Generation of a restriction minus enteropathogenic Escherichia coli E2348/69 strain that is efficiently transformed with large, low copy plasmids

<p>Abstract</p> <p>Background</p> <p>Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic <it>Escherichia coli </it>strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s), with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen.</p> <p>Results</p> <p>Using bioinformatics, we identified genes in the unfinished enteropathogenic <it>Escherichia coli </it>(EPEC) strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP) subunits and type III secreted (T3S) proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S.</p> <p>Conclusion</p> <p>We have shown that EPEC strain E2348/69 utilizes a type I restriction-modification system to limit entry of new DNA. This restriction-modification system does not appear to be involved in virulence determinant expression or infection phenotypes. The <it>hsdR </it>mutant strain should prove useful in genetic analysis of the important diarrheal pathogen EPEC.</p

Research on Elementary, Middle, and Secondary Earth and Space Sciences Teacher Education

In order to fully engage with the vision of the Framework for K-12 Science Education and the NGSS, our nation needs a diverse and well prepared K-12 science teacher workforce. And in order for ESS to gain equal status with other sciences, the geoscience community must ensure that the K-12 science teacher workforce is adequately prepared to teach ESS core knowledge and practices. This is a challenging endeavor and complicated by the fact that the K-12 teacher education landscape is highly variable across institutions in terms of how much ESS content is included, how programs are structured, and how ESS fits into the larger institutional context. Teacher education exists in a complex landscape that involve many domains of research. This theme chapter focuses on teacher education research that most directly aligns to the undergraduate teaching and learning experience. Three grand challenges emerged from discussion and reflections on the existing literature and are poised to guide future research on undergraduate K-12 teacher education

Dinoflagellate Cysts Track Eutrophication In The Northern Gulf Of Mexico

We examined organic-walled dinoflagellate cysts from one 210Pb-dated sediment core and 39 surface sediment samples from the northern Gulf of Mexico to determine the relationship between nutrient enrichment and cyst assemblages in this region characterized by oxygen deficiency. The core spans from 1962 to 1997 and its sampling location is directly influenced by the Mississippi River plume. Surface sediments were collected in 2006, 2007, 2008, and 2014 and represent approximately 1 to 4 years of accumulation. A total of 57 cyst taxa were recorded, and four heterotrophic taxa in particular were found to increase in the top section (1986–1997) of the core—Brigantedinium spp., cysts of Archaeperidinium minutum, cysts of Polykrikos kofoidii, and Quinquecuspis concreta. These taxa show a similar increasing trend with variations in US fertilizer consumption and Mississippi River nitrate concentrations, both of which increased substantially in the 1970s and 1980s. The same four heterotrophic taxa dominated dinoflagellate cyst assemblages collected near the Mississippi River Bird’s Foot Delta where nutrient concentrations were higher, especially in 2014. We propose that these cyst taxa can be used as indicators of eutrophication in the Gulf of Mexico. A canonical correspondence analysis (CCA) supports this proposition. The CCA identified sea-surface nutrient concentrations, sea-surface temperature, and sea-surface salinity as the most important factors influencing the cyst assemblages. In addition, cysts produced by the potentially toxic dinoflagellates Pyrodinium bahamense and Lingulodinium polyedrum were documented, but did not appear to have increased over the past 50 years

Measuring disease-specific quality of life in rare populations: a practical approach to cross-cultural translation

<p>Abstract</p> <p>Background</p> <p>Disease-specific quality of life (QoL) measures have enhanced the capacity of outcome measures to evaluate subtle changes and differences between groups. However, when the specific disease is rare, the cohort of patients is small and international collaboration is often necessary to accomplish meaningful research. As many of the QoL measures have been developed in North American English, they require translation to ensure their usefulness in a multi-cultural and/or international society. Published guidelines provide formal methods to achieve cross-culturally comparable versions of a QoL tool. However, these guidelines describe a rigorous process that is not always feasible, particularly in rare disease groups. The objective of this manuscript is to describe the process that was developed to achieve accurate cross-cultural translations of a disease-specific QoL measure, to overcome the challenges of a small sample size, i.e. children with a rare disorder.</p> <p>Procedure</p> <p>A measurement study was conducted in the United Kingdom (UK), France, Germany and Uruguay, during which the validated measure was translated into the languages of the respective countries.</p> <p>Results</p> <p>This is a report of a modified, child-centric, cross-cultural translation and adaptation process in which culturally appropriate and methodologically valid translations of a disease-specific QoL measure, the Kids' ITP Tools (KIT), were performed in children with immune thrombocytopenic purpura (ITP). The KIT was translated from North American English into UK English, French, German, and Spanish.</p> <p>Conclusion</p> <p>This study was a successful international collaboration. The modified process through which culturally appropriate and methodologically valid translations of QoL measures may be achieved in a pediatric population with a relatively rare disorder is reported.</p

Board of Regents

This report, fifteenth of an annual series, describes 1993 mineral, oil and gas, and geothermal activities and accomplishments in Nevada: production statistics, exploration and development including drilling for petroleum and geothermal resources, discoveries of orebodies, new mines opened, and expansion and other activities of existing mines. Statistics of known gold and silver deposits, and directories of mines and mills are included. For more information contact

Measuring heritable contributions to Alzheimer's disease: polygenic risk score analysis with twins

The heritability of Alzheimer’s disease estimated from twin studies is greater than the heritability derived from genome-based studies, for reasons that remain unclear. We apply both approaches to the same twin sample, considering both Alzheimer’s disease polygenic risk scores and heritability from twin models, to provide insight into the role of measured genetic variants and to quantify uncaptured genetic risk. A population-based heritability and polygenic association study of Alzheimer’s disease was conducted between 1986 and 2016 and is the first study to incorporate polygenic risk scores into biometrical twin models of Alzheimer’s disease. The sample included 1586 twins drawn from the Swedish Twin Registry which were nested within 1137 twin pairs (449 complete pairs and 688 incomplete pairs) with clinically based diagnoses and registry follow-up (Mage = 85.28, SD = 7.02; 44% male; 431 cases and 1155 controls). We report contributions of polygenic risk scores at P < 1 × 10−5, considering a full polygenic risk score (PRS), PRS without the APOE region (PRS.no.APOE) and PRS.no.APOE plus directly measured APOE alleles. Biometric twin models estimated the contribution of environmental influences and measured (PRS) and unmeasured genes to Alzheimer’s disease risk. The full PRS and PRS.no.APOE contributed 10.1 and 2.4% to Alzheimer’s disease risk, respectively. When APOE ɛ4 alleles were added to the model with the PRS.no.APOE, the total contribution was 11.4% to Alzheimer’s disease risk, where APOE ɛ4 explained 9.3% and PRS.no.APOE dropped from 2.4 to 2.1%. The total genetic contribution to Alzheimer’s disease risk, measured and unmeasured, was 71% while environmental influences unique to each twin accounted for 29% of the risk. The APOE region accounts for much of the measurable genetic contribution to Alzheimer’s disease, with a smaller contribution from other measured polygenic influences. Importantly, substantial background genetic influences remain to be understood

Application of Bayesian network structure learning to identify causal variant SNPs from resequencing data

Using single-nucleotide polymorphism (SNP) genotypes from the 1000 Genomes Project pilot3 data provided for Genetic Analysis Workshop 17 (GAW17), we applied Bayesian network structure learning (BNSL) to identify potential causal SNPs associated with the Affected phenotype. We focus on the setting in which target genes that harbor causal variants have already been chosen for resequencing; the goal was to detect true causal SNPs from among the measured variants in these genes. Examining all available SNPs in the known causal genes, BNSL produced a Bayesian network from which subsets of SNPs connected to the Affected outcome were identified and measured for statistical significance using the hypergeometric distribution. The exploratory phase of analysis for pooled replicates sometimes identified a set of involved SNPs that contained more true causal SNPs than expected by chance in the Asian population. Analyses of single replicates gave inconsistent results. No nominally significant results were found in analyses of African or European populations. Overall, the method was not able to identify sets of involved SNPs that included a higher proportion of true causal SNPs than expected by chance alone. We conclude that this method, as currently applied, is not effective for identifying causal SNPs that follow the simulation model for the GAW17 data set, which includes many rare causal SNPs

Glycoengineered outer membrane vesicles: A novel platform for bacterial vaccines

The World Health Organization has indicated that we are entering into a post-antibiotic era in which infections that were routinely and successfully treated with antibiotics can now be lethal due to the global dissemination of multidrug resistant strains. Conjugate vaccines are an effective way to create a long-lasting immune response against bacteria. However, these vaccines present many drawbacks such as slow development, high price, and batch-to-batch inconsistencies. Alternate approaches for vaccine development are urgently needed. Here we present a new vaccine consisting of glycoengineered outer membrane vesicles (geOMVs). This platform exploits the fact that the initial steps in the biosynthesis of most bacterial glycans are similar. Therefore, it is possible to easily engineer non-pathogenic Escherichia coli lab strains to produce geOMVs displaying the glycan of the pathogen of interest. In this work we demonstrate the versatility of this platform by showing the efficacy of geOMVs as vaccines against Streptococcus pneumoniae in mice, and against Campylobacter jejuni in chicken. This cost-effective platform could be employed to generate vaccines to prevent infections caused by a wide variety of microbial agents in human and animals

Investigations into the Toxicology of Spirolides, a Group of Marine Phycotoxins

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD50 value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD50 values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure