The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae.

In most members of the Enterobacteriaceae, including Escherichia coli and Salmonella, the lipopolysaccharide core oligosaccharide backbone is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. Mutants lacking the core heptose region and the phosphate residues display pleiotrophic defects collectively known as the deep-rough phenotype, characterized by changes in outer membrane structure and function. Klebsiella pneumoniae lacks phosphoryl residues in its core, but instead contains galacturonic acid. The goal of this study was to determine the contribution of galacturonic acid as a critical source of negative charge. A mutant was created lacking all galacturonic acid by targeting UDP-galacturonic acid precursor synthesis through a mutation in gla(KP). Gla(KP) is a K. pneumoniae UDP-galacturonic acid C4 epimerase providing UDP-galacturonic acid for core synthesis. The gla(KP) gene was inactivated and the structure of the mutant lipopolysaccharide was determined by mass spectrometry. The mutant displayed characteristics of a deep-rough phenotype, exhibiting a hypersensitivity to hydrophobic compounds and polymyxin B, an altered outer membrane profile, and the release of the periplasmic enzyme beta-lactamase. These results indicate that the negative charge provided by the carboxyl groups of galacturonic acid do play an equivalent role to the core oligosaccharide phosphate residues in establishing outer membrane integrity in E. coli and Salmonella

Crystallization and preliminary crystallographic analysis of the bacterial capsule assembly-regulating tyrosine phosphatases Wzb of Escherichia coli and Cps4B of Streptococcus pneumoniae

The crystallization is reported of two bacterial tyrosine phosphatases which belong to different enzyme families despite their ability to catalyse identical reactions

The assembly system for the lipopolysaccharide R2 core-type of Escherichia coli is a hybrid of those found in Escherichia coli K-12 and Salmonella enterica. Structure and function of the R2 WaaK and WaaL homologs.

In Escherichia coli F632, the 14-kilobase pair chromosomal region located between waaC (formerly rfaC) and waaA (kdtA) contains genes encoding enzymes required for the synthesis of the type R2 core oligosaccharide portion of lipopolysaccharide. Ten of the 13 open reading frames encode predicted products sharing greater than 90% total similarity with homologs in E. coli K-12. However, the products of waaK (rfaK) and waaL (rfaL) each resemble homologs in Salmonella enterica serovar Typhimurium but share little similarity with E. coli K-12. The F632 WaaK and WaaL proteins therefore define differences between the type R2 and K-12 outer core oligosaccharides of E. coli lipopolysaccharides. Based on the chemical structure of the core oligosaccharide of an E. coli F632 waaK::aacC1 mutant and in vitro glycosyltransferase analyses, waaK encodes UDP-N-acetylglucosamine:(glucose) lipopolysaccharide alpha1, 2-N-acetylglucosaminyltransferase. The WaaK enzyme adds a terminal GlcNAc side branch substituent that is crucial for the recognition of core oligosaccharide acceptor by the O-polysaccharide ligase, WaaL. Results of complementation analyses of E. coli K-12 and F632 waaL mutants suggest that structural differences between the WaaL proteins play a role in recognition of, and interaction with, terminal lipopolysaccharide core moieties

Who is a Student: Completion in Coursera Courses at Duke University

Much of the interest in MOOCs centers on questions about who completes them. Duke’s Coursera-based Massive Open Online Courses (MOOCs) confirm many demographic trends previously delineated by researchers at peer institutions. As found in previous research, this study found individuals who speak English as a first language and who already earned at least a bachelor’s degree are the most likely to complete a Coursera course. MOOC researchers to date have not, however, developed clear operational definitions about who constitutes a learner at the outset of the course. This paper proposes some possible definitions to standardize future research. Further, this study looked at factors that predict different learner participation levels and investigated which activities predict Coursera course completion. Study results indicated that viewing online forums and participation in online discussions are both predictive of course completion. The findings suggest that the socio-demographic composition of the group being investigated will depend on how researchers elect to define what a student is. Thus, while any of the definitions presented in this paper may be appropriate, depending on what is being studied, the decision of which definition to use should be intentional

Chromosomal and plasmid-encoded enzymes are required for assembly of the R3-type core oligosaccharide in the lipopolysaccharide of Escherichia coli O157:H7.

The type R3 core oligosaccharide predominates in the lipopolysaccharides from enterohemorrhagic Escherichia coli isolates including O157:H7. The R3 core biosynthesis (waa) genetic locus contains two genes, waaD and waaJ, that are predicted to encode glycosyltransferases involved in completion of the outer core. Through determination of the structures of the lipopolysaccharide core in precise mutants and biochemical analyses of enzyme activities, WaaJ was shown to be a UDP-glucose:(galactosyl) lipopolysaccharide alpha-1,2-glucosyltransferase, and WaaD was shown to be a UDP-glucose:(glucosyl)lipopolysaccharide alpha-1,2-glucosyltransferase. The residue added by WaaJ was identified as the ligation site for O polysaccharide, and this was confirmed by determination of the structure of the linkage region in serotype O157 lipopolysaccharide. The initial O157 repeat unit begins with an N-acetylgalactosamine residue in a beta-anomeric configuration, whereas the biological repeat unit for O157 contains alpha-linked N-acetylgalactosamine residues. With the characterization of WaaJ and WaaD, the activities of all of the enzymes encoded by the R3 waa locus are either known or predicted from homology data with a high level of confidence. However, when core oligosaccharide structure is considered, the origin of an additional alpha-1,3-linked N-acetylglucosamine residue in the outer core is unknown. The gene responsible for a nonstoichiometric alpha-1,7-linked N-acetylglucosamine substituent in the heptose (inner core) region was identified on the large virulence plasmids of E. coli O157 and Shigella flexneri serotype 2a. This is the first plasmid-encoded core oligosaccharide biosynthesis enzyme reported in E. coli

Sourcing knowledge for innovation:the international dimension

Drawing knowledge from external sources in the UK, or internationally, has become increasingly important to small and medium-sized firms (SMEs). SMEs cannot generate all they need to know to develop new products and processes within their own companies, they need to look elsewhere for new ideas and expertise. This practice is known as knowledge sourcing. This report provides a detailed review of patterns of knowledge sourcing, and the key factors influencing these patterns, particularly from a small business perspective. We present key findings from a survey of 393 UK companies and analyse the results. We also highlight case studies of UK SMEs that work closely with overseas partners and agents to widen their own knowledge

Involvement of waaY, waaQ, and waaP in the Modification of Escherichia coliLipopolysaccharide and Their Role in the Formation of a Stable Outer Membrane *

The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations

Interference of the T cell and antigen-presenting cell costimulatory pathway using CTLA4-Ig (abatacept) prevents Staphylococcal enterotoxin B pathology

Abstract Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds the receptors in the APC/T cell synapse and causes increased proliferation of T cells and a cytokine storm syndrome in vivo. Exposure to the toxin can be lethal and cause significant pathology in humans. The lack of effective therapies for SEB exposure remains an area of concern, particularly in scenarios of acute mass casualties. We hypothesized that blockade of the T cell costimulatory signal by the CTLA4-Ig synthetic protein (abatacept) could prevent SEB-dependent pathology. In this article, we demonstrate mice treated with a single dose of abatacept 8 h post SEB exposure had reduced pathology compared with control SEB-exposed mice. SEB-exposed mice showed significant reductions in body weight between days 4 and 9, whereas mice exposed to SEB and also treated with abatacept showed no weight loss for the duration of the study, suggesting therapeutic mitigation of SEB-induced morbidity. Histopathology and magnetic resonance imaging demonstrated that SEB mediated lung damage and edema, which were absent after treatment with abatacept. Analysis of plasma and lung tissues from SEB-exposed mice treated with abatacept demonstrated significantly lower levels of IL-6 and IFN-γ (p &amp;lt; 0.0001), which is likely to have resulted in less pathology. In addition, exposure of human and mouse PBMCs to SEB in vitro showed a significant reduction in levels of IL-2 (p &amp;lt; 0.0001) after treatment with abatacept, indicating that T cell proliferation is the main target for intervention. Our findings demonstrate that abatacept is a robust and potentially credible drug to prevent toxic effects from SEB exposure.</jats:p

Crystallization and preliminary diffraction analysis of Wzi, a member of the capsule export and assembly pathway in Escherichia coli

Wzi is a membrane protein from E. coli thought to be involved in the attachment of capsular polysaccharides to the bacterial surface. This reports describes recombinant Wzi’s purification, crystallization and the results of initial diffraction studies

Social science quantitative methods capacity building in Wales: ESRC/HEFCW scoping study

Previous research undertaken by the ESRC has revealed a “deficit” of quantitative social science researchers and identified that this should be tackled early in the academic life course. However, there is substantial heterogeneity across disciplines with previous studies indicating what while some subjects suffer serious deficits in quantitative methods research capacity, other disciplines such as economics and psychology are perceived to have strengths in quantitative methods training and research. There may be a particular problem in quantitative social science in Wales (possibly relating to the visible “Welsh deficit” in social science funding); however it is difficult to identify the configuration, strengths and weaknesses of quantitative social science in Wales from routine data. To provide more data on the current position of quantitative social science in Wales, and to identify potential ways forward to improve the situation in Wales, ESRC and HEFCW jointly funded this scoping study. The study mapped quantitative social science research (and training) expertise in Wales by undertaking an all-Wales questionnaire survey of social scientists in Higher Education Institutions in Wales. This was complemented by a number of semi-structured interviews with key stakeholders with an interest or expertise in quantitative social science research methods. A workshop including key stakeholders was then held to discuss the outcomes of the survey and interviews and recommendations for future action. This report presents the main findings of the study, which include the need for future actions to recognise the differences between disciplines, to not solely focus on advanced quantitative methods, and to be linked with wider initiatives to improve social science research methods training and capacity more generally. Many issues identified were not Wales specific and optimal solutions include increasing access to and participation in wider UK initiatives rather than solely Wales based actions. The report recommends the creation of a Centre in Wales to co-ordinate and deliver Wales-based solutions, link with UK initiatives and break down disciplinary and methodological barriers. Other recommendations address the deficit at different stages in the academic life course; undergraduate, postgraduate, post-doctoral and continued professional development, as well as suggestions for building wider quantitative capacity in Wales, monitoring and further research