The use of immobilised metal affinity chromatography (IMAC) to compare expression of copper-binding proteins in control and copper-exposed marine microalgae

Toxicity of metals to aquatic organisms is dependent on both external factors, such as exposure concentration and water quality parameters, and intracellular processes including specific metal-binding sites and detoxification. Current models used to predict copper toxicity in microalgae do not adequately consider these intracellular processes. This study compared the copper-binding proteins from four species of marine microalgae, Dunaliella tertiolecta, Tetraselmis sp., Phaedactylum tricornutum and Ceratoneis closterium, in controls (no added copper) and following a 72-h exposure to copper (sufficient to inhibit growth by approximately 50 %). Cells were lysed by sonication, which was optimised to obtain 54–94 % cell rupture for the different algae. Cell lysates were processed by immobilised metal affinity chromatography (IMAC) using Cu2+ as the bound metal (i.e. Cu-IMAC). Bound proteins were subsequently analysed by SDS-PAGE, comparing proteins recovered from algae that were exposed to copper versus untreated control cells. Individual proteins for which copper exposure resulted in changes to proteins present were excised from gels and further analysed by nano LC ESI-MS/MS; proteins were identified using the Mascot database. Proteins identified in this way included heat-shock proteins, rubisco, α- and β-tubulins and ATP synthase (β subunit). The results established that Cu-IMAC is a useful approach to identify proteins involved in copper binding in algae. This study identified several proteins that may play an active role in responses to copper toxicity in marine microalgae

Lymphocyte subsets in healthy Malawians: Implications for immunologic assessment of HIV infection in Africa

BackgroundCD4+T lymphocyte measurements are the most important indicator of mortality in HIV-infected individuals in resource-limited settings. There is currently a lack of comprehensive immunophenotyping data from African populations to guide the immunologic assessment of HIV infection.ObjectiveTo quantify variation in absolute and relative lymphocyte subsets with age in healthy Malawians.MethodsLymphocyte subsets in peripheral blood of 539 healthy HIV-uninfected Malawians stratified by age were enumerated by flow cytometry.ResultsB and T–lymphocyte and T-lymphocyte subset absolute concentrations peaked in early childhood then decreased to adult levels, whereas lymphocyte subset proportions demonstrated much less variation with age. Adult lymphocyte subsets were similar to those in developed countries. In contrast, high B-lymphocyte and CD8+T-lymphocyte levels among children under 2 years, relative to those in developed countries, resulted in low CD4+T-lymphocyte percentages that varied little between 0 and 5 years (35% to 39%). The CD4+T-lymphocyte percentages in 35% of healthy children under 1 year and 18% of children age 1 to 3 years were below the World Health Organization threshold defining immunodeficiency in HIV-infected children in resource-limited settings. Thirteen percent of healthy children under 18 months old had a CD4:CD8T-lymphocyte ratio <1.0, which is commonly associated with HIV infection. All immunologic parameters except absolute natural killer lymphocyte concentration varied significantly with age, and percentage and overall absolute CD4+T-lymphocyte counts were higher in females than males.ConclusionAlthough lymphocyte subsets in Malawian adults are similar to those from developed countries, CD4+T-lymphocyte percentages in young children are comparatively low. These findings need to be considered when assessing the severity of HIV-related immunodeficiency in African children under 3 years

Training recruiters to randomized trials to facilitate recruitment and informed consent by exploring patients' treatment preferences

BACKGROUND: Patients’ treatment preferences are often cited as barriers to recruitment in randomized controlled trials (RCTs). We investigated how RCT recruiters reacted to patients’ treatment preferences and identified key strategies to improve informed decision-making and trial recruitment. METHODS: Audio-recordings of 103 RCT recruitment appointments with 96 participants in three UK multicenter pragmatic RCTs were analyzed using content and thematic analysis. Recruiters’ responses to expressed treatment preferences were assessed in one RCT (ProtecT - Prostate testing for cancer and Treatment) in which training on exploring preferences had been given, and compared with two other RCTs where this specific training had not been given. RESULTS: Recruiters elicited treatment preferences similarly in all RCTs but responses to expressed preferences differed substantially. In the ProtecT RCT, patients’ preferences were not accepted at face value but were explored and discussed at length in three key ways: eliciting and acknowledging the preference rationale, balancing treatment views, and emphasizing the need to keep an open mind and consider all treatments. By exploring preferences, recruiters enabled participants to become clearer about whether their views were robust enough to be sustained or were sufficiently weak that participation in the RCT became possible. Conversely, in the other RCTs, treatment preferences were often readily accepted without further discussion or understanding the reasoning behind them, suggesting that patients were not given the opportunity to fully consider all treatments and trial participation. CONCLUSIONS: Recruiters can be trained to elicit and address patients’ treatment preferences, enabling those who may not have considered trial participation to do so. Without specific guidance, some RCT recruiters are likely to accept initial preferences at face value, missing opportunities to promote more informed decision-making. Training interventions for recruiters that incorporate key strategies to manage treatment preferences, as in the ProtecT study, are required to facilitate recruitment and informed consent. TRIAL REGISTRATION: ProtecT RCT: Current Controlled Trials ISRCTN20141297. The other two trials are registered but have asked to be anonymized

Optimising recruitment and informed consent in randomised controlled trials:the development and implementation of the QuinteT Recruitment Intervention (QRI)

BACKGROUND: Pragmatic randomised controlled trials (RCTs) are considered essential to determine effective interventions for routine clinical practice, but many fail to recruit participants efficiently, and some really important RCTs are not undertaken because recruitment is thought to be too difficult. The ‘QuinteT Recruitment Intervention’ (QRI) aims to facilitate informed decision making by patients about RCT participation and to increase recruitment. This paper presents the development and implementation of the QRI. METHODS: The QRI developed iteratively as a complex intervention. It emerged from the National Institute for Health Research (NIHR) ProtecT trial and has been developed further in 13 RCTs. The final version of the QRI uses a combination of standard and innovative qualitative research methods with some simple quantification to understand recruitment and identify sources of difficulties. RESULTS: The QRI has two major phases: understanding recruitment as it happens and then developing a plan of action to address identified difficulties and optimise informed consent in collaboration with the RCT chief investigator (CI) and the Clinical Trials Unit (CTU). The plan of action usually includes RCT-specific, as well as generic, aspects. The QRI can be used in two ways: it can be integrated into the feasibility/pilot or main phase of an RCT to prevent difficulties developing and optimise recruitment from the start, or it can be applied to an ongoing RCT experiencing recruitment shortfalls, with a view to rapidly improving recruitment and informed consent or gathering evidence to justify RCT closure. CONCLUSIONS: The QRI provides a flexible way of understanding recruitment difficulties and producing a plan to address them while ensuring engaged and well-informed decision making by patients. It can facilitate recruitment to the most controversial and important RCTs. QRIs are likely to be of interest to the CIs and CTUs developing proposals for ‘difficult’ RCTs or for RCTs with lower than expected recruitment and to the funding bodies wishing to promote efficient recruitment in pragmatic RCTs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13063-016-1391-4) contains supplementary material, which is available to authorized users

Inclusion of people with multiple long-term conditions in pregnancy research:patient, public and stakeholder involvement and engagement in a randomised controlled trial

Background: Both pregnant women and those with multiple long-term conditions are under-served groups in clinical research. Informing and improving research through patient and public involvement, including pregnant women with two or more long-term health conditions, is critical to increasing their inclusion in maternity research. Giant PANDA is a randomised controlled trial, evaluating the effect of a treatment initiation strategy with nifedipine versus labetalol on severe maternal hypertension and a composite outcome of fetal/neonatal death, or neonatal unit admission. We aimed to undertake a mixed methods study-within-a-project within the Giant PANDA trial to understand barriers and facilitators to participation, understand and optimise current representativeness of clinical trial delivery of those with multiple long-term conditions and co-create a checklist to support their inclusion in pregnancy research. Methods: We undertook online workshops with women with lived experience and hybrid workshops with healthcare professionals who look after women with multiple long-term conditions. A site audit of Giant PANDA sites provided insights into research delivery capacity and health system set-up, and how this influences inclusion. An extension to the Giant PANDA screening log captured data on multiple long-term conditions enabling analysis of the impact of these health conditions on women’s inclusion in the trial. We co-created a checklist of recommendations for those designing and recruiting to similar clinical trials. Results: Five key recommendations were identified including a need to (1) involve women with multiple long-term conditions as partners in maternity research and (2) minimise barriers that stop them from taking part through (3) designing and delivering research that is flexible in time and place (4) consider research as part of care for everyone, including those with multiple long-term conditions and (5) measure and report inclusion of those with two or more health conditions in maternity research. Multiple long-term conditions were not a barrier to recruitment or randomisation in the Giant PANDA trial. Conclusion: Women with multiple long-term conditions would like opportunities to find out about and participate in research which accounts for their needs. Our checklist aims to support those designing and delivering maternity research to optimise inclusion of individuals with multiple-long term conditions. Trial registration: Giant PANDA: EudraCT number: 2020-003410-12, ISRCTN: 12,792,616

Physiological effects of major genes affecting ovulation rate in sheep

Genetic mutations with major effects on ovulation rate in sheep were recently identified in two genes of the transforming growth factor (TGFβ) superfamily and a TGFβ receptor, namely bone morphogenetic protein 15 (BMP15), otherwise known as the growth differentiation factor 9b (GDF9b), GDF9 and activin-like kinase 6 (ALK6) otherwise known as the BMP receptor type IB (BMPRIB). Animals homozygous for the BMP15 or GDF9 mutations are anovulatory whereas animals heterozygous for BMP15 or GDF9 or heterozygous or homozygous for ALK6 have higher than normal ovulation rates. Immunisation of ewes against BMP15 or GDF9 shows that both are essential for normal follicular development and control of ovulation rate. Common features of fertile animals with the BMP15, ALK6 (and possibly GDF9) mutations are changes in oocyte development during early preantral follicular growth, earlier maturation of granulosa cells and ovulation of mature follicles at smaller diameters. In summary, these findings have led to a new paradigm in reproductive biology, namely that the oocyte plays a key role in regulating the ovulation rate

Exploring the psychological rewards of a familiar semirural landscape: connecting to local nature through a mindful approach

This study analyses a 53,000 word diary of a year engaging with nature through over 200 trips to a semi-rural landscape. Thematic analysis revealed two themes; the transition from observer to nature connectedness and the ways in which the natural environment was experienced once a connection was made. These themes are discussed in relation to theories that seek to explain the positive effect of nature and nature connectedness. The findings are important as they suggest that repeated engagement with local semi-rural countryside can lead to a mindful approach and psychological rewards that do not require travel into the wilderness. The work informs further research into outcomes and processes of nature based interventions such as: trip frequency, duration and diary keeping

The HIF Signaling Pathway in Osteoblasts Directly Modulates Erythropoiesis through the Production of EPO

SummaryOsteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.PaperCli

A Prospective Multicenter Registry on Feasibility, Safety, and Outcome of Endovascular Recanalization in Childhood Stroke (Save ChildS Pro).

Rationale: Early evidence for the benefit of mechanical thrombectomy (MT) in pediatric patients with intracranial large vessel occlusion has been shown in previous retrospective cohorts. Higher-level evidence is needed to overcome the limitations of these studies such as the lack of a control group and the retrospective design. Randomized trials will very likely not be feasible, and several open questions remain, for example, the impact of arteriopathic etiologies or a possible lower age limit for MT. Save ChildS Pro therefore aims to demonstrate the safety and effectiveness of MT in pediatric patients compared to the best medical management and intravenous thrombolysis. Design: Save ChildS Pro is designed as a worldwide multicenter prospective registry comparing the safety and effectiveness of MT to the best medical care alone in the treatment of pediatric arterial ischemic stroke (AIS). It will include pediatric patients (<18 years) with symptomatic acute intracranial arterial occlusion who underwent either MT or best medical treatment including intravenous thrombolysis. Outcomes: The primary endpoint of Save ChildS Pro is the modified Rankin Scale score at 90 days post-stroke. Secondary endpoints will comprise the decrease of the Pediatric National Institutes of Health Stroke Scale score from admission to discharge and rate of complications. Discussion: Save ChildS Pro aims to provide high-level evidence for MT for pediatric patients with AIS, thereby improving functional outcome and quality of life and reducing the individual, societal, and economic burden of death and disability resulting from pediatric stroke. Clinical Trial Registration: Save ChildS Pro is registered at the German Clinical Trials Registry (DRKS; identifier: DRKS00018960)

NLRP12 provides a critical checkpoint for osteoclast differentiation

Members of the nucleotide-binding leucine-rich repeat-containing receptor (NLR) family are generally thought of as initiators of inflammation and are important in a number of inflammatory diseases. However, recent evidence has started to emerge that several NLRs can serve as checkpoint proteins against specific inflammatory pathways. Although checkpoint proteins are well accepted for their importance in adaptive immunity, their roles in innate immunity are still nascent. Receptor activator of nuclear factor kappa B ligand (RANKL), a tumor necrosis factor family cytokine responsible for basal and most forms of pathologic osteoclastogenesis, sends important differentiation signals through the alternative nuclear factor kappa B pathway. This report shows that an NLR member, nucleotide-binding leucine-rich repeat and pyrin domain-containing receptor 12, provides a brake on the activity of RANKL even in noninflammatory settings, extending the role for this type of NLR beyond inflammation-related disease