Preventing broken Borrelia telomeres: ResT couples dual hairpin telomere formation with product release

Spirochetes of the genus Borrelia include the tick-transmitted causative agents of Lyme disease and relapsing fever. They possess unusual genomes composed mainly of linear replicons terminated by closed DNA hairpins. Hairpin telomeres are formed from inverted repeat replicated telomere junctions (rTels) by the telomere resolvase ResT. ResT uses a reaction mechanism similar to that of the type IB topoisomerases and tyrosine recombinases. ResT can catalyze three distinct reactions: telomere resolution, telomere fusion, and Holliday junction (HJ) formation. HJ formation is known to occur only in the context of a synapsed pair of rTels. To test whether telomere resolution was synapsis-dependent, we performed experiments with rTel substrates immobilized on streptavidin-coated beads. We report that telomere resolution by ResT is synapsis-independent, indicating that alternative complexes are formed for telomere resolution and HJ formation. We also present evidence that dual hairpin telomere formation precedes product release. This mechanism of telomere resolution prevents the appearance of broken telomeres. We compare and contrast this mechanism with that proposed for TelK, the telomere resolvase of φKO2.     

Mucosal lymphatic-derived gammadelta T cells respond early to experimental Salmonella enterocolitis by increasing expression of IL-2R alpha

To better understand the roles of gammadelta T cells in mucosal infection, we utilized Salmonella enterica serovar Typhimurium (Salmonella serovar Typhimurium) infection in cattle as it closely approximates Salmonella serovar Typhimurium-induced enterocolitis in humans. Protein and gene expression in alphabeta and gammadelta T cells derived from lymphatic ducts draining the gut mucosa in Salmonella serovar Typhimurium-infected calves were analyzed. In calves with enterocolitis, general gene expression trends in gammadelta T cells suggested subtle activation and innate response, whereas alphabeta T cells were relatively quiescent following Salmonella serovar Typhimurium infection. An increase in IL-2R alpha expression on gammadelta T cells from infected calves and results from in vitro assays suggested that gammadelta T cells were primed by Salmonella serovar Typhimurium LPS to better respond to IL-2 and IL-15. Together with gene expression trends in vivo, these data support early priming activation of target tissue gammadelta T cells during Salmonella serovar Typhimurium infection.     

Effect of myostatin depletion on weight gain, hyperglycemia, and hepatic steatosis during five months of high-fat feeding in mice

The marked hypermuscularity in mice with constitutive myostatin deficiency reduces fat accumulation and hyperglycemia induced by high-fat feeding, but it is unclear whether the smaller increase in muscle mass caused by postdevelopmental loss of myostatin activity has beneficial metabolic effects during high-fat feeding. We therefore examined how postdevelopmental myostatin knockout influenced effects of high-fat feeding. Male mice with ubiquitous expression of tamoxifen-inducible Cre recombinase were fed tamoxifen for 2 weeks at 4 months of age. This depleted myostatin in mice with floxed myostatin genes, but not in control mice with normal myostatin genes. Some mice were fed a high-fat diet (60% of energy) for 22 weeks, starting 2 weeks after cessation of tamoxifen feeding. Myostatin depletion increased skeletal muscle mass ～30%. Hypermuscular mice had ～50% less weight gain than control mice over the first 8 weeks of high-fat feeding. During the subsequent 3 months of high-fat feeding, additional weight gain was similar in control and myostatin-deficient mice. After 5 months of high-fat feeding, the mass of epididymal and retroperitoneal fat pads was similar in control and myostatin-deficient mice even though myostatin depletion reduced the weight gain attributable to the high-fat diet (mean weight with high-fat diet minus mean weight with low-fat diet: 19.9 g in control mice, 14.1 g in myostatin-deficient mice). Myostatin depletion did not alter fasting blood glucose levels after 3 or 5 months of high-fat feeding, but reduced glucose levels measured 90 min after intraperitoneal glucose injection. Myostatin depletion also attenuated hepatic steatosis and accumulation of fat in muscle tissue. We conclude that blocking myostatin signaling after maturity can attenuate some of the adverse effects of a high-fat diet.     

Multiple loci are associated with white blood cell phenotypes

White blood cell (WBC) count is a common clinical measure from complete blood count assays, and it varies widely among healthy individuals. Total WBC count and its constituent subtypes have been shown to be moderately heritable, with the heritability estimates varying across cell types. We studied 19,509 subjects from seven cohorts in a discovery analysis, and 11,823 subjects from ten cohorts for replication analyses, to determine genetic factors influencing variability within the normal hematological range for total WBC count and five WBC subtype measures. Cohort specific data was supplied by the CHARGE, HeamGen, and INGI consortia, as well as independent collaborative studies. We identified and replicated ten associations with total WBC count and five WBC subtypes at seven different genomic loci (total WBC count-6p21 in the HLA region, 17q21 near ORMDL3, and CSF3; neutrophil count-17q21; basophil count- 3p21 near RPN1 and C3orf27; lymphocyte count-6p21, 19p13 at EPS15L1; monocyte count-2q31 at ITGA4, 3q21, 8q24 an intergenic region, 9q31 near EDG2), including three previously reported associations and seven novel associations. To investigate functional relationships among variants contributing to variability in the six WBC traits, we utilized gene expression- and pathways-based analyses. We implemented gene-clustering algorithms to evaluate functional connectivity among implicated loci and showed functional relationships across cell types. Gene expression data from whole blood was utilized to show that significant biological consequences can be extracted from our genome-wide analyses, with effect estimates for significant loci from the meta-analyses being highly corellated with the proximal gene expression. In addition, collaborative efforts between the groups contributing to this study and related studies conducted by the COGENT and RIKEN groups allowed for the examination of effect homogeneity for genome-wide significant associations across populations of diverse ancestral backgrounds.     

Blubber Cortisol: A Potential Tool for Assessing Stress Response in Free-Ranging Dolphins without Effects due to Sampling

When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measurements exhibited appropriate quality characteristics when analyzed via a bootstraped stepwise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 – 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75–150mg; r² = 0.012, p = 0.78, slope = 0.022ng_{cortisol deviation}/ul_{tissue extract added}). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality type (e.g., stranded or bycaught), 2) specimen condition (state of decomposition), 3) total body length, 4) sex, 5) sexual maturity state, 6) pregnancy status, 7) lactation state, and 8) adrenal mass, were assessed using a Bayesian generalized linear model averaging technique. Fatality type was the only factor correlated with blubber cortisol, and the magnitude of the effect size was substantial: beach-stranded individuals had on average 6.1-fold higher cortisol levels than those of bycaught individuals. Because of the difference in conditions surrounding these two fatality types, we interpret this relationship as evidence that blubber cortisol is indicative of stress response. We found no evidence of seasonal variation or a relationship between cortisol and the remaining cofactors.     

Cryptosporidium genotypes in wildlife from a new york watershed

To identify the animal sources for Cryptosporidium contamination, we genotyped Cryptosporidium spp. in wildlife from the watershed of the New York City drinking water supply, using a small-subunit rRNA gene-based PCR-restriction fragment length polymorphism analysis and DNA sequencing. A total of 541 specimens from 38 species of wildlife were analyzed. One hundred and eleven (20.5%) of the wildlife specimens were PCR positive. Altogether, 21 Cryptosporidium genotypes were found in wildlife samples, 11 of which were previously found in storm runoff in the watershed, and six of these 11 were from storm water genotypes of unknown animal origin. Four new genotypes were found, and the animal hosts for four storm water genotypes were expanded. With the exception of the cervine genotype, most genotypes were found in a limited number of animal species and have no major public health significance.     

Three-dimensional structure of a macromolecular assembly that regulates type III secretion in Yersinia pestis

Yersinia pestis, the causative agent of plague, utilizes a type III secretion system (T3SS) to inject effector proteins directly into the cytosol of mammalian cells where they interfere with signal transduction pathways that regulate actin cytoskeleton dynamics and inflammation, thereby enabling the bacterium to avoid engulfment and destruction by macrophages. Type III secretion normally does not occur in the absence of close contact with eukaryotic cells. Negative regulation is mediated in part by a multiprotein complex that has been proposed to act as a physical impediment to type III secretion by blocking the entrance to the secretion apparatus prior to contact with mammalian cells. This complex is composed of YopN, its heterodimeric secretion chaperone SycN-YscB, and TyeA. Here, we report two crystal structures of YopN in complex with its heterodimeric secretion chaperone SycN-YscB and the co-regulatory protein TyeA, respectively. By merging these two overlapping structures, it was possible to construct a credible theoretical model of the YopN-SycN-YscB-TyeA complex. The modeled assembly features the secretion signaling elements of YopN at one end of an elongated structure and the secretion regulating TyeA binding site at the other. A patch of highly conserved residues on the surface of the C-terminal alpha-helix of TyeA may mediate its interaction with structural components of the secretion apparatus. Conserved arginine residues that reside inside a prominent cavity at the dimer interface of SycN-YscB were mutated in order to investigate whether they play a role in targeting the YopN-chaperone complex to the type III secretion apparatus. One of the mutants exhibited a phenotype that is consistent with this hypothesis.     

Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.