVID-19 par rapport au Gold standard. Méthodes : La recherche a été réalisée sur les bases de données PubMed et Scopus, jusqu'au 26/04/2020. Ont été inclues les études ayant comparé les performances des tests sérologiques et/ou antigéniques (Sensibilité (Se) et Spécificité (Sp), valeur prédictive positive (VPP) et valeur prédictive négative (VPN), efficacité diagnostique) avec le test de référence RT-PCR dans le diagnostic du Covid-19. Résultats : Treize études remplissaient les critères de recherche sur un total de 2247 participants. Sept études ont été menées en Asie et six en Europe. Ont été identifiés 25 tests diagnostiques dont 22 étaient sérologiques et 3 antigéniques. Leur Se variait entre 18,4 et 100% , tandis que leur Sp variait entre 14,2 et 100%. Leur efficacité diagnostique variait quant à elle entre 66,5 et 99%. Le test de Hoffman et al. basé sur la détection des anticorps anti-Sars-Cov2 de type IgG avait la meilleure sensibilité et spécificité avec des pourcentages respectifs de 93,1% et 99,2%. Conclusion : L'amélioration des performances de ces tests permettrait d'éclairer les stratégies de riposte futures en matière de diagnostic, de prise en charge et de surveillance face à cette urgence sanitaire Mots-clés Covid-19, performance diagnostique, tests sérologiques, tests antigéniques, tests rapides SUMMARY: Background: Covid-19 is the first pandemic of the 21st century. It caused by an emerging virus SARS-CoV-2. Diagnosis of suspected cases is confirmed by nucleic acid assays with real-time PCR (RT-PCR). The objective of this systematic review was to describe the performance of different antigenic and serological tests in the diagnosis of Covid-19 compared to the Gold standard. Methods: The search was performed on the PubMed and Scopus databases up to 26/04/2020. Only studies are comparing the diagnostic performance of serological and/or antigenic tests (Sensitivity (Se) and Specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV), diagnostic efficiency) with the reference test RT-PCR in the diagnosis of Covid-19 were included. Results: Thirteen studies met the search criteria out of a total of 2247 participants. Seven studies were conducted in Asia and six in Europe. Twenty-five diagnostic tests were identified, 22 of which were serological and 3 antigenic. Their Se ranged from 18.4 to 100%, while their Sp ranged from 14.2 to 100%. Their diagnostic efficacy ranged from 66.5 to 99%. The test by Hoffman et al. based on the detection of anti-Sars-Cov2 antibodies of the IgG type had the best sensitivity and specificity with percentages of 93.1% and 99.2% respectively. Improving the performance of these tests would inform future response strategies for diagnosis, management and surveillance in the face of this health emergency.

The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of mRNA fractions transcribed from hFUT2, hGLA and HLA-E transgenes as compared to a lack of such transcriptional activities in non-transgenic ACFC variants. Furthermore, although TSA-based epigenomic modulation has given rise to a remarkable increase in the expression levels of Galα1→3Gal (α-Gal) epitopes that have been determined by lectin blotting analysis, their semi-quantitative profiles have dwindled profoundly in both TSA-exposed and unexposed 3×TG ACFCs as compared to their nTG counterparts. In conclusion, thoroughly exploring proteomic signatures in such epigenetically modulated ex vivo models devised on hFUT2×hGLA×HLA-E triple-transgenic ACFCs that display augmented reprogrammability of translational activities of two mRNA transcripts coding for rhα-Gal A and HLA-E proteins might provide a completely novel and powerful research tool for the panel of further studies. The objective of these future studies should be to multiply the tri-transgenic pigs with the aid of somatic cell nuclear transfer (SCNT)-based cloning for the purposes of both xenografting the porcine cutaneous bioprostheses and dermoplasty-mediated surgical treatments in human patients.

The present study sought to establish the mitotically stable adult cutaneous fibroblast cell (ACFC) lines stemming from hFUT2×hGLA×HLA-E triple-transgenic pigs followed by trichostatin A (TSA)-assisted epigenetically modulating the reprogrammability of the transgenes permanently incorporated into the host genome and subsequent comprehensive analysis of molecular signatures related to proteomically profiling the generated ACFC lines. The results of Western blot and immunofluorescence analyses have proved that the profiles of relative abundance (RA) noticed for both recombinant human α-galactosidase A (rhα-Gal A) and human leukocyte antigen-E (HLA-E) underwent significant upregulations in tri-transgenic (3×TG) ACFCs subjected to TSA-mediated epigenetic transformation as compared to not only their TSA-unexposed counterparts but also TSA-treated and untreated non-transgenic (nTG) cells. The RT-qPCR-based analysis of porcine tri-genetically engineered ACFCs revealed stable expression of ...

The SCAR (Sequence Characterized Amplified Region) 'Sw-421' molecular marker is located at 1.0 cM from the Sw-5 allele, originated from Lycopersicon peruvianum (L.), which confers resistance to the tomato spotted wilt virus (TSWV). However, it had not been tested yet in advanced tomato populations. The goal of this study was to distinguish resistant homozygotes (Sw-5/Sw-5) and heterozygotes (Sw-5/Sw-5+) from susceptible (Sw-5+/Sw-5+) plants in crossing populations with the Stevens cultivar and advanced backcrossing populations by using 'Sw421' SCAR marker. The amplification of 940 bp and 900 bp bands characterized the resistant homozygotes and susceptible controls, respectively. A two band pattern (900 bp and 940 bp) was observed in heterozygote genotypes (Sw-5/Sw-5+), which confirmed the co-dominant inheritance mechanism of the marker. Fifty seven plants from the isogenic progenies were characterized based on bands pattern: 18 plants (31.6%) were identified as resis...

Background: The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated. Results: RT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite-and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2'-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells. Conclusions: GBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.

We report here the effect of replacing one or both of the purine or pyrimidine blocks of a diblock stilbene donor-acceptor capped hairpin with locked nucleic acid (LNA) bases on the dynamics and efficiency of hole transport. The structures of the DNA and LNA:DNA hybrids are tentatively assigned to B-or A-type structures on the basis of their circular dichroism spectra. Replacing the bases in either the A-block or the G-block of the diblock DNA hairpin with LNA bases results in a modest decrease in the base-to-base hopping rate constant and quantum yield for charge separation. Somewhat larger decreases are observed when all of the purine or pyrimidine bases are replaced by LNA bases.

Grasses of the genus Setaria occur in natural and agricultural eco systems worldwide, from the tropical forage S. sphacelata (golden timothygrass) to the invasive S. viridis (green foxtail) and S. verticillata (hooked bristlegrass) populations that can be found in the farthest northern reaches of Canada. Five Setaria species, S. faberii (giant fox tail), S. viridis, S. pumila (yellow foxtail), S. geniculata (knotroot foxtail) and S. verticillata, are problematic weeds 1 . S. viridis is among the most widespread plant species on the planet, and is problematic for crop production due to its repeated evolution of herbicide resistance 2 . In Northern China, around 6,000 BC, foxtail millet was domesticated from S. viridis, and together with proso millet (Panicum miliaceum), became a major cereal crop 3 . Foxtail millet remains a significant crop and dietary staple in this region. It has been suggested, but not proven, that foxtail millet was independently domesticated in Europe a few millennia later . Although the flower structure of S. italica creates a strong bias for inbreeding, the great diversity in S. italica morphology and adaptation suggests that gene flow between S. italica and S. viridis may continue when these two species are grown in sympatry 9,10 . More recently, directed crosses between S. viridis and S. italica have been used for genetic mapping 11 and to introduce herbicide resistance from S. viridis into S. italica breeding germplasm . Recent interest in the S. italica-S. viridis pair (hereafter referred to as "Setaria") has centered on their potential as model species for understanding basic biological processes . Some accessions of S. viridis have short cycling times (6 weeks, seed to seed) and tiny statures (20 cm at maturity), and can be efficiently transformed using Agrobacteriumbased methods. Setaria is also a diploid, tractable model for polyploid biofuel crops like switchgrass (Panicum virgatum) and Napier grass (Pennisetum purpureum). Multiple mapping populations, fosmid libraries and mutagenized populations have been developed for the genus Setaria (some for S. italica, others for S. viridis). Here, the Setaria toolkit is expanded with a highquality, wellannotated reference genome sequence based on Sanger shotgun sequencing, a highresolution genetic map, an endsequenced bacterial artificial chromosome (BAC) library, expressed sequence tags (ESTs) from 13 cDNA libraries and 4 RNASeq libraries. We compared these sequences with the genomes of S. viridis, switchgrass, sorghum and rice to uncover both shared and unique properties of the independent adaptations of several grasses to life on earth. Detailed comparisons of the diploid S. italica with available polyploid switchgrass genome sequences demonstrated the utility of using a closely related diploid genome sequence to aid assembly of a polyploid plant genome.

Agradecer en primer lugar a mis Tutores, Eduardo Osinaga y Agustín Correa, por su dedicación y apoyo durante todo este camino, por compartir sus conocimientos y por siempre estar con buena disposición para sobrellevar dificultades propias del camino de la investigación. En segundo lugar, agradecer a todos los integrantes del laboratorio de Glicobiología e Inmunología Tumoral, por su constante apoyo y ayuda tanto en tareas de laboratorio, como en el apoyo motivacional. Todos han hecho el laboratorio un lugar de trabajo ameno y agradable. También agradecer a los integrantes de la Unidad de proteínas Recombinantes del Instituto Pasteur, quienes siempre estaban dispuestos a ayudar frente a dificultades técnicas o de laboratorio. Al grupo del Instituto de Radiología InRad, Hospital das Clínicas, HCFMUSP, por su colaboración en los trabajos in vivo y por sus aportes que fueron esenciales para el proyecto. Por último, agradecer a todas las personas que de una u otra manera transitaron este camino conmigo. Un especial agradecimiento a mis padres, Verónica Lima y Walker Flores, por siempre acompañar mis decisiones profesionales, y por regalarme un futuro. A Johann Schuller, por siempre alentarme a seguir creciendo y avanzando en lo que me gusta, y por siempre tener buena disposición para ayudarme. Y a mis amigas que siempre me acompañan, aunque sea desde la distancia. Gracias.

Thy28, also known as ThyN1, is a highly conserved nuclear protein. We previously showed that in a chicken mature B cell line, Thy28 binds to the promoter of the gene encoding Pax5, a transcription factor essential for B cell development, and positively regulates its expression. Here, we generated a Thy28-deficient mouse line to analyze its potential role in B cell development in mice. Thy28-deficient mice showed normal development of B cells, and the expression of Pax5 was comparable between wild-type and Thy28-deficient primary B cells. Thus, species-specific mechanisms regulate Pax5 expression and B cell development.

SUMMARYHIV-1 replicates in activated T cells at significantly higher levels than in resting cells. Thus, certain molecules up-regulated during T cell activation appear to be important for HIV-1 replication. In this study, we present evidence suggesting that expression of MHC class II (class II) molecules on CD4+ T cells facilitate HIV-1 replication. T cells that expressed class II supported greater virus replication than T cells lacking class II. The class II+ cells, when either infected with HIV-1 or transfected with an env-minus HIV-1 provirus plasmid, produced 10–20-fold greater virus expression than class II− cells. Anti-class II antibody markedly inhibited virus expression in class II+ cells (but not class II− cells) and also decreased the nuclear binding activity of AP-1, an inducible transcription factor important in T cell activation and HIV-1 expression. Most importantly, the induction of class II expression by transfection of the MHC class II transactivator (CIITA) stimula...

Introduction-Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na + /H + exchange and H 2 O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. Methods-We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. Results-piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. Conclusions-Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

Background: For last five years, as technological advancement occurs, novel kinds of human generation viruses’ discovery have been increased. A rare human cancer associated virus Merkel’s Cell polyomavirus (MCPyV or MCV) got remarkable attraction among the newly discovered viruses. As a common human virus MCPyV infection frequently found in skin and also occurs at other anatomical sites.  Methods:  In this study, the in-silico screening of miRNAs from MCPyV was done, as the computational screening procedures are functionally vital, efficient and inexpensive for the said purpose.  Results:  Primarily 52 sequences, possessing possible hairpin-like structures, were extracted from MCPyV genome by searching through VMir software using various filters. 17 nominees were confirmed with real pre-miRNA like hairpin organizations by iMiRNA-SSF program. Further seven nominees were excluded by free energy measurement and other parameters. 10 mature miRNAs were affirmed in 10 impending candidates...

Major clinical challenges associated with islet transplantation for type 1 diabetes include shortage of donor organs, poor engraftment due to ischemia, and need for immunosuppressive medications. Semipermeable membrane capsules can immunoprotect transplanted islets by blocking passage of the host's immune components while providing exchange of glucose, insulin, and other small molecules. However, capsules-based diffusive transport often exacerbates ischemic injury to islets by reducing the rate of oxygen and nutrient transport. We previously reported the efficacy of a newly developed semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM) under convective-driven transport, in limiting the passage of pro-inflammatory cytokines while overcoming the mass transfer limitations associated with diffusion through nanometer-scale pores. In this study, we report that SNM-encapsulated mouse islets perfused in culture solution under convection outperformed those under diffusive conditions in terms of magnitude (1.49-fold increase in stimulation index and 3.86-fold decrease in shutdown index) and rate of insulin secretion (1.19-fold increase and 6.45-fold decrease during high and low glucose challenges), respectively. Moreover, SNM-encapsulated mouse islets under convection demonstrated rapid glucose-insulin sensing within a physiologically relevant time-scale while retaining healthy islet viability even under cytokine exposure. We conclude that encapsulation of islets with SNM under convection improves islet in vitro functionality. This approach may provide a novel strategy for islet transplantation in the clinical setting.

Many infectious agents can cause central nervous system (CNS) diseases in humans. Since microbial agents infecting CNS are numerous and have different features, conventional laboratory tests may not be sensitive enough to identify and characterise viruses and bacteria in human biological specimens. Thus, the need to define methods for the diagnosis of infectious neurological diseases, such as progressive multifocal leukoencephalopathy (PML), is urgent, in order to improve the outcome of the diseases with rapid and accurate detection of the pathogens.

The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, DacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H 2 O 2 ), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.

The aim of this study was to determine the macrophage migration inhibitory factor (MIF) protein and mRNA expression in esophageal squamous cell carcinoma (ESCC), and the effect of bile acids, aspirin and a selective cyclooxygenase-2 (COX-2) inhibitor, NS398, on MIF expression in ESCC cells in vitro. Specimens from tumors and the adjacent non-cancerous tissues were obtained from 52 ESCC patients. Western blotting was used for the detection of MIF protein expression, and reverse transcription--polymerase chain reaction (RT--PCR) for MIF mRNA expression. Cells of an ESCC cell line, Eca-109, were treated with chenodeoxycholate (CD, 100 mM), glycochenodeoxycholate (GCD, 1 mM), aspirin (1 mM) or NS398 (1 mM). Enzyme-linked immunosorbent assay (ELISA) and RT--PCR were used to detect the expression of the MIF protein and mRNA, respectively, in the supernatant and cultured cells. Western blotting demonstrated that levels of MIF protein were increased in tumors versus non-malignant tissues, with the expression ratio of MIF over b-actin of 0.93 AE 0.21 and 0.57 AE 0.08, respectively (P ¼ 0.012). In vitro, both CD and GCD induced a dramatic increase in MIF protein and mRNA in ESCC cells. On the other hand, aspirin and NS398 significantly decreased MIF protein and mRNA expression, and completely blocked bile acid-induced MIF synthesis in the presence or absence of prostaglandin E 2 . In conclusion, MIF expression is increased in ESCC. Whereas bile acids induce MIF expression in ESCC cells, aspirin and NS398 significantly inhibit MIF expression, even in the presence of bile acids, via a COX-independent mechanism.

Prescribing individualized moderate exercise intensity is a useful method to reach positive effects on health status in obese adults. This study aimed to establish a practical reference equation to estimate the heart rate (HR) at individual ventilatory threshold (IVT) (HR IVT ). One hundred sixty-one obese subjects were clinically evaluated and characterized by anthropometric and body composition. Participants performed the six-minute walking test (6-MWT) and the cardiopulmonary exercise test to assess IVT. Multiple regression analysis for HR IVT , including 6-MWT, anthropometric, and body composition parameters, as independent variables, was performed for both gender separately. A cross-validation study was also performed to determine the accuracy of the prediction equation. Whereas HR IVT was not significantly different between males (121.5±18.3 bpm) and females (117.6±17.1 bpm), it differently correlated with physical and performance parameters. Therefore, two sex-specific equations were developed including 6-MWT HR and HR rest (R 2 = 0.69 and 0.65 and root mean square errors of 8.8 and 10.1 bpm for females and males, respectively). In conclusion, in female and male obese adults, the 6-MWT might be used to predict HR at IVT. These outcomes are useful to prescribe optimal physical activity intensity when gold standard methods (e.g. gas exchange analysis) are unavailable.

The use of the Individual Ventilatory Threshold (IVT), as parameter to prescribe exercise intensity in individuals with obesity, has become more frequent during the last years. This study aimed to evaluate the relationship between IVT and Maximal Fat Oxidation (MFO) in women with obesity. Fifty-two obese female adults (age = 43.6±10.9 years; BMI = 38.5±5.2 kg/m 2 ) were included in this study. According to the BMI classification, subjects were divided into three groups: Obese Class I (OBI, n = 16); Obese Class II (OBII, n = 20) and Obese Class III (OBIII, n = 16). All subjects performed an incremental graded exercise test to evaluate peak oxygen uptake (VO 2peak ), IVT and MFO. MFO was evaluated using a stoichiometric equation. Fat max zone was determined for each subject within 10% of fat oxidation rates at MFO. For each HR, %HR max , VO 2 and %VO 2peak variable, Pearson's correlation test was done between IVT and MFO exercise intensity. When statistical correlation was found we used a comparative statistical analysis to assess differences between IVT and MFO. Statistical significance was set at P � 0.05. For each HR, %HR max , VO 2 and %VO 2peak variable there was a positive significant correlation (P<0.01) between IVT and MFO. No significant differences were found for HR, % HR max , and VO 2 between IVT and MFO. %VO 2peak was significantly higher at IVT than at MFO (P = 0.03).

Prescribing individualized moderate exercise intensity is a useful method to reach positive effects on health status in obese adults. This study aimed to establish a practical reference equation to estimate the heart rate (HR) at individual ventilatory threshold (IVT) (HRIVT). One hundred sixty-one obese subjects were clinically evaluated and characterized by anthropometric and body composition. Participants performed the six-minute walking test (6-MWT) and the cardiopulmonary exercise test to assess IVT. Multiple regression analysis for HRIVT, including 6-MWT, anthropometric, and body composition parameters, as independent variables, was performed for both gender separately. A cross-validation study was also performed to determine the accuracy of the prediction equation. Whereas HRIVT was not significantly different between males (121.5±18.3 bpm) and females (117.6±17.1 bpm), it differently correlated with physical and performance parameters. Therefore, two sex-specific equations w...

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university, Amsterdam, the ~etherlakls. Liver is the main organ in the elimination of 5-fluorouracil (5FU). Rat hepatocytes and hepatoma cells (H35) were incubated in monolayer in the presence of 0, 17, 50 and 100 pM 5 N for 4 hrs. The metabolism of 5 N was monitored with [3H]5FU, the protein synthesis was determined by the incorporation of ["~Ileucine and glycoconjugate synthesis by incorporation of ['HIG~CNH and ['~Jfucose. In rat hepatocytes the amount of soluble fluironucleotides was dependent on the concentration of 5 N . With 50 pM 5 N the concentrations of NDP-HexNAc, NDP-hexose and FLPrP were 17, 5 and 9 p1/106 hepatocytes. In the presence of 0.5 mM thymine to inhibit the catabolism of 5 N , these concentrations were 23, 29 and 49 pml/lOC hepatocytes, resp. The effect of thymine at 17 and 100 PM 5 N was comparable. The incorporation of 5 N into FaiA was also concentration dependent and was 72 pl/106 hepatocytes at 50 pM 5 N . In contrast to nucleotide synthesis thymine enhanced the incorporation of 5 N into RNA only less than 1.5 fold at all 5 N concentrations. In H35 cells both synthesis of soluble fluoronucleotides and the incorporation of 5 N into FaiA was linear with the concentration of 5 N . At 50 pM 5 N the amounts of NDP-HexNAC, FUDP-hexose and NTP were 30, 68 and 106 pmol/106 cells, and 345 p l 5FU/106 cells was incorporated into RNR. In both cell types incubation with 5 N had no effect on the incorporation of leucine into proteins and of sugars into glycoconjugates.

AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit crossspecies immunoreactivities (AMPD1, rat isoform A and human isoform M ; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5h ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89 % and 93 % identical respectively, although the rat open reading frame is lacking two

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = -164±5 mV, pKox1,2=7.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 ≈ -150 mV, pK...

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins 1-4 . In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N 2 ; <2 ppm O 2 ) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Unfavorable neuroblastomas (NBLs) achieve telomere stabilization via telomerase activation through MYCN amplification, TERT promoter region (TERT-PR) rearrangements, or alternative telomere lengthening of telomeres. No well-established methods are available for investigating TERT-PR rearrangements. We examined the relationship between and prognosis by fluorescence in situ hybridization (FISH) upstream and downstream of TERT to establish a simple analysis method. TERT-PR rearrangements were analyzed in 3 M MYCN amplified cases and, 11MYCN non-amplified cases (1 MS case, 1 L2 case and 2 M cases less than 18 months, and 1 L2 case and 6 M cases over 18 months old at diagnosis) to determine if MYCN and TERT-PR rearrangement were independent prognostic factors. In total, 14 patients (11 males, 3 females; median age 36.4 months, range 1–122 months) with NBLs were evaluated at Hiroshima University. We identified MYCN amplification, TERT expression, and TERT-PR rearrangements. TERT-PR rearra...

Single-cell sequencing of circulating tumor cells can precisely represent tumor heterogeneity and provide useful information for cancer treatment and research. After spiking TGW neuroblastoma cells into blood derived from healthy volunteer, the cells were isolated by fluorescence-activated cell sorting. DNA and mRNA were amplified by four different whole-genome amplifications (WGA) and three whole-transcriptome amplifications (WTA) methods, followed by single-cell DNA and RNA sequencing. Multiple displacement amplification (MDA)-based WGA methods showed higher amplification efficiency than other methods with a comparable depth of coverage as the bulk sample. The uniformity of coverage greatly differed among samples (12.5–89.2%), with some samples evaluated by the MDA-based WGA method using phi29 DNA polymerase and random primers showing a high (&gt; 80%) uniformity of coverage. The MDA-based WTA method less effectively amplified mRNA and showed non-specific gene expression patterns....

Une série d’expérimentations a été conduite pour mesurer l’impact de la monotraite à l’échelle de la lactation de brebis laitières de race Lacaune. La production de lait et sa composition, la physiologie et la morphologie de la mamelle, l’ingestion et le métabolisme énergétique ont été mesurés tout au long de la période de traite, du sevrage au tarissement. L’impact de la monotraite a été modélisé à l&#39;échelle de l&#39;exploitation et du bassin de production. La perte de lait a été de 14 % chez les multipares comme les primipares. Selon la ration offerte, le taux butyreux a diminué ou a eu tendance à augmenter, alors que le taux protéique n’a pas été modifié ou a augmenté en lien avec une augmentation des protéines solubles. Les quantités ingérées n’étaient pas différentes entre brebis en traite biquotidienne et monotraite, ce qui a entraîné un bilan énergétique positif supérieur pour ces dernières. Cela s’est traduit par des taux d’acides gras non estérifiés rapidement plus bas ...

RESUME -L'effet de la monotraite a été testé sur deux périodes successives de 9 semaines (P1 et P2) chez 48 chèvres Alpine réparties en 4 lots (un lot 2 traites/j et un lot monotraite sur les 2 périodes ; un lot 2 traites/j en P1 puis monotraite en P2 et un lot conduit à l'inverse). Aucun trouble de comportement ni aucune perte de lait au sol n'a été observé en monotraite. La réduction de production laitière est estimée à 15 % (406 vs. 345 l sur P1+P2). La perte est de 10 % environ en matière totale produite suite à un enrichissement du lait (+2 g/l [p<0,03] et +3,5 g/l [P<0,001] de TP et -1,2 g/l [NS] et +3,5 g/l [P<0,06] de TB respectivement sur P1 et P2). Les CCS moyens en monotraite restent faibles (<604 000 cellules/ml), mais ont tendance à s'accroître par rapport à 2 traites/j (+135 000 cellules/ml en P1 ; P=0,07 et +237 000 cellules/ml en P2 ; P<0,10). La monotraite induit une réduction de la lipolyse (1,09 à 0,54 meq/l ; P=0,02) alors que le rendement fromager augmente de 14,6 à 16 % (P<0,02) en P2. Les faibles modifications de concentration en ions Na/K et en albumine dans le lait et le bon taux de recouvrement du potentiel initial lors du retour à 2 traites/j en P2 (98 %) suggèrent une faible atteinte de l'intégrité de la mamelle et de son potentiel sécrétoire lors de la monotraite. L'aptitude des chèvres Alpine à la monotraite, sa facilité de mise en oeuvre et son faible impact économique font de la monotraite une solution d'avenir aux problèmes de temps de travail.

High-throughput sequencing was applied to investigate the mutation/methylation patterns on 1q and gene expression profiles in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) with/without (w/wo) dup(1q). Sequencing of the breakpoint regions and all exons on 1q in seven dup(1q)-positive cases revealed non-synonymous somatic single nucleotide variants (SNVs) in BLZF1, FMN2, KCNT2, LCE1C, NES, and PARP1. Deep sequencing of these in a validation cohort w (n = 17)/wo (n = 94) dup(1q) revealed similar SNV frequencies in the two groups (47% vs. 35%; P = 0.42). Only 0.6% of the 36,259 CpGs on 1q were differentially methylated between cases w (n = 14)/wo (n = 13) dup(1q). RNA sequencing of high hyperdiploid (HeH) and t(1;19)(q23;p13)-positive cases w (n = 14)/wo (n = 52) dup(1q) identified 252 and 424 differentially expressed genes, respectively; only seven overlapped. Of the overexpressed genes in the HeH and t(1;19) groups, 23 and 31%, respectively, mapped to 1q; 60-80% of these encode nucleic acid/protein binding factors or proteins with catalytic activity. We conclude that the pathogenetically important consequence of dup(1q) in BCP ALL is a genedosage effect, with the deregulated genes differing between genetic subtypes, but involving similar molecular functions, biological processes, and protein classes.

High-throughput sequencing was applied to investigate the mutation/methylation patterns on 1q and gene expression profiles in pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) with/without (w/wo) dup(1q). Sequencing of the breakpoint regions and all exons on 1q in seven dup(1q)-positive cases revealed non-synonymous somatic single nucleotide variants (SNVs) in BLZF1, FMN2, KCNT2, LCE1C, NES, and PARP1. Deep sequencing of these in a validation cohort w (n = 17)/wo (n = 94) dup(1q) revealed similar SNV frequencies in the two groups (47% vs. 35%; P = 0.42). Only 0.6% of the 36,259 CpGs on 1q were differentially methylated between cases w (n = 14)/wo (n = 13) dup(1q). RNA sequencing of high hyperdiploid (HeH) and t(1;19)(q23;p13)-positive cases w (n = 14)/wo (n = 52) dup(1q) identified 252 and 424 differentially expressed genes, respectively; only seven overlapped. Of the overexpressed genes in the HeH and t(1;19) groups, 23 and 31%, respectively, mapped to 1q; 60-80% of...

Antibiotics are the chemical or biochemical moieties that specifically and effectively inhibit the growth of a pathogen but not the host organism and thus are employed in the treatment of the infection by the pathogen. However, the survival pressure in the presence of antimicrobial agents forces a minor fraction of the bacterial population to evolve mechanisms that evade the inhibitory effects of the administered antibiological agent, thereby emerging as a drug-resistant variety and consequently challenging the treatment regime. Additionally, the emergence of multidrug-resistant (MDR) variants of the pathogens that are capable of resisting several structurally dissimilar drugs is becoming very common and resulting in the infections that are difficult or impossible to treat. To counter the vast array of chemically and structurally dissimilar antibiotics within their lifestyle boundaries, different bacteria have developed different antibiotic-and pathogen-specific resistance mechanisms. Although distinct and involving several molecular events, these mechanisms can be broadly classified into two modes, the intrinsic and the acquired modes of resistance, which are further classified into subclasses. This chapter reviews recent advances and current understanding of the molecular details of these mechanisms.

Accurate diagnosis of tuberculosis, especially by using rapid molecular assays, can reduce transmission of drug resistant tuberculosis in communities. However, the frequency of resistance conferring mutations varies with geographic location of Mycobacterium tuberculosis, and this affects the efficiency of rapid molecular assays in detecting resistance. This has created need for characterizing drug resistant isolates from different settings to investigate frequencies of resistance conferring mutations. Here, we describe the prevalence and patterns of rifampicin-and isoniazid-resistance conferring mutations in isolates from Uganda, which could be useful in the management of MDR-TB patients in Uganda and other countries in sub-Saharan Africa. Ninety seven M. tuberculosis isolates were characterized, of which 38 were MDR, seven rifampicin-resistant, 12 isoniazid-mono-resistant, and 40 susceptible to rifampicin and isoniazid. Sequence analysis of the rpoB rifampicin-resistance determining region (rpoB/RRDR) revealed mutations in six codons: 588, 531, 526, 516, 513, and 511, of which Ser531Leu was the most frequent (40%, 18/45). Overall, the three mutations (Ser531Leu, His526Tyr, Asp516Tyr) frequently associated with rifampicin-resistance occurred in 76% of the rifampicin resistant isolates while 18% (8/45) of the rifampicin-resistant isolates lacked mutations in rpoB/RRDR. Furthermore, sequence analysis of katG and inhA gene promoter revealed mainly the Ser315Thr (76%, 38/50) and C(-15)T (8%, 4/50) mutations, respectively. These two mutations combined, which are frequently associated with isoniazid-resistance,

Monilethrix is a rare autosomal dominant disease characterized by hair fragility and hyperkeratotic papules. Mutations in type‐II hair specific keratins hHb6 and hHb1 have recently been reported. We describe a large family with a E410D mutation in the evolutionary conserved helix termination motif of keratin hHb6 that was variably expressed among 12 heterozygous members, and severely expressed among 3 homozygous members. These 3 patients had essentially complete lack of scalp hair since the age of 2 months with no improvement over time as well as follicular keratotic involvement extensively expressed over the scalp and large body areas. The variability seen in heterozygous patients, along with seasonal and pregnancy‐related improvement suggest that other genetic or environmental factors may modify keratin gene expression. This represents the first report of a co‐dominant keratin hHb6 mutation resulting in severe disease.

The cowpea (Vigna unguiculata L. Walp.) is important pulse crop in India and recognised as vegetarian meat due to its immense nutrition al value. Phytoparasitic nematodes are the major constraints in the production of these pulses. Root knot nematode, Meloidogyne incognita is major nematode pest infecting pulses and cowpea is another host of this pest. Host Giant cells are formed by obligate parasite, M. incognita inside the host root that incites the physiological and biochemical changes in plant. In the present study pre-confirmed resistant and susceptible cultivar cowpea used as test plant to assess the starch changes in infected and non infected plants. The Starch content in both leaves and shoots showed a decrease from uninoculated to inoculated cowpea varieties. Higher reduction was observed in susceptible cultivars KM-5 and Gomati i.e. 34.74% and 31.33% respectively in leaves; 31.75% and 38.24% respectively in roots. Likewise, lowest reduction in starch i.e. 12.12% and 10.47%...

Purpose-Photoreceptor mitochondrial oxidative stress is the initial pathologic event in experimental autoimmune uveitis. In this study, the authors determined alterations in retinal mitochondrial protein levels in response to oxidative stress during the early phase of experimental autoimmune uveitis (EAU). Methods-Retinal mitochondrial fractions during early EAU were prepared and subjected to twodimensional difference in gel electrophoresis (2D-DIGE). Protein spots showing differential expression were excised and subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for peptide identification. Levels of these proteins were also confirmed by Western blot analysis. mRNA expression of these proteins was confirmed by real-time PCR. TUNEL staining was performed to detect apoptosis. Results-2D-DIGE analysis revealed differential expression of 13 proteins. Ten proteins were overexpressed, including manganese-SOD, αA crystallin, β crystallin, and four proteins were downregulated, including adenosine triphosphate (ATP) synthase, malate dehydrogenase, and calretinin. Increased levels of αA crystallin, βB2 crystallin, MnSOD, and aconitase and decreased levels of ATP synthase were confirmed by Western blot analysis. qPCR also confirmed the increased expression of αA crystallin, βB2 crystallin, MnSOD, and Hsp70. Apoptosis was absent during this phase. Conclusions-The presence of mitochondrial-specific oxidative stress-related proteins in the early EAU retina along with the downregulation of ATP synthase provides early evidence of stress-related retinal damage. The presence of high levels of αA and βB2 crystallin in the mitochondria may prevent cell death during early EAU.

In early S-antigen induced experimental uveitis (EAU), photoreceptor mitochondrial proteins are nitrated prior to macrophage infiltration of the retina, suggesting that oxidative stress is an initial event in the development of EAU. We attempted to detect the oxidative stress and localise it in the EAU retina. Methods: Lewis rats were immunised with S-antigen in complete Freund's adjuvant (CFA). Animals were injected with CFA alone and non-immunised animals served as controls. Immunised and non-immunised animals were killed on day 5 and subsequent days. Isolated retinas were processed for inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)a, interferon (IFN)c, interleukin (IL)Ia and CD28 expression by real time polymerase chain reaction. In addition, iNOS was colocalised with cytochrome c oxidase on day 5 of EAU. Oxidative stress was detected by 29, 79-dichlorodihydrofluorescein diacetate and localised by a mitochondrial specific marker. Leucocyte and T cell infiltration in the retina/choroid was evaluated by immunohistochemistry. Results: The iNOS, TNFa, IFNc, IL1a and CD28 transcripts were significantly upregulated on day 5 in EAU, and iNOS was colocalised with cytochrome c oxidase in the photoreceptor mitochondria. Oxidative stress was seen primarily in the photoreceptor mitochondria. Occasional T cells were present in the retina at this stage. Conclusions: During early EAU, mitochondrial oxidative stress is selectively noted in the photoreceptor inner segments. The oxidative stress appears to result from iNOS upregulation in the photoreceptor mitochondria and cytokine generation in the retina by a few antigen specific infiltrating T cells.

To assess knowledge, pregnancy attitudes and contraceptive practices in relation to the Zika virus outbreak in Brazil. We interviewed 526 women 18 to 49 years old in primary health services in a Northeastern capital of Brazil, in 2016. They provided information about their knowledge of Zika transmission and health consequences, their receipt of counseling related to sexual and perinatal transmission of Zika, their pregnancy intentions and reassessment of contraceptive options in the context of the Zika virus outbreak. Awareness about Zika congenital syndrome was high, but knowledge about sexual transmission was low. Few women had changed pregnancy intentions or contraceptive practices in response to Zika. Pregnant women were more likely to access counseling about family planning, condom use and pregnancy postponement due to Zika virus than non-pregnant women, which may suggest that health system responses followed pregnancy occurrence. We observed missed opportunities for prevention...

ETS transcription factor ETV2 / Etsrp functions as a key regulator of embryonic vascular development in multiple vertebrates. However, its role in pathological vascular development has not been previously investigated. To analyze its role in tumor angiogenesis, we utilized a zebrafish xenotransplantation model. Using a photoconvertible kdrl:NLS-KikGR line we demonstrated that all tumor vessels originate from the existing embryonic vasculature by the mechanism of angiogenesis. Xenotransplantation of mouse B16 melanoma cells resulted in a significant increase in expression of the ETS transcription factors etv2 and fli1b expression throughout the embryonic vasculature. etv2 null mutants which undergo significant recovery of embryonic angiogenesis during later developmental stages displayed a strong inhibition of tumor angiogenesis. We utilized highly specific and fully validated photoactivatable morpholinos to inhibit Etv2 function after embryonic vasculogenesis has completed. Inducible inhibition of Etv2 function resulted in a significant reduction of tumor angiogenesis and inhibition of tumor growth. Furthermore, inducible inhibition of Etv2 function in fli1b mutant embryos resulted in even stronger reduction in tumor angiogenesis and growth, demonstrating that Etv2 and Fli1b have a partially redundant requirement during tumor angiogenesis. These results demonstrate the requirement for Etv2 and Fli1b in tumor angiogenesis and suggest that inhibition of these ETS factors may present a novel strategy to inhibit tumor angiogenesis and reduce tumor growth.

Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase b, elongation factor-1a, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida + Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta + Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta + Archiheterodonta + Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.

To re-evaluate the relationships of the major bivalve lineages, we amassed detailed morpho-anatomical, ultrastructural and molecular sequence data for a targeted selection of exemplar bivalves spanning the phylogenetic diversity of the class. We included molecular data for 103 bivalve species (up to five markers) and also analysed a subset of taxa with four additional nuclear protein-encoding genes. Novel as well as historically employed morphological characters were explored, and we systematically disassembled widely used descriptors such as gill and stomach ‘types’. Phylogenetic analyses, conducted using parsimony direct optimisation and probabilistic methods on static alignments (maximum likelihood and Bayesian inference) of the molecular data, both alone and in combination with morphological characters, offer a robust test of bivalve relationships. A calibrated phylogeny also provided insights into the tempo of bivalve evolution. Finally, an analysis of the informativeness of mo...

The melanocortin-4 receptor (MC4R) has been indicated as a therapeutic target for metabolic disorders such as anorexia, cachexia, and obesity. The current study investigates the in vivo effects on energy homeostasis of a 15 nM MC4R antagonist SKY2-23-7, Ac-Trp-DPhe(p-I)-Arg-Trp-NH 2 , that is a 3,700 nM melanocortin-3 receptor (MC3R) antagonist with minimal MC3R and MC4R agonist activity. When monitoring both male and female mice in TSE metabolic cages, sexspecific responses were observed in food intake, respiratory exchange ratio (RER), and energy expenditure. A 7.5 nmol dose of SKY2-23-7 increased food intake, increased RER, and trended towards decreasing energy expenditure in male mice. However, this compound had minimal effect on female mice's food intake and RER at the 7.5 nmol dose. A 2.5 nmol dose of SKY2-23-7 significantly increased female food intake, RER, and energy expenditure while having a minimal effect on male mice at this dose. The observed sex differences of SKY2-23-7 administration result in the discovery of a novel chemical probe for elucidating the molecular mechanisms of the sexual dimorphism present within the melanocortin pathway. To further explore the melanocortin sexual dimorphism, hypothalamic gene expression was examined. The mRNA expression of the MC3R and proopiomelanocortin (POMC) were not significantly different between sexes. However, the expression of agouti-related peptide (AGRP) was significantly higher in female mice which may be a possible mechanism for the sex-specific effects observed with SKY2-23-7.

The purpose of the current study is to assess changes in the expression of Acid-Sensing Ion Channel (ASIC)1a and ASIC2 in retinal ganglion cells (RGCs) after retinal ischemia and reperfusion (I/R) injury and to test if inhibition of ASIC1a provides RGC neuroprotection. Methods: Transient ischemia was induced in one eye of C57BL/6 mice by raising intraocular pressure to 120 mmHg for 60 min followed by retinal reperfusion by restoring normal pressure. RGC function was measured by Pattern electroretinography (PERG). In addition, retinal ASIC1a and ASIC2 were observed by immunohistochemistry and western blot. Changes in calpain, fodrin, heat shock protein 70 (HSP70), Brn3a, super oxide dismutase-1 (SOD1), catalase, and glutathione perioxidase-4 (GPX4) protein levels were assessed by western blot. RGC numbers were measured by immunohistochemistry on whole retinal flat mounts using anti-RNA binding protein with multiple splicing (RBPMS) antibodies. Intravitreal injection of psalmotoxin-1, a selective ASIC1a blocker, was used to assess the neuroprotective effect of ASIC1a inhibition. Results: Levels of ASIC1a and ASIC2 after I/R increased in RGCs. Upregulation of ASIC1a but not ASIC2 was attenuated by intravitreal injection of psalmotoxin-1. I/R induced activation of calpain and degradation of fodrin, HSP70, and reduction in Brn3a. In contrast, while psalmotoxin-1 attenuated calpain activation and increased Brn3a levels, it failed to block HSP70 degradation. Unlike SOD1 protein which was reduced, catalase protein levels increased after I/R. Psalmotoxin-1, although not affecting SOD1 and GPX4, increased catalase levels significantly. Psalmotoxin-1 also increased RBPMS-labeled RGCs following I/R as judged by immunohistochemistry of retinal flat mounts. Finally, psalmotoxin-1 enhanced the amplitude of PERG following I/R, suggesting partial rescue of RGC function. Psalmotoxin-1 appears to exert a neuroprotective effect under ischemic insults and targeting inhibition of ASICs may represent a new therapeutic approach in ischemic retinal diseases.