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.     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pseudomonas fluorescens' view of the periodic table

Growth in a biofilm modulates microbial metal susceptibility, sometimes increasing the ability of microorganisms to withstand toxic metal species by several orders of magnitude. In this study, a high-throughput metal toxicity screen was initiated with the aim of correlating biological toxicity data in planktonic and biofilm cells to the physiochemical properties of metal ions. To this end, Pseudomonas fluorescens ATCC 13525 was grown in the Calgary Biofilm Device (CBD) and biofilms and planktonic cells of this microorganism were exposed to gradient arrays of different metal ions. These arrays included 44 different metals with representative compounds that spanned every group of the periodic table (except for the halogens and noble gases). The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentration (MBEC) values were obtained after exposing the biofilms to metal ions for 4 h. Using these values, metal ion toxicity was correlated to the following ion-specific physicochemical parameters: standard reduction-oxidation potential, electronegativity, the solubility product of the corresponding metal–sulfide complex, the Pearson softness index, electron density and the covalent index. When the ions were grouped according to outer shell electron structure, we found that heavy metal ions gave the strongest correlations to these parameters and were more toxic on average than the other classes of the ions. Correlations were different for biofilms than for planktonic cells, indicating that chemical mechanisms of metal ion toxicity differ between the two modes of growth. We suggest that biofilms can specifically counter the toxic effects of certain physicochemical parameters, which may contribute to the increased ability of biofilms to withstand metal toxicity.     

Using the longest significance run to estimate region-specific p-values in genetic association mapping studies

Background  

Association testing is a powerful tool for identifying disease susceptibility genes underlying complex diseases. Technological advances have yielded a dramatic increase in the density of available genetic markers, necessitating an increase in the number of association tests required for the analysis of disease susceptibility genes. As such, multiple-tests corrections have become a critical issue. However the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. Alternatively, we propose here the application of the longest significant run (LSR) method to estimate a region-specific p-value to provide an index for the most likely candidate region.     

MPDA: Microarray pooled DNA analyzer

Background  

Microarray-based pooled DNA experiments that combine the merits of DNA pooling and gene chip technology constitute a pivotal advance in biotechnology. This new technique uses pooled DNA, thereby reducing costs associated with the typing of DNA from numerous individuals. Moreover, use of an oligonucleotide gene chip reduces costs related to processing various DNA segments (e.g., primers, reagents). Thus, the technique provides an overall cost-effective solution for large-scale genomic/genetic research. However, few publicly shared tools are available to systematically analyze the rapidly accumulating volume of whole-genome pooled DNA data.     

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Rational design of novel glycomimetics: inhibitors of concanavalin A

A virtual screening approach was used to identify new glycomimetics. The National Cancer Institute Diversity Set was docked into the carbohydrate binding site of the lectin concanavalin A (ConA). The resulting poses were analyzed and 19 molecules were tested for inhibition with an enzyme-linked lectin assay (ELLA). Eight of the 19 molecules inhibited ConA-carbohydrate binding. The two most potent inhibitors have IC(50) values that are an order of magnitude smaller than the monosaccharide methyl alpha-D-mannopyranoside.     

A new take on prions: preventing Alzheimer's disease

Alzheimer's disease is a major neurodegenerative disease of the brain, the incidence of which increases dramatically in old age. Currently, no drugs are available to halt or slow the progression of this disease, which poses an ever-expanding burden on health services, families and society. The prion protein has become infamous owing to its role as the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans. However, our view of the prion protein as an unwanted, harmful entity has been challenged recently. New data indicate that the normal cellular form of the prion protein might have a crucial role in suppressing the production of the amyloid-beta peptide, the neurotoxic molecule involved in the pathogenesis of Alzheimer's disease.     

Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation

The Hsp70 molecular chaperones are ATPases that play critical roles in the pathogenesis of many human diseases, including breast cancer. Hsp70 ATP hydrolysis is relatively weak but is stimulated by J domain-containing proteins. We identified pyrimidinone-peptoid hybrid molecules that inhibit cell proliferation with greater potency than previously described Hsp70 modulators. In many cases, anti-proliferative activity correlated with inhibition of J domain stimulation of Hsp70.