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Fresh Stable Isotope-Resolved Metabolomics Way of a Small Number of Cellular material Employing Chip-Based Nanoelectrospray Bulk Spectrometry.
Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https//www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2 186-200, 406-411; PB1 135-143, 538-546; PA 515-523; NP 61-68; M1 76-84; M2 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.The methods for expansion of human cytomegalovirus (HCMV)-specific T lymphocytes are limited due to the complex culture process, long culture duration, and human leukocyte antigen (HLA) restriction. Here, we report that in vitro stimulation with pp65 kDa phosphoprotein (pp65)-derived overlapping synthetic peptides rapidly generates large numbers of HCMV-specific cytotoxic T lymphocytes from peripheral blood mononuclear cells (PBMCs) regardless of HLA type. Treatment of PBMCs from healthy volunteers expressing HLA-A*0201 or HLA-A*2402 with 138 pp65 overlapping peptides (OLP) resulted in an expansion of HCMV pp65 NLVPMVATV (NLV) pentamer-specific CD8+ T lymphocytes that expressed interferon (IFN)-γ, but the pp65 NLV peptide did not generate HCMV-specific CD8+ T lymphocytes in PBMCs obtained from an HLA-A*2402 donor due to HLA restriction. The OLP-induced T lymphocytes specific for HCMV derived from PBMCs of HLA-A*0201- and HLA-A*2402-expressing donors showed effective cytolytic responses against target cells loaded with OLP or the NLV epitope, but pp65 NLV peptide-induced T lymphocytes did not. Phenotypic analyses demonstrated that OLP increased the frequency of CD3+ CD8+ cells, but not CD3+ CD4+, CD14+, or CD56+ cells, in donor PBMCs. Thus, this study provides evidence that in vitro stimulation with OLP efficiently generates sufficient numbers of HCMV pp65-specific cytotoxic T lymphocytes for adoptive cell therapy. Keywords human cytomegalovirus; cytotoxic T lymphocyte; overlapping peptides; pp65; cytotoxicity.Avian infectious bronchitis virus (IBV) and avian pathogenic Escherichia coli (APEC) are two important respiratory pathogens in the chicken. The co-infection can lead to chronic complications and considerable economic losses in the poultry industry worldwide. In the present study, we compared differential transcriptional profiles in the trachea tissue of three infected groups (IBV, APEC, and co-infection) with the control group to investigate transcriptome profile changes at the early stage of the infection. After the challenge of SPF chickens with IBV IS-1494 like (GI-23) and APEC, serotype O78 K80, or co-infection, the trachea tissue was used for RNA extraction, and changes in the transcriptome were investigated by Illumina RNA-seq technique. Up-regulated and down-regulated differentially expressed genes (DEGs) in the transcriptome of each group's trachea were identified. Gene ontology category, KEGG pathway, and gene interaction networks (STRING analysis) were analyzed to identify relationships among differentially expressed genes. In general, the numbers of up-regulated genes were higher than of down-regulated genes. In the co-infection group, a more severe immune response and macrophage infiltration occurred; an important cluster of pathway signaling in this group's up-regulated genes was an apoptotic cluster, cytokine-mediated signaling cluster, and the PAMPs recognizing cluster. This is the first study to provide a general overview of transcriptome changes in the trachea at the early stage of infection with these pathogens. Keywords avian infectious bronchitis virus; avian pathogenic E. coli; transcriptome; RNA-Seq.Bovine leukemia virus (BLV) is a retrovirus that affects primarily milky cows. Animals serologically positive to BLV show a Th1 cytokine profile with a predominance of interferon gamma (IFN-γ). IFN-γ has antiviral activity through mechanisms such as resistance to infection, inhibition of viral replication and apoptosis. The objective of this work was to determine the transcription levels of IFN-γ and its relationship with proviral load and persistent lymphocytosis in a population of Holstein cows of the province of Antioquia, Colombia. VS-4718 clinical trial IFN-γ transcription levels were evaluated by qPCR in 140 Holstein cows. A one-way analysis of variance and a Student's t test were used to evaluate the differences between the means. The amount of IFN-γ mRNA found in BLV-positive cows was lower than in BLV-negative cows. Moreover, in the group of infected cows a lower level of IFN-γ mRNA expression was found in BLV and persistent lymphocytosis cows (BLV+PL) compared with BLV and aleukemia cows (BLV+AL). The level of IFN-γ mRNA expression was lower in cows with high proviral load (HPL) compared to cows with low proviral load (LPL).
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