Does DNA replication direct locus-specific recombination during host immune evasion by antigenic variation in the African trypanosome?

All pathogens must survive host immune attack and, amongst the survival strategies that have evolved, antigenic variation is a particularly widespread reaction to thwart adaptive immunity. Though the reactions that underlie antigenic variation are highly varied, recombination by gene conversion is a widespread approach to immune survival in bacterial and eukaryotic pathogens. In the African trypanosome, antigenic variation involves gene conversion-catalysed movement of a huge number of variant surface glycoprotein (VSG) genes into a few telomeric sites for VSG expression, amongst which only a single site is actively transcribed at one time. Genetic evidence indicates VSG gene conversion has co-opted the general genome maintenance reaction of homologous recombination, aligning the reaction strategy with targeted rearrangements found in many organisms. What is less clear is how gene conversion might be initiated within the locality of the VSG expression sites. Here, we discuss three emerging models for VSG switching initiation and ask how these compare with processes for adaptive genome change found in other organisms.</p

Leptin modifies the prosecretory and prokinetic effects of the inflammatory cytokine interleukin-6 on colonic function in Sprague–Dawley rats

Leptin ameliorates the prosecretory and prokinetic effects of the pro-inflammatory cytokine interleukin-6 on rat colon. Leptin also suppresses the neurostimulatory effects of irritable bowel syndrome plasma, which has elevated concentrations of interleukin-6, on enteric neurons. This may indicate a regulatory role for leptin in immune-mediated bowel dysfunction. In addition to its role in regulating energy homeostasis, the adipokine leptin modifies gastrointestinal (GI) function. Indeed, leptin-resistant obese humans and leptin-deficient obese mice exhibit altered GI motility. In the functional GI disorder irritable bowel syndrome (IBS), circulating leptin concentrations are reported to differ from those of healthy control subjects. Additionally, IBS patients display altered cytokine profiles, including elevated circulating concentrations of the pro-inflammatory cytokine interleukin-6 (IL-6), which bears structural homology and similarities in intracellular signalling to leptin. This study aimed to investigate interactions between leptin and IL-6 in colonic neurons and their possible contribution to IBS pathophysiology. The functional effects of leptin and IL-6 on colonic contractility and absorptosecretory function were assessed in organ baths and Ussing chambers in Sprague–Dawley rat colon. Calcium imaging and immunohistochemical techniques were used to investigate the neural regulation of GI function by these signalling molecules. Our findings provide a neuromodulatory role for leptin in submucosal neurons, where it inhibited the stimulatory effects of IL-6. Functionally, this translated to suppression of IL-6-evoked potentiation of veratridine-induced secretory currents. Leptin also attenuated IL-6-induced colonic contractions, although it had little direct effect on myenteric neurons. Calcium responses evoked by IBS plasma in both myenteric and submucosal neurons were also suppressed by leptin, possibly through interactions with IL-6, which is elevated in IBS plasma. As leptin has the capacity to ameliorate the neurostimulatory effects of soluble mediators in IBS plasma and modulated IL-6-evoked changes in bowel function, leptin may have a role in immune-mediated bowel dysfunction in IBS patients

An investigation into the initiation of VSG switching in Trypanosoma brucei

Trypanosoma brucei, the eukaryotic parasite that causes human African trypanosomiasis in humans, evades the immune system through antigenic variation. T. brucei antigenic variation involves the periodic switching of the variant surface glycoprotein (VSG) coat to an antigenically distinct variant. A single VSG is expressed on the cell surface at any one time, but the T. brucei genome contains a vast number of silent VSGs. To be expressed, a VSG must be located in a specialised VSG blood stream form expression site (VSG BES). Silent VSGs are copied into VSG BES by homologous recombination. Several proteins have been demonstrated to be involved in this process but how VSG switching is initiated remains unclear. Four putative DNA repair factors were identified in T. brucei, whose eukaryotic homologues play a range of roles in DNA repair and other aspects of genome maintenance. These were two RecQ-like helicases, a Mus81 endonuclease and a Pif1 family helicase (PIF6). To examine whether these factors play a role in DNA repair and VSG switching, mutants were generated in blood stream form T. brucei cells. Analysis of RecQ1 by RNAi knockdown revealed it to be an essential gene in bloodstream form T. brucei, possibly involved in nuclear DNA replication. Phenotypic analysis of recq2 mutants suggests that RECQ2 is involved in the repair of a range of DNA damaging agents. Furthermore, analysis of survival following DSB induction suggests RECQ2 is involved in the repair of DNA DSBs, including those in the active VSG BES. VSG switching analysis showed that recq2-/- mutants have an elevated VSG switching rate and increase in recombination events upstream of the active VSG. These analyses suggest that RECQ2 suppresses VSG switching in T. brucei by suppressing recombination events near the active VSG. Analysis of mus81 mutants showed mus81-/- mutants to be sensitive to agents inducing replication stalling and DNA breaks, and that MUS81 is important in the repair of DSBs. PIF6 appears to be a complicated DNA repair factor, different from MUS81 and RECQ2. pif6+/- and pif6-/- mutants appear to be more resistant to MMS than wild type cells, though more sensitive to the replication stalling agent hydroxyurea. pif6 mutants do not appear to be more sensitive to DSBs than wild type cells and may even be more resistant. It is unclear whether PIF6 is involved in VSG switching and more work is required on this factor to attempt to understand its DNA repair and VSG switching function in T. brucei. These analyses shed light on the DNA repair functions of four previously uncharacterised T. brucei proteins. In particular, observations that RECQ2 is deficient in repairing DSBs upstream of the active VSG and mutants exhibit an elevated VSG switching rate cannot be reconciled with current thinking that direct formation of DSBs in this location initiates VSG switching. This suggests that the initiation of VSG switching is more complex than currently thought and requires careful further study and consideration of the relevance of using direct DSBs in this location to model VSG switching

A New Era in Extragalactic Background Light Measurements: The Cosmic History of Accretion, Nucleosynthesis and Reionization

(Brief Summary) What is the total radiative content of the Universe since the epoch of recombination? The extragalactic background light (EBL) spectrum captures the redshifted energy released from the first stellar objects, protogalaxies, and galaxies throughout cosmic history. Yet, we have not determined the brightness of the extragalactic sky from UV/optical to far-infrared wavelengths with sufficient accuracy to establish the radiative content of the Universe to better than an order of magnitude. Among many science topics, an accurate measurement of the EBL spectrum from optical to far-IR wavelengths, will address: What is the total energy released by stellar nucleosynthesis over cosmic history? Was significant energy released by non-stellar processes? Is there a diffuse component to the EBL anywhere from optical to sub-millimeter? When did first stars appear and how luminous was the reionization epoch? Absolute optical to mid-IR EBL spectrum to an astrophysically interesting accuracy can be established by wide field imagingat a distance of 5 AU or above the ecliptic plane where the zodiacal foreground is reduced by more than two orders of magnitude.Comment: 7 pages; Science White Paper for the US Astro 2010-2020 Decadal Survey. If interested in further community-wide efforts on this topic please contact the first autho

Research Exploring Physical Activity in Care Homes (REACH): study protocol for a randomised controlled trial

Background: As life expectancy increases and the number of older people, particularly those aged 85 years and over, expands there is an increase in demand for long-term care. A large proportion of people in a care home setting spend most of their time sedentary, and this is one of the leading preventable causes of death. Encouraging residents to engage in more physical activity could deliver benefits in terms of physical and psychological health, and quality of life. This study is the final stage of a programme of research to develop and preliminarily test an evidence-based intervention designed to enhance opportunities for movement amongst care home residents, thereby increasing levels of physical activity. Methods/design: This is a cluster randomised feasibility trial, aiming to recruit at least 8–12 residents at each of 12 residential care homes across Yorkshire, UK. Care homes will be randomly allocated on a 1:1 basis to receive either the intervention alongside usual care, or to continue to provide usual care alone. Assessment will be undertaken with participating residents at baseline (prior to care home randomisation) and at 3, 6, and 9 months post-randomisation. Data relating to changes in physical activity, physical function, level of cognitive impairment, mood, perceived health and wellbeing, and quality of life will be collected. Data at the level of the home will also be collected and will include staff experience of care, and changes in the numbers and types of adverse events residents experience (for example, hospital admissions, falls). Details of National Health Service (NHS) usage will be collected to inform the economic analysis. An embedded process evaluation will obtain information to test out the theory of change underpinning the intervention and its acceptability to staff and residents. Discussion: This feasibility trial with embedded process evaluation and collection of health economic data will allow us to undertake detailed feasibility work to inform a future large-scale trial. It will provide valuable information to inform research procedures in this important but challenging area

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

Functional delineation of the luminal epithelial microenvironment in breast using cell-based screening in combinatorial microenvironments

Microenvironment signals are potent determinants of cell fate and arbiters of tissue homeostasis, however understanding how different microenvironment factors coordinately regulate cellular phenotype has been experimentally challenging. Here we used a high-throughput microenvironment microarray comprised of 2640 unique pairwise signals to identify factors that support proliferation and maintenance of primary human mammary luminal epithelial cells. Multiple microenvironment factors that modulated luminal cell number were identified, including: HGF, NRG1, BMP2, CXCL1, TGFB1, FGF2, PDGFB, RANKL, WNT3A, SPP1, HA, VTN, and OMD. All of these factors were previously shown to modulate luminal cell numbers in painstaking mouse genetics experiments, or were shown to have a role in breast cancer, demonstrating the relevance and power of our high-dimensional approach to dissect key microenvironmental signals. RNA-sequencing of primary epithelial and stromal cell lineages identified the cell types that express these signals and the cognate receptors in vivo. Cell-based functional studies confirmed which effects from microenvironment factors were reproducible and robust to individual variation. Hepatocyte growth factor (HGF) was the factor most robust to individual variation and drove expansion of luminal cells via cKit+ progenitor cells, which expressed abundant MET receptor. Luminal cells from women who are genetically high risk for breast cancer had significantly more MET receptor and may explain the characteristic expansion of the luminal lineage in those women. In ensemble, our approach provides proof of principle that microenvironment signals that control specific cellular states can be dissected with high-dimensional cell-based approaches.publishedVersio

Pembrolizumab in Patients With Advanced Clear Cell Gynecological Cancer:A Phase 2 Nonrandomized Clinical Trial

Importance Advanced clear cell gynecological cancers (CCGCs) have a poor prognosis, with response rates to second-line chemotherapy less than 8%. Preliminary clinical activity with programmed cell death 1 protein (PD-1) inhibitors reported in CCGC merits further investigation.Objective To assess the clinical benefit of pembrolizumab in patients with previously treated advanced CCGC.Design, Setting, and Participants The PEACOCC trial is a single-arm multicenter phase 2 trial conducted at 5 UK centers investigating the clinical benefit and safety of pembrolizumab. PD-1 inhibitor–naive patients with histologically confirmed advanced CCGC, radiological disease progression following 1 or more prior courses of chemotherapy, and an Eastern Cooperative Oncology Group (ECOG) performance status (PS) score of 0 to 1 were included. Patients were enrolled from March 2019 to October 2021, with data collected until July 2024.Interventions Pembrolizumab, 200 mg, intravenously every 21 days up to 2 years until progression, discontinuation due to toxic effects, or patient/clinician decision. Up to 1 year of retreatment on diseases progression, if stable disease, partial response, or complete response at 2 years.Main Outcomes and Measures The primary end point was progression-free survival (PFS) rate at 12 weeks using Response Evaluation Criteria in Solid Tumors version 1.1 to detect a 12-week PFS rate of 33% or greater and exclude a PFS rate of less than 15%, with 90% power and 1-sided 5% significance level. Secondary end points included objective response rate, duration of response, PFS, overall survival, safety, and quality of life.Results A total of 48 patients were eligible. The median (range) age was 58.5 (32-77) years, and 26 (54%) had an ECOG PS score of 0 and 22 (46%) had an ECOG PS score of 1; 41 (85%) had ovarian, 6 (13%) had endometrial, and 1 (2%) had cervical advanced CCGC. The median (range) courses prior therapy was 3 (1-6); 19 patients (40%) received prior anti-angiogenic therapy, and 19 (40%) had a platinum-free interval of more than 12 months. Grade 3 treatment-related adverse events were observed in 9 patients (19%), and no patients had grade 4 or 5 adverse events. A total of 45 of 46 patients (98%) had mismatch repair–proficient tumors. The 12-week PFS rate was 42% (95% CI, 28-57), and the best objective response rate was 25% (95% CI, 14-40), with 12 partial responses. After a median follow-up of 46.9 months (95% CI, 43.4-55.0), the median PFS was 2.7 months (95% CI, 1.3-5.4), and the median overall survival was 14.8 months (95% CI, 6.7-28.2).Conclusions and Relevance The PEACOCC trial showed clinical benefit with pembrolizumab in patients with previously treated advanced CCGC, of whom all except 1 had MMR-proficient disease. Clinical outcomes were durable with an overall tolerable safety profile, justifying further evaluation of pembrolizumab monotherapy for advanced CCGC in a randomized clinical trial.Trial Registration ClinicalTrials.gov Identifier: NCT0342556