BACKGROUND: Physical inactivity impairs insulin sensitivity, which is exacerbated with aging. We examined the impact of 2 wk of acute inactivity and recovery on glycemic control, and integrated rates of muscle protein synthesis in older men and women.
METHODS: Twenty-two overweight, prediabetic older adults (12 men, 10 women, 69 ± 4 y) undertook 7 d of habitual activity (baseline; BL), step reduction (SR; <1,000 steps.d-1 for 14 d), followed by 14 d of recovery (RC). An oral glucose tolerance test was used to assess glycemic control and deuterated water ingestion to measure integrated rates of muscle protein synthesis.
RESULTS: Daily step count was reduced (all p < .05) from BL at SR (7362 ± 3294 to 991 ± 97) and returned to BL levels at RC (7117 ± 3819). Homeostasis model assessment-insulin resistance increased from BL to SR and Matsuda insulin sensitivity index decreased and did not return to BL in RC. Glucose and insulin area under the curve were elevated from BL to SR and did not recover in RC. Integrated muscle protein synthesis was reduced during SR and did not return to BL in RC.
CONCLUSIONS: Our findings demonstrate that 2 wk of SR leads to lowered rates of muscle protein synthesis and a worsening of glycemic control that unlike younger adults is not recovered during return to normal activity in overweight, prediabetic elderly humans.
Round whitefish (Prosopium cylindraceum) have a broad, disjunct range across northern North America and Eurasia, and little is known about their genetic population structure. We performed genetic analyses of round whitefish from 17 sites across its range using nine microsatellites, two mitochondrial DNA (mtDNA) loci, and 4918 to 8835 single-nucleotide polymorphism (SNP) loci. Our analyses identified deep phylogenetic division between eastern and western portions of the range, likely indicative of origins from at least two separate Pleistocene glacial refugia. Regionally, microsatellites and SNPs identified congruent patterns in subdivision, and population structure was consistent with expectations based on hydrologic connectivity. Within the Laurentian Great Lakes, Lake Huron and Lake Ontario were identified as key areas of interest. Lake Huron appears to be a contemporary source population for several other Great Lakes, and Lake Ontario contains a genetically discrete group of round whitefish. In all cases, multiple genetic markers yielded similar patterns, but SNPs offered substantially enhanced resolution. We conclude that round whitefish have population subdivision on several scales important for understanding their evolutionary history and conservation planning.
Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically divided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic ‘tip complex’ translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.
The loss of effective antimicrobials is reducing our ability to protect the global population from infectious disease. However, the field of antibiotic drug discovery and the public health monitoring of antimicrobial resistance (AMR) is beginning to exploit the power of genome and metagenome sequencing. The creation of novel AMR bioinformatics tools and databases and their continued development will advance our understanding of the molecular mechanisms and threat severity of antibiotic resistance, while simultaneously improving our ability to accurately predict and screen for antibiotic resistance genes within environmental, agricultural, and clinical settings. To do so, efforts must be focused toward exploiting the advancements of genome sequencing and information technology. Currently, AMR bioinformatics software and databases reflect different scopes and functions, each with its own strengths and weaknesses. A review of the available tools reveals common approaches and reference data but also reveals gaps in our curated data, models, algorithms, and data-sharing tools that must be addressed to conquer the limitations and areas of unmet need within the AMR research field before DNA sequencing can be fully exploited for AMR surveillance and improved clinical outcomes.
Jia B, Raphenya AR, Alcock B, Waglechner N, Guo P, Tsang KK, Lago BA, Dave BM, Pereira S, Sharma AN, Doshi S, Courtot M, Lo R, Williams LE, Frye JG, Elsayegh T, Sardar D, Westman EL, Pawlowski AC, Johnson TA, Brinkman FS, Wright GD, & McArthur AG.
The Comprehensive Antibiotic Resistance Database (CARD; http://arpcard.mcmaster.ca) is a manually curated resource containing high quality reference data on the molecular basis of antimicrobial resistance (AMR), with an emphasis on the genes, proteins and mutations involved in AMR. CARD is ontologically structured, model centric, and spans the breadth of AMR drug classes and resistance mechanisms, including intrinsic, mutation-driven and acquired resistance. It is built upon the Antibiotic Resistance Ontology (ARO), a custom built, interconnected and hierarchical controlled vocabulary allowing advanced data sharing and organization. Its design allows the development of novel genome analysis tools, such as the Resistance Gene Identifier (RGI) for resistome prediction from raw genome sequence. Recent improvements include extensive curation of additional reference sequences and mutations, development of a unique Model Ontology and accompanying AMR detection models to power sequence analysis, new visualization tools, and expansion of the RGI for detection of emergent AMR threats. CARD curation is updated monthly based on an interplay of manual literature curation, computational text mining, and genome analysis.
Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore reservoirs of resistance elements and reflective of the natural history of antibiotics and resistance. In a previous study, we discovered that multi-drug resistance is common in bacteria isolated from Lechuguilla Cave, an underground ecosystem that has been isolated from the surface for over 4 Myr. Here we use whole-genome sequencing, functional genomics and biochemical assays to reveal the intrinsic resistome of Paenibacillus sp. LC231, a cave bacterial isolate that is resistant to most clinically used antibiotics. We systematically link resistance phenotype to genotype and in doing so, identify 18 chromosomal resistance elements, including five determinants without characterized homologues and three mechanisms not previously shown to be involved in antibiotic resistance. A resistome comparison across related surface Paenibacillus affirms the conservation of resistance over millions of years and establishes the longevity of these genes in this genus.
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The McArthur lab and the Comprehensive Antibiotic Resistance Database are proud to have contributed to the Genome Canada – Canadian Food Inspection Agency Forum on Genomics and Antimicrobial Resistance. The two-day event brought together over sixty leading experts from academic, government, industry and commodities groups to address the challenge of AMR and discuss a path forward. A summary of the Forum and the Workshop Report are now online.
YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5-6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.
Development is a complex and well-defined process characterized by rapid cell proliferation and apoptosis. At this stage in life, a developmentally young organism is more sensitive to toxicants as compared to an adult. In response to pro-oxidant exposure, members of the Cap’n’Collar (CNC) basic leucine zipper (b-ZIP) transcription factor family (including Nfe2 and Nfe2-related factors, Nrfs) activate the expression of genes whose protein products contribute to reduced toxicity. Here, we studied the role of the CNC protein, Nfe2, in the developmental response to pro-oxidant exposure in the zebrafish (Danio rerio). Following acute waterborne exposures to diquat or tert-buytlhydroperoxide (tBOOH) at one of three developmental stages, wildtype (WT) and nfe2 knockout (KO) embryos and larvae were morphologically scored and their transcriptomes sequenced. Early in development, KO animals suffered from hypochromia that was made more severe through exposure to pro-oxidants; this phenotype in the KO may be linked to decreased expression of alas2, a gene involved in heme synthesis. WT and KO eleutheroembryos and larvae were phenotypically equally affected by exposure to pro-oxidants, where tBOOH caused more pronounced phenotypes as compared to diquat. Comparing diquat and tBOOH exposed embryos relative to the WT untreated control, a greater number of genes were up-regulated in the tBOOH condition as compared to diquat (tBOOH: 304 vs diquat: 148), including those commonly found to be differentially regulated in the vertebrate oxidative stress response (OSR) (e.g. hsp70.2, txn1, and gsr). When comparing WT and KO across all treatments and times, there were 1170 genes that were differentially expressed, of which 33 are known targets of the Nrf proteins Nrf1 and Nrf2. More specifically, in animals exposed to pro-oxidants a total of 968 genes were differentially expressed between WT and KO across developmental time, representing pathways involved in coagulation, embryonic organ development, body fluid level regulation, erythrocyte differentiation, and oxidation-reduction, amongst others. The greatest number of genes that changed in expression between WT and KO occurred in animals exposed to diquat at 2h post fertilization (hpf). Across time and treatment, there were six genes (dhx40, cfap70, dnajb9b, slc35f4, spi-c, and gpr19) that were significantly up-regulated in KO compared to WT and four genes (fhad1, cyp4v7, nlrp12, and slc16a6a) that were significantly down-regulated. None of these genes have been previously identified as targets of Nfe2 or the Nrf family. These results demonstrate that the zebrafish Nfe2 may be a regulator of both primitive erythropoiesis and the OSR during development.
Genes of the major histocompatibility complex (MHC) exhibit heterozygote advantage in immune defence, which in turn can select for MHC-disassortative mate choice. However, many species lack this expected pattern of MHC-disassortative mating. A possible explanation lies in evolutionary processes following gene duplication: if two duplicated MHC genes become functionally diverged from each other, offspring will inherit diverse multilocus genotypes even under random mating. We used locus-specific primers for high-throughput sequencing of two expressed MHC Class II B genes in Leach’s storm-petrels, Oceanodroma leucorhoa, and found that exon 2 alleles fall into two gene-specific monophyletic clades. We tested for disassortative vs. random mating at these two functionally diverged Class II B genes, using multiple metrics and different subsets of exon 2 sequence data. With good statistical power, we consistently found random assortment of mates at MHC. Despite random mating, birds had MHC genotypes with functionally diverged alleles, averaging 13 amino acid differences in pairwise comparisons of exon 2 alleles within individuals. To test whether this high MHC diversity in individuals is driven by evolutionary divergence of the two duplicated genes, we built a phylogenetic permutation model. The model showed that genotypic diversity was strongly impacted by sequence divergence between the most common allele of each gene, with a smaller additional impact of monophyly of the two genes. Divergence of allele sequences between genes may have reduced the benefits of actively seeking MHC-dissimilar mates, in which case the evolutionary history of duplicated genes is shaping the adaptive landscape of sexual selection.