CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression1. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR–Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.M.L.B. is supported by a Cancer Research UK Clinician Scientist Fellowship (C53779/A20097), Addenbrooke’s Charitable Trust award and NIHR fellowship. P.J.L. is supported by a Wellcome Trust PRF (101835/Z/13/Z) and work in the Lehner laboratory is supported by NHSBT, NIHR Cambridge BRC, a Wellcome Trust Strategic Award to CIMR, and the Addenbrooke’s Charitable Trust M.A.D. is supported by a Senior Leukaemia Foundation Australia Fellowship and work in the Dawson laboratory is supported by the NHMRC (Grants 1085015, 1106444 and 1106447) Cancer Council Victoria and Leukaemia Foundation Australia. Cancer Research UK (C53779/A20097

Antibody-free magnetic cell sorting of genetically modified primary human CD4+ T cells by one-step streptavidin affinity purification.

Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing

An Insertion Sequence-Dependent Plasmid Rearrangement in Aeromonas salmonicida Causes the Loss of the Type Three Secretion System

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida

Bayesian paternity analysis and mating patterns in a parasitic nematode, Trichostrongylus tenuis

Mating behaviour is a fundamental aspect of the evolutionary ecology of sexually reproducing species, but one that has been under-researched in parasitic nematodes. We analysed mating behaviour in the parasitic nematode Trichostrongylus tenuis by performing a paternity analysis in a population from a single red grouse host. Paternity of the 150 larval offspring of 25 mothers (sampled from one of the two host caeca) was assigned among 294 candidate fathers (sampled from both caeca). Each candidate father's probability of paternity of each offspring was estimated from 10-locus microsatellite genotypes. Seventy-six (51%) offspring were assigned a father with a probability of >0.8, and the estimated number of unsampled males was 136 (95% credible interval (CI) 77-219). The probability of a male from one caecum fathering an offspring in the other caecum was estimated as 0.024 (95% CI 0.003-0.077), indicating that the junction of the caeca is a strong barrier to dispersal. Levels of promiscuity (defined as the probability of two of an adult's offspring sharing only one parent) were high for both sexes. Variance in male reproductive success was moderately high, possibly because of a combination of random mating and high variance in post-copulatory reproductive success. These results provide the first data on individual mating behaviour among parasitic nematodes

The air quality impacts of road closures associated with the 2004 Democratic National Convention in Boston

BACKGROUND: The Democratic National Convention (DNC) in Boston, Massachusetts in 2004 provided an opportunity to evaluate the impacts of a localized and short-term but potentially significant change in traffic patterns on air quality, and to determine the optimal monitoring approach to address events of this nature. It was anticipated that the road closures associated with the DNC would both influence the overall air pollution level and the distribution of concentrations across the city, through shifts in traffic patterns. METHODS: To capture these effects, we placed passive nitrogen dioxide badges at 40 sites around metropolitan Boston before, during, and after the DNC, with the goal of capturing the array of hypothesized impacts. In addition, we continuously measured elemental carbon at three sites, and gathered continuous air pollution data from US EPA fixed-site monitors and traffic count data from the Massachusetts Highway Department. RESULTS: There were significant reductions in traffic volume on the highway with closures north of Boston, with relatively little change along other highways, indicating a more isolated traffic reduction rather than an across-the-board decrease. For our nitrogen dioxide samples, while there was a relatively small change in mean concentrations, there was significant heterogeneity across sites, which corresponded with our a priori classifications of road segments. The median ratio of nitrogen dioxide concentrations during the DNC relative to non-DNC sampling periods was 0.58 at sites with hypothesized traffic reductions, versus 0.88 for sites with no changes hypothesized and 1.15 for sites with hypothesized traffic increases. Continuous monitors measured slightly lower concentrations of elemental carbon and nitrogen dioxide during road closure periods at monitors proximate to closed highway segments, but not for PM(2.5 )or further from major highways. CONCLUSION: We conclude that there was a small but measurable influence of DNC-related road closures on air quality patterns in the Boston area, and that a low-cost monitoring study combining passive badges for spatial heterogeneity and continuous monitors for temporal heterogeneity can provide useful insight for community air quality assessments

Identification of adults with symptoms suggestive of obstructive airways disease: Validation of a postal respiratory questionnaire

BACKGROUND: Two simples scoring systems for a self-completed postal respiratory questionnaire were developed to identify adults who may have obstructive airways disease. The objective of this study was to validate these scoring systems. METHOD: A two-stage design was used. All adults in two practice populations were sent the questionnaire and a stratified random sample of respondents was selected to undergo full clinical evaluation. Three respiratory physicians reviewed the results of each evaluation. A majority decision was reached as to whether the subject merited a trial of obstructive airways disease medication. This clinical decision was compared with two scoring systems based on the questionnaire in order to determine their positive predictive value, sensitivity and specificity. RESULTS: The PPV (positive predictive value) of the first scoring system was 75.1% (95% CI 68.6–82.3), whilst that of the second system was 82.3% (95% CI 75.9–89.2). The more stringent second system had the greater specificity, 97.1% (95% CI 96.0–98.2) versus 95.3% (95% CI 94.0–96.7), but poorer sensitivity 46.9% (95% CI 33.0–66.8) versus 50.3% (95% CI 35.3–71.6). CONCLUSION: This scoring system based on the number of symptoms/risk factors reported via a postal questionnaire could be used to identify adults who would benefit from a trial of treatment for obstructive airways disease

Omega-3 fatty acids in high-risk cardiovascular patients: a meta-analysis of randomized controlled trials

<p>Abstract</p> <p>Background</p> <p>Multiple randomized controlled trials (RCTs) have examined the cardiovascular effects of omega-3 fatty acids and have provided unexplained conflicting results. A meta-analysis of these RCTs to estimate efficacy and safety and potential sources of heterogeneity may be helpful.</p> <p>Methods</p> <p>The Cochrane library, MEDLINE, and EMBASE were systematically searched to identify all interventional trials of omega-3 fatty acids compared to placebo or usual diet in high-risk cardiovascular patients. The primary outcome was all-cause mortality and secondary outcomes were coronary restenosis following percutaneous coronary intervention and safety. Meta-analyses were carried out using Bayesian random-effects models, and heterogeneity was examined using meta-regression.</p> <p>Results</p> <p>A total of 29 RCTs (n = 35,144) met our inclusion criteria, with 25 reporting mortality and 14 reporting restenosis. Omega-3 fatty acids were not associated with a statistically significant decreased mortality (relative risk [RR] = 0.88, 95% Credible Interval [CrI] = 0.64, 1.03) or with restenosis prevention (RR = 0.89, 95% CrI = 0.72, 1.06), though the probability of some benefit remains high (0.93 and 0.90, respectively). However in meta-regressions, there was a >90% probability that larger studies and those with longer follow-up were associated with smaller benefits. No serious safety issues were identified.</p> <p>Conclusions</p> <p>Although not reaching conventional statistical significance, the evidence to date suggests that omega-3 fatty acids may result in a modest reduction in mortality and restenosis. However, caution must be exercised in interpreting these benefits as results were attenuated in higher quality studies, suggesting that bias may be at least partially responsible. Additional high quality studies are required to clarify the role of omega-3 fatty acid supplementation for the secondary prevention of cardiovascular disease.</p

A new approach to comprehensively evaluate the morphological properties of the human femoral head : example of application to osteoarthritic joint

Osteoarthritis affects the morphological properties of the femoral head. The goal of this study was to develop a method to elucidate whether these changes are localised to discrete regions, or if the reported trends in microstructural changes may be identified throughout the subchondral bone of the human femoral head. Whole femoral heads extracted from osteoarthritic (n = 5) and healthy controls (n = 5) underwent microCT imaging 39 μm voxel size. The subchondral bone plate was virtually isolated to evaluate the plate thickness and plate porosity. The trabecular bone region was divided into 37 volumes of interest spatially distributed in the femoral head, and bone morphometric properties were determined in each region. The study showed how the developed approach can be used to study the heterogeneous properties of the human femoral head affected by a disease such as osteoarthritis. As example, in the superior femoral head osteoarthritic specimens exhibited a more heterogeneous micro-architecture, with trends towards thicker cortical bone plate, higher trabecular connectivity density, higher trabecular bone density and thicker structures, something that could only be observed with the newly developed approach. Bone cysts were mostly confined to the postero-lateral quadrants extending from the subchondral region into the mid trabecular region. Nevertheless, in order to generalise these findings, a larger sample size should be analysed in the future. This novel method allowed a comprehensive evaluation of the heterogeneous micro-architectural properties of the human femoral head, highlighting effects of OA in the superior subchondral cortical and trabecular bone. Further investigations on different stages of OA would be needed to identify early changes in the bone