Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta.

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.     

Land cover change and carbon emissions over 100 years in an African biodiversity hotspot

Agricultural expansion has resulted in both land use and land cover change (LULCC) across the tropics. However, the spatial and temporal patterns of such change and their resulting impacts are poorly understood, particularly for the presatellite era. Here, we quantify the LULCC history across the 33.9 million ha watershed of Tanzania's Eastern Arc Mountains, using geo‐referenced and digitized historical land cover maps (dated 1908, 1923, 1949 and 2000). Our time series from this biodiversity hotspot shows that forest and savanna area both declined, by 74% (2.8 million ha) and 10% (2.9 million ha), respectively, between 1908 and 2000. This vegetation was replaced by a fivefold increase in cropland, from 1.2 million ha to 6.7 million ha. This LULCC implies a committed release of 0.9 Pg C (95% CI: 0.4–1.5) across the watershed for the same period, equivalent to 0.3 Mg C ha^?1 yr^?1. This is at least threefold higher than previous estimates from global models for the same study area. We then used the LULCC data from before and after protected area creation, as well as from areas where no protection was established, to analyse the effectiveness of legal protection on land cover change despite the underlying spatial variation in protected areas. We found that, between 1949 and 2000, forest expanded within legally protected areas, resulting in carbon uptake of 4.8 (3.8–5.7) Mg C ha^?1, compared to a committed loss of 11.9 (7.2–16.6) Mg C ha^?1 within areas lacking such protection. Furthermore, for nine protected areas where LULCC data are available prior to and following establishment, we show that protection reduces deforestation rates by 150% relative to unprotected portions of the watershed. Our results highlight that considerable LULCC occurred prior to the satellite era, thus other data sources are required to better understand long‐term land cover trends in the tropics.     

Predation of artificial nests by introduced rhesus macaques (<Emphasis Type="Italic">Macaca mulatta</Emphasis>) in Florida,USA

Native throughout Asia, rhesus macaques are believed to have the widest native range of any non-human primate and are capable of adapting to an extensive diversity of habitats. Rhesus macaques have caused environmental degradation in introduced habitats, including decreasing bird populations through nest predation. In the 1930s, rhesus macaques were intentionally introduced into what is today Silver Springs State Park (SSSP), central Florida, in an effort to increase tourism. Our objective was to determine whether introduced rhesus macaques in SSSP would consume eggs presented in artificial nests. We used camera traps adjacent to 100 open-cupped artificial bird nests baited with quail eggs near the Silver River. Nests were placed in shrubs and left in the field site for 12 days, representative of the incubation period of native passerine species. Twenty-one nests were depredated by rhesus macaques, nine by nest predators other than macaques, and five nests by an unidentified predator. Nests were more likely to be depredated by macaques when located in areas of high macaque relative abundance. This study suggests introduced rhesus macaques may influence nest predation rates of native bird species in natural areas.     

An Evolutionary View of the Arms Race between Protein Kinase R and Large DNA Viruses

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.     

Intrapopulation genomics in a model mutualist: Population structure and candidate symbiosis genes under selection in Medicago truncatula

Bottom‐up evolutionary approaches, including geographically explicit population genomic analyses, have the power to reveal the mechanistic basis of adaptation. Here, we conduct a population genomic analysis in the model legume, Medicago truncatula, to characterize population genetic structure and identify symbiosis‐related genes showing evidence of spatially variable selection. Using RAD‐seq, we generated over 26,000 SNPs from 191 accessions from within three regions of the native range in Europe. Results from STRUCTURE analysis identify five distinct genetic clusters with divisions that separate east and west regions in the Mediterranean basin. Much of the genetic variation is maintained within sampling sites, and there is evidence for isolation by distance. Extensive linkage disequilibrium was identified, particularly within populations. We conducted genetic outlier analysis with F_ST‐based genome scans and a Bayesian modeling approach (PCAdapt). There were 70 core outlier loci shared between these distinct methods with one clear candidate symbiosis related gene, DMI1. This work sets that stage for functional experiments to determine the important phenotypes that selection has acted upon and complementary efforts in rhizobium populations.     

Regulators of complement activity mediate inhibitory mechanisms through a common C3b‐binding mode

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.     

Evaluation of artificial selection in Standard Poodles using whole-genome sequencing

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.     

Targeted imputation of sequence variants and gene expression profiling identifies twelve candidate genes associated with lactation volume,composition and calving interval in dairy cattle

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.     

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling

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, 9–11). 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 (15–21). 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/).     

South Asia as a Reservoir for the Global Spread of Ciprofloxacin-Resistant Shigella sonnei: A Cross-Sectional Study

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.