Exome sequencing offers the potential to study the population-genomic variables that underlie patterns of deleterious variation. Runs of homozygosity (ROH) are long stretches of consecutive homozygous genotypes probably reflecting segments shared identically by descent as the result of processes such as consanguinity, population size reduction, and natural selection. The relationship between ROH and patterns of predicted deleterious variation can provide insight into the way in which these processes contribute to the maintenance of deleterious variants. Here, we use exome sequencing to examine ROH in relation to the distribution of deleterious variation in 27 individuals of varying levels of apparent inbreeding from 6 human populations. A significantly greater fraction of all genome-wide predicted damaging homozygotes fall in ROH than would be expected from the corresponding fraction of nondamaging homozygotes in ROH (p < 0.001). This pattern is strongest for long ROH (p < 0.05). ROH, and especially long ROH, harbor disproportionately more deleterious homozygotes than would be expected on the basis of the total ROH coverage of the genome and the genomic distribution of nondamaging homozygotes. The results accord with a hypothesis that recent inbreeding, which generates long ROH, enables rare deleterious variants to exist in homozygous form. Thus, just as inbreeding can elevate the occurrence of rare recessive diseases that represent homozygotes for strongly deleterious mutations, inbreeding magnifies the occurrence of mildly deleterious variants as well.
SummaryExosomes are membranous nanovesicles of endocytic origin that carry and transfer regulatory bioactive molecules and mediate intercellular communication between cells and tissues. Although seminal exosomes have been identified in human seminal plasma, their exact composition and possible physiologic function remain unknown. The objective of this study was to perform a comprehensive proteomics analysis of exosomes derived from human seminal plasma. Seminal exosomes were isolated and purified from 12 healthy donors using a 30% sucrose cushion‐based exosome‐isolation protocol, followed by characterization by western blot, transmission electron microscopy, and nanoparticle tracking analysis before performing extensive liquid chromatography tandem mass spectrometry proteomics analysis. The identified proteins were analyzed by bioinformatics analysis, and seminal exosomes‐associated proteins were selectively validated by western blot. A total of 1474 proteins were identified in all seminal exosomes samples, with Gene Ontology analysis demonstrating that these identified seminal exosomes‐associated proteins were mostly linked to ‘exosomes,’ ‘cytoplasm,’ and ‘cytosol.’ Bioinformatics analysis indicated that these proteins were mainly involved in biologic processes, including metabolism, energy pathways, protein metabolism, cell growth and maintenance, and transport. Of these identified proteins, PHGDH, LGALS3BP, SEMG1, ACTB, GAPDH, and the exosomal‐marker protein ALIX were validated by western blot. This study provided a more comprehensive description of the seminal exosomes proteome and could also be a resource for further screening of biomarkers and comparative proteomics studies, including those associated with male infertility and prostate cancer.
Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.
Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 µmol/L, a selectivity index >20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.
The latency-associated transcript (LAT) is the only herpes simplex virus type 1 (HSV-1) gene that is abundantly transcribed during latency. Plasmids expressing LAT inhibit apoptosis induced by etoposide and ceramide in transiently transfected cells. LAT also inhibits apoptosis in trigeminal ganglia of rabbits and promotes spontaneous reactivation, suggesting these events are coupled. In this study, we compared caspase cleavage (activation) in cells infected with dLAT2903 (LAT-null mutant) versus wild-type McKrae or the rescued LAT-null mutant (dLAT2903R). Neuro-2A cells (mouse neuroblastoma), but not NIH3T3 cells infected with dLAT2903, contained higher levels of cleaved caspase 9 compared to cells infected with McKrae. Cleaved caspase 9 was also readily detected in neuro-2A cells, but not NIH3T3 cells, after ultraviolet (UV) light treatment, suggesting that the ability of cells to process caspases and undergo apoptosis influences the antiapoptotic properties of LAT. HSV-1 expresses numerous genes in addition to LAT that can block apoptosis during productive infection of cultured cells. Because these genes may mask the effects of LAT on apoptosis, transient transfection assays were performed to test whether LAT can inhibit caspase 8- and caspase 9-induced apoptosis. A plasmid expressing nucleotides 1 to 4658 of LAT efficiently inhibited caspase 8- and caspase 9-induced apoptosis in transiently transfected neuro-2A cells. These studies indicate that LAT has the potential to inhibit the two major pathways of apoptosis in the absence of other viral genes. Furthermore, these studies support a role for the antiapoptotic properties of LAT in the latency-reactivation cycle.
Innate cell-autonomous antiviral responses are essential first lines of defense against central nervous system infections but may also contribute to neuropathogenesis. We investigated the relationships between innate immunity and neuronal differentiation using an in vitro culture system with human cell lines to analyze cellular responses to the neurotropic alphavirus western equine encephalitis virus. Human neuronal cells displayed a maturation-dependent reduction in virus-induced cytopathology that was independent of autocrine interferon alpha or beta activity. In addition, maturation was associated with enhanced responsiveness to exogenous stimuli, such that differentiated neurons required five- to ten-fold less type I interferon to suppress viral replication or virus-induced cytopathology compared to immature cells, although this enhanced responsiveness extended to only a subset of unique type I interferons. These results demonstrate that maturation-dependent changes in human neuronal cells may be key determinants in the innate immune response to infections with neurotropic alphaviruses.
The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for the high spontaneous and induced reactivation phenotype of HSV-1 in the rabbit ocular model and for the high induced reactivation phenotype in the mouse ocular model. Recently we showed that LAT has an antiapoptosis function, and we hypothesized that LAT's ability to inhibit apoptosis played an important role in LAT's ability to enhance the reactivation phenotype. Expression of just the first 1.5 kb of the 8.3-kb LAT gene is sufficient for both inhibition of apoptosis in an in vitro transient-transfection assay and the high spontaneous reactivation phenotype in vivo. Here we show the results of more complex mapping studies in which inhibition of apoptosis and the enhanced spontaneous reactivation phenotype also appear to be linked. The HSV-1 mutant virus dLAT371 has a high spontaneous reactivation phenotype in rabbits, suggesting that the LAT region deleted in this mutant (LAT nucleotides 76 to 447) is not required for this phenotype. The LAT3.3A viral mutant (which expresses LAT nucleotides 1 to 1499) also has a high spontaneous reactivation phenotype, suggesting that the region of LAT not expressed by this mutant (LAT nucleotide 1500 to the end of LAT) is also not required for this phenotype. Surprisingly, LAT2.9A, which is a combination of dLAT371 and LAT3.3A (i.e., it expresses LAT nucleotides 1 to 76 and 447 to 1499), has a low spontaneous reactivation phenotype indistinguishable from that of LAT null mutants. We report here that consistent with the low spontaneous reactivation phenotype of LAT2.9A, a plasmid expressing the identical LAT RNA did not inhibit caspase 9-induced apoptosis. In contrast, plasmids containing the same deletion but able to transcribe up to or past LAT nucleotide 2850 (rather than just up to LAT nucleotide 1499) inhibited caspase 9-induced apoptosis, consistent with the high spontaneous reactivation phenotype of dLAT371. Thus, LAT2.9A may have a low spontaneous reactivation phenotype because the LAT RNA that is made cannot block apoptosis, and dLAT371 apparently has a high spontaneous reactivation phenotype because the LAT RNA made has significant antiapoptosis activity. Furthermore, LAT appeared to have at least two regions capable of interfering with caspase 9-induced apoptosis. One region partially overlaps LAT nucleotides 76 to 447. The second region is partially (or completely) downstream of LAT nucleotide 1499.
Objective: To elucidate the genetic cause of a rare recessive ataxia presented by 2 siblings from a consanguineous Turkish family with a nonprogressive, congenital ataxia with mental retardation of unknown etiology.Methods: Whole-exome sequencing was combined with homozygosity mapping, linkage, and expression analysis to identify candidate genes, confirmed by Sanger sequencing. Reverse transcription-PCR and immunoblotting were used to determine the functional consequences of the gene variant. A zebrafish model was developed using morpholino-mediated knockdown.Results: We identified a homozygous mutation at the invariant 11 position (c.96411G.A) in intron 9 of the CWF19L1 (complexed with cdc5 protein 19-like 1) gene. This mutation is absent in .6,500 European and African American individuals and 200 Turkish control DNAs. The mutation causes exon skipping, reduction in messenger RNA levels, and protein loss in cell lines of affected individuals. Morpholino-mediated knockdown in a zebrafish model demonstrates that loss of the evolutionarily highly conserved CWF19L1, whose normal biological function is unknown, alters cerebellar morphology and causes movement abnormalities. Conclusions:Our results suggest that CWF19L1 mutations may be a novel cause of recessive ataxia with developmental delay. Our research may help with diagnosis, especially in Turkey, identify causes of other ataxias, and may lead to novel therapies. Neurology ® 2014;83:2175-2182 GLOSSARY CWF19L1 5 complexed with cdc5 protein 19-like 1; GAPDH 5 glyceraldehyde 3-phosphate dehydrogenase; LCL 5 lymphoblastoid cell line; MO 5 morpholino oligonucleotide; mRNA 5 messenger RNA; qRT 5 quantitative reverse transcription.
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