Structural and Biophysical Investigations of Two Negative Regulators of DNA Replication Initiation in Bacillus subtilis: YabA and SirA

DNA replication initiation is strictly controlled to maintain chromosome copy number. Over- or under-replication of an organism's genome is detrimental to survival, thus initiation events are tightly regulated to ensure only one round of DNA replication occurs per cell cycle. In prokaryotes, DNA replication commences when a protein initiator, DnaA, forms a helical filament at the origin of replication, oriC, inducing localised DNA unwinding and the recruitment of the replication machinery. The work described here sought to elucidate how DNA replication initiation is regulated in the Gram-positive model organism Bacillus subtilis. In particular, this work aimed to offer insight into the mechanisms of two negative regulators of DNA replication initiation, SirA and YabA; proteins that play significant roles in regulating replication initiation in sporulating and vegetatively growing cells, respectively. Both SirA and YabA interact directly with the initiator DnaA, and YabA has additionally been shown to interact with the DNA polymerase β-clamp, DnaN (an essential component of the replication machinery). Detailed here are structural and biophysical studies of SirA and YabA. This includes the X-ray crystal structure of SirA bound to the N-terminal domain of DnaA in an inhibitory complex, and characterisation of the SirA-DnaADI interface using an in vitro assay. When coupled with in vivo localisation studies carried out by our collaborators, the work on SirA suggests a mechanism for its inhibition of DNA replication. Also detailed are biophysical studies used to characterise the architecture of YabA, guided by the use of in silico models, and the X-ray crystal structure of YabA's N-terminal domain. These results are discussed alongside structural and mutational studies of YabA carried out by our collaborators; collectively the results delineate the full-length structure of YabA and offer insight into its interactions with DnaA and DnaN

In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay

Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues

cDNA Sequence and Fab Crystal Structure of HL4E10, a Hamster IgG Lambda Light Chain Antibody Stimulatory for γδ T Cells

Hamsters are widely used to generate monoclonal antibodies against mouse, rat, and human antigens, but sequence and structural information for hamster immunoglobulins is sparse. To our knowledge, only three hamster IgG sequences have been published, all of which use kappa light chains, and no three-dimensional structure of a hamster antibody has been reported. We generated antibody HL4E10 as a probe to identify novel costimulatory molecules on the surface of γδ T cells which lack the traditional αβ T cell co-receptors CD4, CD8, and the costimulatory molecule CD28. HL4E10 binding to γδ T cell, surface-expressed, Junctional Adhesion Molecule-Like (JAML) protein leads to potent costimulation via activation of MAP kinase pathways and cytokine production, resulting in cell proliferation. The cDNA sequence of HL4E10 is the first example of a hamster lambda light chain and only the second known complete hamster heavy chain sequence. The crystal structure of the HL4E10 Fab at 2.95 Å resolution reveals a rigid combining site with pockets faceted by solvent-exposed tyrosine residues, which are structurally optimized for JAML binding. The characterization of HL4E10 thus comprises a valuable addition to the spartan database of hamster immunoglobulin genes and structures. As the HL4E10 antibody is uniquely costimulatory for γδ T cells, humanized versions thereof may be of clinical relevance in treating γδ T cell dysfunction-associated diseases, such as chronic non-healing wounds and cancer

Linking soil microbial community structure to potential carbon mineralization: A continental scale assessment of reduced tillage

Potential carbon mineralization (Cmin) is a commonly used indicator of soil health, with greater Cmin values interpreted as healthier soil. While Cmin values are typically greater in agricultural soils managed with minimal physical disturbance, the mechanisms driving the increases remain poorly understood. This study assessed bacterial and archaeal community structure and potential microbial drivers of Cmin in soils maintained under various degrees of physical disturbance. Potential carbon mineralization, 16S rRNA sequences, and soil characterization data were collected as part of the North American Project to Evaluate Soil Health Measurements (NAPESHM). Results showed that type of cropping system, intensity of physical disturbance, and soil pH influenced microbial sensitivity to physical disturbance. Furthermore, 28% of amplicon sequence variants (ASVs), which were important in modeling Cmin, were enriched under soils managed with minimal physical disturbance. Sequences identified as enriched under minimal disturbance and important for modeling Cmin, were linked to organisms which could produce extracellular polymeric substances and contained metabolic strategies suited for tolerating environmental stressors. Understanding how physical disturbance shapes microbial communities across climates and inherent soil properties and drives changes in Cmin provides the context necessary to evaluate management impacts on standardized measures of soil microbial activity

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Formation of functional E3 ligase complexes with UBC2 and UEV1 of Leishmania mexicana

In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation

PROMIS - Prostate MR imaging study: a paired validating cohort study evaluating the role of multi-parametric MRI in men with clinical suspicion of prostate cancer

Background: Transrectal ultrasound-guided prostate biopsies are prone to detection errors. Multi-parametric MRI (MP-MRI) may improve the diagnostic pathway. Methods: PROMIS is a prospective validating paired-cohort study that meets criteria for level 1 evidence in diagnostic test evaluation. PROMIS will investigate whether multi-parametric (MP)-MRI can discriminate between men with and without clinically-significant prostate cancer who are at risk prior to first biopsy. Up to 714 men will have MP-MRI (index), 10-12 core TRUS-biopsy (standard) and 5. mm transperineal template mapping (TPM) biopsies (reference). The conduct and reporting of each test will be blinded to the others. Results: PROMIS will measure and compare sensitivity, specificity, and positive and negative predictive values of both MP-MRI and TRUS-biopsy against TPM biopsies. The MP-MRI results will be used to determine the proportion of men who could safely avoid biopsy without compromising detection of clinically-significant cancers