Drosophila RecQ4 Is Directly Involved in Both DNA Replication and the Response to UV Damage in S2 Cells.

The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.Here we show that depletion of DmRecQ4 by dsRNA interference in S2 cells causes defects consistent with a replication function for the protein. The cells show reduced proliferation associated with an S phase block, reduced BrdU incorporation, and an increase in cells with a subG1 DNA content. At the molecular level we observe reduced chromatin association of DNA polymerase-alpha and PCNA. We also observe increased chromatin association of phosphorylated H2AvD--consistent with the presence of DNA damage and increased apoptosis.Analysis of DmRecQ4 repair function suggests a direct role in NER, as the protein shows rapid but transient nuclear localisation after UV treatment. Re-localisation is not observed after etoposide or H₂O₂ treatment, indicating that the involvement of DmRecQ4 in repair is likely to be pathway specific.Deletion analysis of DmRecQ4 suggests that the SLD2 domain was essential, but not sufficient, for replication function. In addition a DmRecQ4 N-terminal deletion could efficiently re-localise on UV treatment, suggesting that the determinants for this response are contained in the C terminus of the protein. Finally several deletions show differential rescue of dsRNA generated replication and proliferation phenotypes. These will be useful for a molecular analysis of the specific role of DmRecQ4 in different cellular pathways

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference.

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment

A simple bypass assay for DNA polymerases shows that cancer-associated hypermutating variants exhibit differences in vitro.

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease-defective and two hypermutating human DNA polymerase ε variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild-type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed misincorporation rate comparable to the exonuclease dead enzyme rather than P286R, suggesting that there may be some differences in the way that these variants cause hypermutation. Using this assay, misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C-to-T transitions. This is consistent with the observation that C-to-T transitions are commonly seen in DNA polymerase ε mutant tumours

Innate Immune Recognition of Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a major health problem, with 10 million new cases diagnosed each year. Innate immunity plays an important role in the host defense against M. tuberculosis, and the first step in this process is recognition of MTB by cells of the innate immune system. Several classes of pattern recognition receptors (PPRs) are involved in the recognition of M. tuberculosis, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and Nod-like receptors (NLRs). Among the TLR family, TLR2, TLR4, and TLR9 and their adaptor molecule MyD88 play the most prominent roles in the initiation of the immune response against tuberculosis. In addition to TLRs, other PRRs such as NOD2, Dectin-1, Mannose receptor, and DC-SIGN are also involved in the recognition of M. tuberculosis. Human epidemiological studies revealed that genetic variation in genes encoding for PRRs and downstream signaling products influence disease susceptibility, severity, and outcome. More insight into PRRs and the recognition of mycobacteria, combined with immunogenetic studies in TB patients, does not only lead to a better understanding of the pathogenesis of tuberculosis but also may contribute to the design of novel immunotherapeutic strategies

The Human TPR Protein TTC4 Is a Putative Hsp90 Co-Chaperone Which Interacts with CDC6 and Shows Alterations in Transformed Cells

BACKGROUND: The human TTC4 protein is a TPR (tetratricopeptide repeat) motif-containing protein. The gene was originally identified as being localized in a genomic region linked to breast cancer and subsequent studies on melanoma cell lines revealed point mutations in the TTC4 protein that may be associated with the progression of malignant melanoma. METHODOLOGY/PRINCIPLE FINDINGS: Here we show that TTC4 is a nucleoplasmic protein which interacts with HSP90 and HSP70, and also with the replication protein CDC6. It has significant structural and functional similarities with a previously characterised Drosophila protein Dpit47. We show that TTC4 protein levels are raised in malignant melanoma cell lines compared to melanocytes. We also see increased TTC4 expression in a variety of tumour lines derived from other tissues. In addition we show that TTC4 proteins bearing some of the mutations previously identified from patient samples lose their interaction with the CDC6 protein. CONCLUSIONS/SIGNIFICANCE: Based on these results and our previous work with the Drosophila Dpit47 protein we suggest that TTC4 is an HSP90 co-chaperone protein which forms a link between HSP90 chaperone activity and DNA replication. We further suggest that the loss of the interaction with CDC6 or with additional client proteins could provide one route through which TTC4 could influence malignant development of cells

Accuracy of diabetes screening methods used for people with tuberculosis, Indonesia, Peru, Romania, South Africa

Objective To evaluate the performance of diagnostic tools for diabetes mellitus, including laboratory methods and clinical risk scores, in newly-diagnosed pulmonary tuberculosis patients from four middle-income countries. Methods In a multicentre, prospective study, we recruited 2185 patients with pulmonary tuberculosis from sites in Indonesia, Peru, Romania and South Africa from January 2014 to September 2016. Using laboratory-measured glycated haemoglobin (HbA1c) as the gold standard, we measured the diagnostic accuracy of random plasma glucose, point-of-care HbA1c, fasting blood glucose, urine dipstick, published and newly derived diabetes mellitus risk scores and anthropometric measurements. We also analysed combinations of tests, including a two-step test using point-of-care HbA1cwhen initial random plasma glucose was ≥ 6.1 mmol/L. Findings The overall crude prevalence of diabetes mellitus among newly diagnosed tuberculosis patients was 283/2185 (13.0%; 95% confidence interval, CI: 11.6–14.4). The marker with the best diagnostic accuracy was point-of-care HbA1c (area under receiver operating characteristic curve: 0.81; 95% CI: 0.75–0.86). A risk score derived using age, point-of-care HbA1c and random plasma glucose had the best overall diagnostic accuracy (area under curve: 0.85; 95% CI: 0.81–0.90). There was substantial heterogeneity between sites for all markers, but the two-step combination test performed well in Indonesia and Peru. Conclusion Random plasma glucose followed by point-of-care HbA1c testing can accurately diagnose diabetes in tuberculosis patients, particularly those with substantial hyperglycaemia, while reducing the need for more expensive point-of-care HbA1c testing. Risk scores with or without biochemical data may be useful but require validation

The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

Open Access funded by Wellcome Trust: Under a Creative Commons license Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. Acknowledgments We would like to thank S. Hardison, P. Redelinghuys, J. Taylor, C. Wallace, A. Richmond, S. Hadebe, A. Plato, F. Abbass, L. Fick, N. Allie, R. Wilkinson, K. Wilkinson, S. Cooper, D. Lang, and V. Kumar for reagents and assistance, and the animal facility staff for the care of our animals. This work was supported by the MRC (UK) and Wellcome Trust (G.D.B.); MRC (South Africa) and Sydney Brenner Fellowship (M.J.M.); Vici (M.G.N.), Vidi (R.v.C.), and Veni grants (T.S.P.) from the Netherlands Organization for Scientific Research; the Royal Netherlands Academy of Arts and Sciences (T.H.M.O.); EC FP7 projects (NEWTBVAC, ADITEC; T.H.M.O.); Carnegie Corporation and CIDRI (J.C.H.); and the University of Aberdeen (B.K.).Peer reviewedPublisher PD

Tissue metabolic changes drive cytokine responses to Mycobacterium tuberculosis

Cellular metabolism can influence host immune responses to Mycobacterium tuberculosis (Mtb). Using a systems biology approach, differential expression of 292 metabolic genes involved in glycolysis, glutathione, pyrimidine and inositol phosphate pathways was evident at the site of a human tuberculin skin test challenge in patients with active tuberculosis infection. For 28 metabolic genes, we identified single nucleotide polymorphisms (SNPs) that were trans-acting for in vitro cytokine responses to Mtb stimulation, including glutathione and pyrimidine metabolism genes that alter production of Th1 and Th17 cytokines. Our findings identify novel therapeutic targets in host metabolism that may shape protective immunity to tuberculosis

Ectopic A-lattice seams destabilize microtubules

Natural microtubules typically include one A-lattice seam within an otherwise helically symmetric B-lattice tube. It is currently unclear how A-lattice seams influence microtubule dynamic instability. Here we find that including extra A-lattice seams in GMPCPP microtubules, structural analogues of the GTP caps of dynamic microtubules, destabilizes them, enhancing their median shrinkage rate by >20-fold. Dynamic microtubules nucleated by seeds containing extra A-lattice seams have growth rates similar to microtubules nucleated by B-lattice seeds, yet have increased catastrophe frequencies at both ends. Furthermore, binding B-lattice GDP microtubules to a rigor kinesin surface stabilizes them against shrinkage, whereas microtubules with extra A-lattice seams are stabilized only slightly. Our data suggest that introducing extra A-lattice seams into dynamic microtubules destabilizes them by destabilizing their GTP caps. On this basis, we propose that the single A-lattice seam of natural B-lattice MTs may act as a trigger point, and potentially a regulation point, for catastrophe

Transport by molecular motors in the presence of static defects

The transport by molecular motors along cytoskeletal filaments is studied theoretically in the presence of static defects. The movements of single motors are described as biased random walks along the filament as well as binding to and unbinding from the filament. Three basic types of defects are distinguished, which differ from normal filament sites only in one of the motors' transition probabilities. Both stepping defects with a reduced probability for forward steps and unbinding defects with an increased probability for motor unbinding strongly reduce the velocities and the run lengths of the motors with increasing defect density. For transport by single motors, binding defects with a reduced probability for motor binding have a relatively small effect on the transport properties. For cargo transport by motors teams, binding defects also change the effective unbinding rate of the cargo particles and are expected to have a stronger effect.Comment: 20 pages, latex, 7 figures, 1 tabl