Molecular Epidemiology of Antimicrobial-Resistant Commensal Escherichia coli Strains in a Cohort of Newborn Calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp^r) and nalidixic acid-resistant (Nal^r) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp^r population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal^r population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp^r genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp^r genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp^r isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

Fundulus heteroclitus: ovarian reproductive physiology and the impact of environmental contaminants

Fundulus heteroclitus, the mummichog or Atlantic killifish, is the dominant small-bodied fish species of the east coast estuaries and salt marshes of Canada and the USA, where it is present as two subspecies, the northern F. h. macrolepidotus and the southern F. h. heteroclitus. Recently identified as the premier teleost model in environmental biology, the species has long been of value in understanding evolved tolerance to toxicants and more lately in adding to our knowledge about reproductive effects of environmental endocrine disruptors. The body of literature on F. heteroclitus ovarian physiology and reproduction, from both field and laboratory studies, provides the foundation for present work focused on understanding the reproductive effects and modes of action of environmental toxicants. In this paper, we review the environmental and endocrine factors controlling ovarian and reproductive cycling in F. heteroclitus, noting specifics related to field and laboratory studies on the two subspecies as well as key research gaps compared to other fish species. We also summarize recent development of methodologies to study the effects of environmental contaminants on endocrine signalling and egg production in F. heteroclitus. Continued efforts to progress both our fundamental understanding of reproductive physiology in mummichog, coupled with studies focused on the modes of action of environmental contaminants, have high potential to further develop this teleost model. While the model may presently lag behind those based on other species of fish, the unique biochemical and physiological adaptations which allow F. heteroclitus to adapt to changing environmental and toxic conditions provide a valuable experimental system for comparative physiologists, ecotoxicologists and evolutionary biologists.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Osteoarchaeological Studies of Human Systemic Stress of Early Urbanization in Late Shang at Anyang,China

Through the analysis of human skeletal remains and mortuary practice in Yinxu, this study investigates the impact of early urbanization on the commoners during the Late Shang dynasty (ca. 1250–1046 B.C.). A total of 347 individuals examined in this study represent non-elites who were recovered from two different burial contexts (formally buried in lineage cemeteries and randomly scattered in refuse pits). Frequencies of enamel hypoplasia (childhood stress), cribra orbitalia (childhood stress and frailty) and osteoperiostitis (adult stress) were examined to assess systemic stress exposure. Our results reveal that there was no significant difference in the frequency of enamel hypoplasia between two burial groups and between sexes, suggesting these urban commoners experienced similar stresses during childhood, but significantly elevated levels of cribra orbitalia and osteoperiostitis were observed in the refuse pit female cohort. Theoretically, urbanization would have resulted in increased population density in the urban centre, declining sanitary conditions, and increased risk of resource shortage. Biologically, children would be more vulnerable to such physiological disturbance; as a result, high percentages of enamel hypoplasia (80.9% overall) and cribra orbitalia (30.3% overall) are observed in Yin commoners. Adults continued to suffer from stress, resulting in high frequencies of osteoperiostitis (40.0% total adults); in particular, in the refuse pit females who may also reflect a compound impact of gender inequality. Our data show that the non-elite urban population in the capital city of Late Shang Dynasty had experienced extensive stress exposure due to early urbanization with further social stratification only worsening the situation, and eventually contributing to collapse of the Shang Dynasty.     

Neurospora Trehalase and Its Structural Gene

We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.     

Nitrosylation of ISG15 prevents the disulfide bond-mediated dimerization of ISG15 and contributes to effective ISGylation

The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.