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991.
Real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Botrytis cinerea, suitable for a wide range of different host plant species. Assays were designed to the beta-tubulin gene, the intergenic spacer (IGS) region of the nuclear ribosomal DNA and also to a previously published, species-specific sequence characterised amplified region (SCAR) marker; the assays were compared to a published method based on SYBR Green I technology. The assays designed to the IGS region and SCAR marker proved to be highly specific for B. cinerea but assays designed to the beta-tubulin gene and the previously published assay designed to the cutinase-A gene both cross-react with B. fabae. The assay designed to the IGS region was the most sensitive and was able to reliably detect and quantify as little as 20 fg of B. cinerea DNA. The method incorporates the detection of a gene from the plant host to compensate for variations in extraction efficiency and size of sample tested. The assays designed were used to follow the progression of infection of B. cinerea in plant material inoculated with spores to the point of symptom induction. They should be ideally suited to investigating infection processes in-planta and could be used to investigate aspects of infection/plant pathogenesis, by B. cinerea and are particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.  相似文献   
992.
To establish productive infections, viruses must counteract numerous cellular defenses that are poised to recognize viruses as nonself and to activate antiviral pathways. The opposing goals of host and viral factors lead to evolutionary arms races that can be illuminated by evolutionary and computational methods and tested in experimental models. Here we illustrate how this perspective has been contributing to our understanding of the interactions of the protein kinase R pathway with large DNA viruses.  相似文献   
993.
Regulators of complement activation (RCA) inhibit complement‐induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i–iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b‐binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease‐related mutations and immune evasion.  相似文献   
994.
Identifying regions of artificial selection within dog breeds may provide insights into genetic variation that underlies breed-specific traits or diseases—particularly if these traits or disease predispositions are fixed within a breed. In this study, we searched for runs of homozygosity (ROH) and calculated the d i statistic (which is based upon F ST) to identify regions of artificial selection in Standard Poodles using high-coverage, whole-genome sequencing data of 15 Standard Poodles and 49 dogs across seven other breeds. We identified consensus ROH regions ≥1 Mb in length and common to at least ten Standard Poodles covering 0.6 % of the genome, and d i regions that most distinguish Standard Poodles from other breeds covering 3.7 % of the genome. Within these regions, we identified enriched gene pathways related to olfaction, digestion, and taste, as well as pathways related to adrenal hormone biosynthesis, T cell function, and protein ubiquitination that could contribute to the pathogenesis of some Poodle-prevalent autoimmune diseases. We also validated variants related to hair coat and skull morphology that have previously been identified as being under selective pressure in Poodles, and flagged additional polymorphisms in genes such as ITGA2B, CBX4, and TNXB that may represent strong candidates for other common Poodle disorders.  相似文献   
995.
Dairy cattle are an interesting model for gaining insights into the genes responsible for the large variation between and within mammalian species in the protein and fat content of their milk and their milk volume. Large numbers of phenotypes for these traits are available, as well as full genome sequence of key founders of modern dairy cattle populations. In twenty target QTL regions affecting milk production traits, we imputed full genome sequence variant genotypes into a population of 16,721 Holstein and Jersey cattle with excellent phenotypes. Association testing was used to identify variants within each target region, and gene expression data were used to identify possible gene candidates. There was statistical support for imputed sequence variants in or close to BTRC, MGST1, SLC37A1, STAT5A, STAT5B, PAEP, VDR, CSF2RB, MUC1, NCF4, and GHDC associated with milk production, and EPGN for calving interval. Of these candidates, analysis of RNA-Seq data demonstrated that PAEP, VDR, SLC37A1, GHDC, MUC1, CSF2RB, and STAT5A were highly differentially expressed in mammary gland compared to 15 other tissues. For nine of the other target regions, the most significant variants were in non-coding DNA. Genomic predictions in a third dairy breed (Australian Reds) using sequence variants in only these candidate genes were for some traits more accurate than genomic predictions from 632,003 common SNP on the Bovine HD array. The genes identified in this study are interesting candidates for improving milk production in cattle and could be investigated for novel biological mechanisms driving lactation traits in other mammals.  相似文献   
996.
We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).Proteins rarely work as monomers to carry out all the biological processes needed for cells to function. An estimate of the total number of protein-protein interactions within the human proteome, based on currently available data sets, is ∼650,000 (1). This is likely an underestimate, given that many proteins form either transient, or weak interactions within intact cells that may not yet have been detected. This suggests that the majority of human proteins can participate in protein complex formation, at least under some conditions. This includes the many well-studied soluble protein complexes in the cytoplasm, exemplified by the proteasome, ribosomes and cytoskeletal network. It also includes many membrane-associated complexes, for example receptor tyrosine kinase signaling complexes, integrin networks and transmembrane transporters (2). To characterize the many roles of multi-protein complexes in biological regulatory mechanisms, it is important to have convenient methods for the rapid and efficient analysis of their composition and dynamics (3). Ideally, such methods should be applicable to system-wide studies and allow the analysis of endogenous proteins, rather than exclusively use tagged and/or over-expressed baits.The methods available for the proteome-wide analysis of protein interactions have developed swiftly over the last ten years. This field is dominated by affinity-enrichment based approaches, using either tagged constructs, or antibodies specific for endogenous proteins. Another approach is in vivo proximity labeling, based, for example, on the exogenous expression of a protein of interest, fused either to a promiscuous biotin-ligase (BioID) (4), or to a peroxidase enzyme that activates biotin-phenol (APEX) (5). While these data sets have proved very useful, there are some downsides. For example, a large expense in terms of both time and money to generate the thousands of individual “bait” proteins required for global interaction analyses. In addition, each of these affinity enrichments will be performed in only one type of buffer system, which is unlikely to be compatible with the maintenance of all protein-protein interactions. Another dimension to the analytical problem is that many proteins are expressed as different sized isoforms and/or in different post-translationally modified forms, resulting in formation of multiple, related, but functionally distinct complexes, with different combinations of interaction partners (6). Using affinity-enrichment/pull-down methods alone makes it difficult to resolve such mixtures of different forms of related protein complexes, complicating a detailed understanding of biological response mechanisms.An alternative strategy involves protein correlation profiling-MS, i.e. correlating similarities in the fractionation profiles of proteins detected by mass spectrometry, assuming that proteins in a common complex will cofractionate. This approach was previously applied to the analysis of subcellular organelle proteomes (7, 8), and subsequently extended to analyze soluble protein complexes. Thus, recent studies have shown that chromatography-based separation of soluble protein complexes, combined with fraction collection and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS)1, facilitates analysis of many hundreds of soluble complexes from a single experiment (6, 911). A limitation of all of these studies, however, is that the native extraction conditions used to preserve protein-protein interactions isolates predominantly stable, soluble complexes. For example, many proteins that are integral to membranes are not recovered (12). Similarly, soluble protein complexes that have weakly bound protein subunits can dissociate upon cell lysis and the inevitable dilution associated with extraction. Thus, the potential value of this approach for the system-wide analysis of protein complexes is limited without a covalent tether to hold protein-protein interactions intact during extraction and subsequent chromatographic separation (13).Covalent protein crosslinking has been used extensively to stabilize protein complexes, cultured cells and tissues for subsequent analysis, either by microscopy, nucleotide sequencing or mass spectrometry. The agents employed to crosslink proteins to each other include various chemical groups able to react with the side-chains of either amino acids, nucleotides, carbohydrates or lipids (14). These crosslinking agents vary in the efficiency with which they perfuse into unbroken cells/tissues and the speed of their reaction when in proximity to a suitable chemical group. One of the most widely used crosslinkers is formaldehyde, which can reversibly form a covalent crosslink to stabilize both protein-protein and protein-nucleotide interactions (1521). One of the main benefits of using formaldehyde is that because of its small size, it readily permeates intact cells and tissues. Another benefit of using formaldehyde is the easy reversal of the crosslinks by heating and subsequent compatibility with mass spectrometry-based proteome analysis.Here, we describe a mass spectrometry-based proteomic approach for the efficient global analysis of protein complexes, including membrane proteins, using in vivo protein crosslinking combined with denaturing extraction. Using high-resolution, size-exclusion chromatography (SEC) to separate crosslinked complexes under denaturing conditions and MS analysis of fractionated proteins, we could identify membrane bound and membrane associated complexes not accessible in native extracts. We present a detailed comparison of the sets of protein complexes that can be identified using protein correlation profiling MS analysis in conjunction with both formaldehyde crosslinked and native extracts from U2OS cells. We provide access to the entire proteome-wide data sets of both in vivo crosslinked and native U2OScell protein complexes via a searchable online database (http://www.peptracker.com/epd/).  相似文献   
997.
BackgroundAntimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei.ConclusionsThis study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.  相似文献   
998.
We aim to identify candidate brain biomarkers for, and to elucidate the pathophysiology of closed traumatic brain injury (TBI). Nuclear magnetic resonance (NMR) based metabolomic analysis was performed on the whole brain of mice undergoing TBI using a validated technique. There were 10 TBI mice compared to 8 sham operated controls. A total of 45 metabolites were evaluated. There was a statistically significant alteration in concentrations of 29 metabolites in TBI brains as compared to controls (FDR <0.05). Profound disturbances of several metabolic pathways (FDR <1E-07), including pathways associated with purine, alanine, aspartate and glutamine and glutathione metabolism were observed. Also, a significant elevation in glutamate (the main excitatory neurotransmitter) and depression of GABA (the main inhibitory neurotransmitter) was observed. Four metabolites, ADP, AMP, NAD+, and IMP were the most important indicators of TBI, relative to normal controls. All were elevated in the TBI mice. A combination of these 4 biomarkers produced a perfect predictor of TBI status, AUC (95 % CI) = 1.0 (1.0, 1.0). We also detected significant disturbances in mitochondrial function, energy metabolism, neurotransmitter metabolism and other important biochemical pathways in TBI mouse brains. Further studies to assess the utility of metabolomics to detect and classify the severity of and assess the prognosis of TBI is warranted.  相似文献   
999.
Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated.  相似文献   
1000.
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