Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor

Helicobacter pylori Membrane Vesicles Stimulate Innate Pro- and Anti-Inflammatory Responses and Induce Apoptosis in Jurkat T Cells

Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity

High incidence of antibiotic resistance amongst isolates of Helicobacter pylori collected in Nottingham, UK, between 2001 and 2018

Introduction. Helicobacter pylori is the leading cause of peptic ulcers and gastric cancer. The most common treatment regimens use combinations of two or three antibiotics and a proton pump inhibitor (PPI) to suppress stomach acid. The World Health Organization designated clarithromycin-resistant H. pylori as a high priority pathogen for drug development, due to increasing antibiotic resistance globally.Hypothesis/Gap Statement. There is no routine surveillance of H. pylori primary antimicrobial sensitivities in the UK, and published data are lacking.Aim. This study aimed to characterize antimicrobial sensitivities of isolates collected in Nottingham, UK, between 2001 and 2018.Methodology. Gastric biopsy samples were collected, with informed written consent and ethics approval, from 162 patients attending the Queen’s Medical Centre in Nottingham for an upper GI tract endoscopy. Antibiotic sensitivity was assessed using E-Tests and a more cost-effective disc diffusion test.Results. The clarithromycin, amoxicillin and levofloxacin disc diffusion tests provided identical results to E-Tests on a subset of 30 isolates. Disparities were observed in the metronidazole test results, however. In total, 241 isolates from 162 patients were tested using at least one method. Of all isolates, 28 % were resistant to clarithromycin, 62 % to metronidazole and 3 % to amoxicillin, which are used in first-line therapies. For those antibiotics used in second- and third-line therapies, 4 % were resistant to levofloxacin and none of the isolates were resistant to tetracycline. Resistance to more than one antibiotic was found in 27 % of isolates. The frequency of patients with a clarithromycin-resistant strain increased dramatically over time: from 16 % between 2001 and 2005 to 40 % between 2011 and 2018 (P=0.011). For the same time periods, there was also an increase in those with a metronidazole-resistant strain (from 58 to 78 %; P=0.05). The frequencies of clarithromycin and metronidazole resistance were higher in isolates from patients who had previously received eradication therapy, compared to those who had not (40 % versus 77 %, and 80 % versus 92 %, respectively). Of 79 pairs of isolates from the antrum and corpus regions of the same patient’s stomach, only six had differences in their antimicrobial susceptibility profiles.Conclusion. Although there was high and increasing resistance to clarithromycin and metronidazole, there was no resistance to tetracycline and the frequencies of amoxicillin and levofloxacin resistance were very low

The UmuC subunit of the E. coli DNA polymerase V shows a unique interaction with the β-clamp processivity factor

Background Strict regulation of replisome components is essential to ensure the accurate transmission of the genome to the next generation. The sliding clamp processivity factors play a central role in this regulation, interacting with both DNA polymerases and multiple DNA processing and repair proteins. Clamp binding partners share a common peptide binding motif, the nature of which is essentially conserved from phage through to humans. Given the degree of conservation of these motifs, much research effort has focussed on understanding how the temporal and spatial regulation of multiple clamp binding partners is managed. The bacterial sliding clamps have come under scrutiny as potential targets for rational drug design and comprehensive understanding of the structural basis of their interactions is crucial for success. Results In this study we describe the crystal structure of a complex of the E. coli β-clamp with a 12-mer peptide from the UmuC protein. UmuC is the catalytic subunit of the translesion DNA polymerase, Pol V (UmuD’2C). Due to its potentially mutagenic action, Pol V is tightly regulated in the cell to limit access to the replication fork. Atypically for the translesion polymerases, both bacterial and eukaryotic, Pol V is heterotrimeric and its β-clamp binding motif (357 QLNLF 361) is internal to the protein, rather than at the more usual C-terminal position. Our structure shows that the UmuC peptide follows the overall disposition of previously characterised structures with respect to the highly conserved glutamine residue. Despite good agreement with the consensus β-clamp binding motif, distinct variation is shown within the hydrophobic binding pocket. While UmuC Leu-360 interacts as noted in other structures, Phe-361 does not penetrate the pocket at all, sitting above the surface. Conclusion Although the β-clamp binding motif of UmuC conforms to the consensus sequence, variation in its mode of clamp binding is observed compared to related structures, presumably dictated by the proximal aspartate residues that act as linker to the poorly characterised, unique C-terminal domain of UmuC. Additionally, interactions between Asn-359 of UmuC and Arg-152 on the clamp surface may compensate for the reduced interaction of Phe-361

Effects of α-synuclein overexpression in transgenic Caenorhabditis elegans strains

The neural protein α-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson’s disease pathogenesis. We have generated α-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human α-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, gamma-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing α-synuclein showed significant reductions (p < 0.05) in lifespan, motility and pharyngeal pumping, as compared to wildtype worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms

Understanding the roles of economy and society in the relative risks of zoonosis emergence from livestock

The emergence of zoonotic infections that can develop into pathogens of pandemic potential is a major concern for public health. The risks of emergence and transmission relate to multiple factors that range from land use to human-non-human animal contacts. Livestock agriculture plays a potentially significant role in those risks, shaping landscapes and providing hosts that can act as the source or amplifiers of emergent pathogens. The relative risks will be contingent upon the nature of those systems, with comparisons often made between intensive, indoor, biosecure systems and more extensive, outdoor, insecure systems. Microbiological, ecological and veterinary sciences provide useful entry points in specifying and modelling some of the relative risks. Yet, they often do so with little regard for social science inputs and by making assumptions about social and economic conditions. In this article, we respond to recent analyses of relative risks by raising the importance of social and economic drivers of risk. We chart social science insights and research that materially alter the zoonotic risks associated with livestock production. Our purpose is to emphasize the requirement for full appreciation of the social, economic and political components of zoonotic and pandemic risk

Technology-enabled and universally designed assessment: Considering access in measuring the achievement of students with disabilities—A foundation for research

This paper represents one outcome from the Invitational Research Symposium on Technology-Enabled and Universally Designed Assessments, which examined technology-enabled assessments (TEA) and universal design (UD) as they relate to students with disabilities (SWD). It was developed to stimulate research into TEAs designed to make tests appropriate for the full range of the student population through enhanced accessibility. Four themes are explored: (a) a construct-centered approach to developing accessible assessments; (b) how technology and UD can provide access to targeted knowledge, skills, and abilities by embedding access and interactive features directly into systems that deliver TEAs; (c) the possibility of incorporating scaffolding directly into innovative assessment items; and (d) the importance of investigating the validity of inferences from TEAs that incorporate accessibility features designed to maximize validity. Through the paper, symposium participants and contributing authors share their understanding of issues and offer insights to researchers who conduct studies on the design, development, and validation of technology-enabled and universally designed assessments that include SWD. The paper proposes a focused research agenda and makes it clear that a principled program of research is needed to properly develop and use technology-enabled and universally designed educational assessments that encourage the inclusion of SWD. As research progresses, TEAs need to improve how they assess studentsâ understanding of complex academic content and how they provide equitable access to all students including SWD

Technology-enabled and universally designed assessment: Considering access in measuring the achievement of students with disabilities—A foundation for research

Research Symposium on Technology-Enabled and Universally Designed Assessments, which examined technology-enabled assessments (TEA) and universal design (UD) as they relate to students with disabilities (SWD). It was developed to stimulate research into TEAs designed to make tests appropriate for the full range of the student population through enhanced accessibility. Four themes are explored: (a) a construct-centered approach to developing accessible assessments; (b) how technology and UD can provide access to targeted knowledge, skills, and abilities by embedding access and interactive features directly into systems that deliver TEAs; (c) the possibility of incorporating scaffolding directly into innovative assessment items; and (d) the importance of investigating the validity of inferences from TEAs that incorporate accessibility features designed to maximize validity. Through the paper, symposium participants and contributing authors share their understanding of issues and offer insights to researchers who conduct studies on the design, development, and validation of technology-enabled and universally designed assessments that include SWD. The paper proposes a focused research agenda and makes it clear that a principled program of research is needed to properly develop and use technology-enabled and universally designed educational assessments that encourage the inclusion of SWD. As research progresses, TEAs need to improve how they assess studentsâ understanding of complex academic content and how they provide equitable access to all students including SWD