Transcription Factor Competition Allows Embryonic Stem Cells to Distinguish Authentic Signals from Noise

Stem cells occupy variable environments where they must distinguish stochastic fluctuations from developmental cues. Here, we use optogenetics to investigate how the pluripotency network in embryonic stem cells (ESCs) achieves a robust response to differentiation cues but not to gene expression fluctuations. We engineered mouse ESCs to allow quantitative control over the endogenous mechanism of neural differentiation through a light-inducible Brn2 transgene and monitored differentiation status through a genome-integrated Nanog-GFP reporter. By exposing cells to pulses of Brn2, we find that the pluripotency network rejects Brn2 inputs that are below specific magnitude or duration thresholds, but allows rapid differentiation when both thresholds are satisfied. The filtering properties of the network arise through its positive feedback architecture and the intrinsic half-life of Nanog, which determines the duration threshold in the network. Together our results suggest that the dynamic properties of positive feedback networks might determine how inputs are classified as signal or noise by stem cells

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidised low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified 5 synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently-labelled oxLDL by the LOX-1-expressing cell model was inhibited with sub-nanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p<0.01) by pre-incubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease

“Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease

Interaction between Plate Make and Protein in Protein Crystallisation Screening

Background: Protein crystallisation screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate

Race, Age, and Kidney Transplant Waitlisting Among Patients Receiving Incident Dialysis in the United States

Rationale &amp; Objective: Patients with kidney failure from racial and ethnic minority groups and older patients have reduced access to the transplant waitlist relative to White and younger patients. Although racial disparities in the waitlisting group have declined after the 2014 kidney allocation system change, whether there is intersectionality of race and age in waitlisting access is unknown. Study Design: Retrospective cohort study. Setting &amp; Participants: 439,455 non-Hispanic White and non-Hispanic Black US adults initiating dialysis between 2015 and 2019 were identified from the United States Renal Data System, and followed through 2020. Exposures: Patient race and ethnicity (non-Hispanic White and non-Hispanic Black) and age group (18-29, 30-49, 50-64, and 65-80 years). Outcomes: Placement on the United Network for Organ Sharing deceased donor waitlist. Analytical Approach: Age- and race-stratified waitlisting rates were compared. Multivariable Cox proportional hazards models, censored for death, examined the association between race and waitlisting, and included interaction term for race and age. Results: Over a median follow-up period of 1 year, the proportion of non-Hispanic White and non-Hispanic Black patients waitlisted was 20.7% and 20.5%, respectively. In multivariable models, non-Hispanic Black patients were 14% less likely to be waitlisted (aHR, 0.86, 95% CI, 0.77-0.95). Relative differences between non-Hispanic Black and non-Hispanic White patients were different by age group. Non-Hispanic Black patients were 27%, 12%, and 20% less likely to be waitlisted than non-Hispanic White patients for ages 18-29 years (aHR, 0.73; 95% CI, 0.61-0.86), 50-64 (aHR, 0.88; 95% CI, 0.80-0.98), and 65-80 years (aHR, 0.80; 95% CI, 0.71-0.90), respectively, but differences were attenuated among patients aged 30-49 years (aHR, 0.89; 95% CI, 0.77-1.02). Limitations: Race and ethnicity data is physician reported, residual confounding, and analysis is limited to non-Hispanic White and non-Hispanic Black patients. Conclusions: Racial disparities in waitlisting exist between non-Hispanic Black and non-Hispanic White individuals and are most pronounced among younger patients with kidney failure. Results suggest that interventions to address inequalities in waitlisting may need to be targeted to younger patients with kidney failure. Plain-Language Summary: Research has shown that patients from racial and ethnic minority groups and older patients have reduced access to transplant waitlisting relative to White and younger patients; nevertheless, how age impacts racial disparities in waitlisting is unknown. We compared waitlisting between non-Hispanic Black and non-Hispanic White patients with incident kidney failure, within age strata, using registry data for 439,455 US adults starting dialysis (18-80 years) during 2015-2019. Overall, non-Hispanic Black patients were less likely to be waitlisted and relative differences between the two racial groups differed by age. After adjusting for patient-level factors, the largest disparity in waitlisting was observed among adults aged 18-29 years. These results suggest that interventions should target younger adults to reduce disparities in access to kidney transplant waitlisting

Development and evaluation of a de-escalation training intervention in adult acute and forensic units:the EDITION systematic review and feasibility trial

Background: Containment (e.g. physical restraint and seclusion) is used frequently in mental health inpatient settings. Containment is associated with serious psychological and physical harms. De-escalation (psychosocial techniques to manage distress without containment) is recommended to manage aggression and other unsafe behaviours, for example self-harm. All National Health Service staff are trained in de-escalation but there is little to no evidence supporting training’s effectiveness. Objectives: Objectives were to: (1) qualitatively investigate de-escalation and identify barriers and facilitators to use across the range of adult acute and forensic mental health inpatient settings; (2) co-produce with relevant stakeholders an intervention to enhance de-escalation across these settings; (3) evaluate the intervention’s preliminary effect on rates of conflict (e.g. violence, self-harm) and containment (e.g. seclusion and physical restraint) and understand barriers and facilitators to intervention effects. Design: Intervention development informed by Experience-based Co-design and uncontrolled pre and post feasibility evaluation. Systematic reviews and qualitative interviews investigated contextual variation in use and effects of de-escalation. Synthesis of this evidence informed co-design of an intervention to enhance de-escalation. An uncontrolled feasibility trial of the intervention followed. Clinical outcome data were collected over 24 weeks including an 8-week pre-intervention phase, an 8-week embedding and an 8-week post-intervention phase. Setting: Ten inpatient wards (including acute, psychiatric intensive care, low, medium and high secure forensic) in two United Kingdom mental health trusts. Participants: In-patients, clinical staff, managers, carers/relatives and training staff in the target settings. Interventions: Enhancing de-escalation techniques in adult acute and forensic units: Development and evaluation of an evidence-based training intervention (EDITION) interventions included de-escalation training, two novel models of reflective practice, post-incident debriefing and feedback on clinical practice, collaborative prescribing and ward rounds, practice changes around admission, shift handovers and the social and physical environment, and sensory modulation and support planning to reduce patient distress. Main outcome measures: Outcomes measured related to feasibility (recruitment and retention, completion of outcome measures), training outcomes and clinical and safety outcomes. Conflict and containment rates were measured via the Patient–Staff Conflict Checklist. Clinical outcomes were measured using the Attitudes to Containment Measures Questionnaire, Attitudes to Personality Disorder Questionnaire, Violence Prevention Climate Scale, Capabilities, Opportunities, and Motivation Scale, Coercion Experience Scale and Perceived Expressed Emotion in Staff Scale. Results: Completion rates of the proposed primary outcome were very good at 68% overall (excluding remote data collection), which increased to 76% (excluding remote data collection) in the post-intervention period. Secondary outcomes had high completion rates for both staff and patient respondents. Regression analyses indicated that reductions in conflict and containment were both predicted by study phase (pre, embedding, post intervention). There were no adverse events or serious adverse events related to the intervention. Conclusions: Intervention and data-collection procedures were feasible, and there was a signal of an effect on the proposed primary outcome. Limitations: Uncontrolled design and self-selecting sample. Future work: Definitive trial determining intervention effects. Trial registration: This trial is registered as ISRCTN12826685 (closed to recruitment). Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 16/101/02) and is published in full in Health Technology Assessment; Vol. 28, No. 3. See the NIHR Funding and Awards website for further award information. Context: Conflict (a term used to describe a range of potentially unsafe events including violence, self-harm, rule-breaking, medication refusal, illicit drug and alcohol use and absconding) in mental health settings causes serious physical and psychological harm. Containment interventions which are intended to minimise harm from violence (and other conflict behaviours) such as restraint, seclusion and rapid tranquilisation can result in serious injuries to patients and, occasionally, death. Involvement in physical restraint is the most common cause of serious physical injury to National Health Service mental health staff in the United Kingdom. Violence to staff results in substantial costs to the health service in sickness and litigation payments. Containment interventions are also expensive (e.g. physical restraint costs mental health services £6.1 million and enhanced observations £88 million per annum). Despite these harms, recent findings indicate containment interventions such as seclusion and physical restraint continue to be used frequently in mental health settings. Clinical trials have demonstrated that interventions can reduce containment without increasing violence and other conflict behaviours (e.g. verbal aggression, self-harm). Substantial cost-savings result from reducing containment use. De-escalation, as an intervention to manage aggression and potential violence without restrictive practices, is a core intervention. ‘De-escalation’ is a collective term for a range of psychosocial techniques designed to reduce distress and anger without the need to use ‘containment’ interventions (measures to prevent harm through restricting a person’s ability to act independently, such as physical restraint and seclusion). Evidence indicates that de-escalation involves ensuring conditions for safe intervention and effective communication are established, clarifying and attempting to resolve the patient’s concern, conveyance of respect and empathy and regulating unhelpful emotions such as anxiety and anger. Despite featuring prominently in clinical guidelines and training policy domestically and internationally and being a component of mandatory National Health Service training, there is no evidence-based model on which to base training. A systematic review of de-escalation training effectiveness and acceptability conducted in 2015 concluded: (1) no model of training has demonstrated effectiveness in a sufficiently rigorous evaluation, (2) the theoretical underpinning of evaluated models was often unclear and (3) there has been inadequate investigation of the characteristics of training likely to enhance acceptability and uptake. Despite all National Health Service staff being trained in de-escalation there have been no high-quality trials evaluating the effectiveness and cost-effectiveness of training. Feasibility studies are needed to establish whether it is possible to conduct a definitive trial that can determine the clinical, safety and cost-effectiveness of this intervention.</p