Mutational Insight into Allosteric Regulation of Kir Channel Activity.

Potassium (K+) channels are regulated in part by allosteric communication between the helical bundle crossing, or inner gate, and the selectivity filter, or outer gate. This network is triggered by gating stimuli. In concert, there is an allosteric network which is a conjugated set of interactions which correlate long-range structural rearrangements necessary for channel function. Inward-rectifier K+ (Kir) channels favor inward K+ conductance, are ligand-gated, and help establish resting membrane potentials. KirBac1.1 is a bacterial Kir (KirBac) channel homologous to human Kir (hKir) channels. Additionally, KirBac1.1 is gated by the anionic phospholipid ligand phosphatidylglycerol (PG). In this study, we use site-directed mutagenesis to investigate residues involved in the KirBac1.1 gating mechanism and allosteric network we previously proposed using detailed solid-state NMR (SSNMR) measurements. Using fluorescence-based K+ and sodium (Na+) flux assays, we identified channel mutants with impaired function that do not alter selectivity of the channel. In tandem, we performed coarse grain molecular dynamics simulations, observing changes in PG-KirBac1.1 interactions correlated with mutant channel activity and contacts between the two transmembrane helices and pore helix tied to this behavior. Lipid affinity is closely tied to the proximity of two tryptophan residues on neighboring subunits which lure anionic lipids to a cationic pocket formed by a cluster of arginine residues. Thus, these simulations establish a structural and functional basis for the role of each mutated site in the proposed allosteric network. The experimental and simulated data provide insight into key functional residues involved in gating and lipid allostery of K+ channels. Our findings also have direct implications on the physiology of hKir channels due to conservation of many of the residues identified in this work from KirBac1.1.

A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer.

BACKGROUND: miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. METHODS: RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). RESULTS: miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. CONCLUSIONS: A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

UK 2022 Consensus on Normal Tissue Dose-Volume Constraints for Oligometastatic, Primary Lung and Hepatocellular Carcinoma Stereotactic Ablative Radiotherapy.

The use of stereotactic ablative radiotherapy (SABR) in the UK has expanded over the past decade, in part as the result of several UK clinical trials and a recent NHS England Commissioning through Evaluation programme. A UK SABR Consortium consensus for normal tissue constraints for SABR was published in 2017, based on the existing literature at the time. The published literature regarding SABR has increased in volume over the past 5 years and multiple UK centres are currently working to develop new SABR services. A review and update of the previous consensus is therefore appropriate and timely. It is hoped that this document will provide a useful resource to facilitate safe and consistent SABR practice.

What motivates men to donate blood? A systematic review of the evidence.

BACKGROUND AND OBJECTIVES: Effective recruitment and retention of male donors are vital for the ongoing provision of blood products. Compared with females, male donors are less likely to be medically deferred or experience vasovagal reactions and are typically preferred for plasmapheresis donation in voluntary non-remunerated settings. However, females outnumber males among donors aged under 40 years. This systematic review aimed to synthesize evidence and identify key motivators for blood donation among males to inform targeted recruitment/retention campaigns. MATERIALS AND METHODS: Databases (e.g. EBSCOhost, Web of Science) were searched using terms (dona* OR dono*) AND (blood OR aphaeresis OR apheresis OR plasma* OR platelet* OR platlet*) in title AND (male OR gender OR sex OR female) AND (motivat* OR intention OR attitude OR behavi* OR predictor OR barrier OR deter*) NOT (organ OR sperm OR tissue OR autologous OR oocyte) in text. Two researchers independently systematically scanned quantitative, full-text, English language, peer-reviewed publications from 1990 to 2015 that examined males/females separately with outcomes of blood donation or self-reported intention. Two additional researchers resolved discrepancies. RESULTS: Among 28 identified articles, the most frequently cited motivators for male blood product donation were as follows: altruism; positive attitude towards incentives; health check(s); subjective norms. Altruism was less pronounced among males compared with females and was combined with 'warm glow' in novice males (impure altruism). Perceived health benefits and incentives (e.g. coffee mugs) were stronger motivators of males than females. CONCLUSION: Marketing campaigns for recruitment/retention of male donors should focus on identified motivators rather than take a 'one-size-fits-all' approach.

What predicts children's active transport and independent mobility in disadvantaged neighborhoods?

This study examined two year changes in children's active transport and independent mobility and prospective associations between individual, social and physical environmental predictors of interest and these behaviors two years later. Overall, 43.5% of children (12.0±2.1 years) used active transport on the school journey at T1 and at T2 (p=0.77), and 35.3% engaged in independent mobility on the school journey at T1 and 29.6% at T2 (p=0.07). Enjoyment, parental safety concerns, and proximity to walking tracks were associated with independent mobility on the school journey. Road safety and social norms were associated with active transport and independent mobility to local destinations. These factors provide potential targets for interventions.

Objectively measured sedentary behaviour and health and development in children and adolescents: systematic review and meta-analysis.

Sedentary behaviour has emerged as a unique determinant of health in adults. Studies in children and adolescents have been less consistent. We reviewed the evidence to determine if the total volume and patterns (i.e. breaks and bouts) of objectively measured sedentary behaviour were associated with adverse health outcomes in young people, independent of moderate-intensity to vigorous-intensity physical activity. Four electronic databases (EMBASE MEDLINE, Ovid EMBASE, PubMed and Scopus) were searched (up to 12 November 2015) to retrieve studies among 2- to 18-year-olds, which used cross-sectional, longitudinal or experimental designs, and examined associations with health outcomes (adiposity, cardio-metabolic, fitness, respiratory, bone/musculoskeletal, psychosocial, cognition/academic achievement, gross motor development and other outcomes). Based on 88 eligible observational studies, level of evidence grading and quantitative meta-analyses indicated that there is limited available evidence that the total volume or patterns of sedentary behaviour are associated with health in children and adolescents when accounting for moderate-intensity to vigorous-intensity physical activity or focusing on studies with low risk of bias. Quality evidence from studies with robust designs and methods, objective measures of sitting, examining associations for various health outcomes, is needed to better understand if the overall volume or patterns of sedentary behaviour are independent determinants of health in children and adolescents.

Indication of electron neutrino appearance from an accelerator-produced off-axis muon neutrino beam.

The T2K experiment observes indications of ν(µ) → ν(e) appearance in data accumulated with 1.43×10(20) protons on target. Six events pass all selection criteria at the far detector. In a three-flavor neutrino oscillation scenario with |Δm(23)(2)| = 2.4×10(-3) eV(2), sin(2)2θ(23) = 1 and sin(2)2θ(13) = 0, the expected number of such events is 1.5±0.3(syst). Under this hypothesis, the probability to observe six or more candidate events is 7×10(-3), equivalent to 2.5σ significance. At 90% C.L., the data are consistent with 0.03(0.04) < sin(2)2θ(13) < 0.28(0.34) for δ(CP) = 0 and a normal (inverted) hierarchy.

Prohibitin, a protein downregulated by androgens, represses androgen receptor activity.

Prohibitin (PHB) is a cell cycle regulatory protein, known to repress E2F1-mediated gene activation via recruitment of transcriptional regulatory factors such as retinoblastoma and histone deacetylase 1 (HDAC1). We previously identified PHB as a target protein of androgen signaling in prostate cancer cells and showed that downregulation of PHB is required for androgen-induced cell cycle entry in these cells. We now present evidence that PHB, which has 54% homology at the protein level to the oestrogen receptor corepressor REA (repressor of oestrogen receptor activity), can repress androgen receptor (AR)-mediated transcription and androgen-dependent cell growth. Depletion of endogenous PHB resulted in an increase in expression of the androgen-regulated prostate-specific antigen gene. The repression appears to be specific to androgen and closely related receptors, as it is also evident for the glucocorticoid and progesterone, but not oestrogen, receptors. In spite of interaction of PHB with HDAC1, HDAC activity is not required for this repression. Although AR and PHB could be co-immunoprecipitated, no direct interaction was detectable, suggesting that PHB forms part of a repressive complex with the AR. Competition with the co-activator SRC1 further suggests that formation of a complex with AR, PHB and other cofactors is the mechanism by which repression is achieved. It appears then that repression of AR activity is one mechanism by which PHB inhibits androgen-dependent growth of prostate cells. Further, this study implies that the AR itself could, by mediating downregulation of a corepressor, be involved in the progression of prostate tumours to the hormone refractory stage.

Percutaneous endoscopic gastrostomy feeding in a district rehabilitation service.

OBJECTIVES: To examine gender, diagnosis, age, reasons for feeding, nutritional status, complications, outcome and duration of feeding in patients who have required a percutaneous endoscopic gastrostomy (PEG) for nutritional support at a district rehabilitation unit in the six years since the service was established. To identify potential for improvements in the management of future patients. DESIGN: Retrospective case note review of cases from 1992 to 1998. SETTING: District rehabilitation service for ages 16-64 serving the population of Fife, Scotland (population circa 350 000). SUBJECTS: All patients (n = 42) who had been fed via a PEG feeding tube in the previous six years. RESULTS: Forty-four PEG tube insertions had been conducted for 43 episodes of feeding in 42 patients. Five episodes of feeding were because of persistent vegetative state or low awareness state and 38 because of neurological swallowing impairment. Twenty-six (60%) patients were nutritionally depleted when PEG feeding was commenced. Twenty-seven (64%) patients experienced minor complications and 15 (34%) had no complications. At three months post procedure four (9.5%) patients had died and 21 (50%) had been discharged home. The mean duration of feeding on 31 October 1998 of the 20 patients (48%) who continued feeding at that date was 3.19+/-1.89 (mean +/- SD) years. CONCLUSIONS: Patients requiring PEG feeding in a district rehabilitation service have a range of diagnoses and the main indication for intervention is neurological swallowing impairment. The majority of patients were nutritionally depleted when feeding commenced and the reasons for this require further investigation.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Age related changes in inhibitory control as measured by stop signal task performance.

A modified version of the stop signal task (suitable for use with very young children) was administered to a pre primary school group of children (<5 years, 6 months); a young primary group (5 years, 7 months to 7 years, 6 months); a mid primary group (7 years, 7 months to 9 years, 6 months) and a group of adults. Significant age differences in the ability to inhibit responding were found. These results highlight the need for measures of response inhibition which are appropriate for use with very young children, when the first signs of inhibitory skills are emerging. It is also imperative that such measures allow the assessment of skills across a broad range of age groups in order to comprehensively monitor their development.

Further manipulation of the stop-signal task: developmental changes in the ability to inhibit responding with longer stop-signal delays.

The stop-signal task, a measure of inhibitory control, was further modified in order to examine its suitability as a task for very young children. A previous study (Carver et al., 2001) showed that it can be successfully adapted for use with primary school-aged children. The present study manipulated the presentation of the signal to inhibit responding and found that this improved the likelihood of responding. A pre-primary school group of children (< 5 years, 6 months), a young primary school group (5 years, 7 months to 7 years, 6 months), and a mid-primary school group (7 years, 7 months to 9 years, 6 months) participated in the study. The results emphasize the pre- and early school years as a sensitive time for the development of inhibitory skills. Measures of inhibitory control must therefore be age-appropriate and sensitive to these early developmental changes.

Palliative care in neurology.

This article defines the evolving role of the neurologist as a provider of palliative care. As neurologists care for chronically and critically ill, dying patients, and individuals whose diseases are incurable at the time of diagnosis, clinical competence requires expertise in the principles and practice of palliative medicine. Multiple studies suggest that despite available guidelines many patients with neurological disease suffer from pain, dyspnea, and other symptoms at or near the end of life. Recommendations from the American Academy of Neurology and Institute of Medicine are provided and the many ongoing educational efforts aimed at closing the existing gap in knowledge and improving patient care are reviewed.

Symptom assessment and management.

This article reviews how to assess and manage several symptoms commonly encountered by neurologists who care for patients with advanced illness. Scientifically validated guidelines are reviewed and practical advice is offered on how to manage pain, nausea and vomiting, dyspnea, and respiratory secretions at the end of life. The role of the neurologist as a provider of end of life care is discussed including suggestions for communicating with patients and families. This article concludes with a review of when sedation may be offered within the purview of good palliative care to patients who are imminently dying.

Terminal care and bereavement.

Care of patients at the end of life requires a high level of clinical vigilance, compassion and skill. The involvement of the patient's primary neurologist in end-of-life care and into bereavement can be an invaluable comfort to the patient and family. An understanding of the techniques for assessing and anticipating patient and family needs and knowledge of the resources available is essential if the neurologist is to provide guidance in their care.

End-of-life care: a survey of US neurologists' attitudes, behavior, and knowledge.

OBJECTIVE: The American Academy of Neurology (AAN) surveyed the attitudes, behavior, and knowledge of its members regarding care at the end of life. Three groups of AAN members were surveyed: neuro-oncologists, ALS specialists, and a representative sample of US neurologists. METHODS: The survey presented two clinical scenarios involving end-of-life care. Neurologists were asked a series of questions to assess their knowledge of existing medical, ethical, and legal guidelines; their willingness to participate in physician-assisted suicide (PAS) or carry out voluntary euthanasia (VE); and their general attitudes regarding end-of-life care. RESULTS: Neurologists support a patient's right to refuse life-sustaining treatment, but many believe that they are killing their patients in supporting such refusals. Thirty-seven percent think it is illegal to administer analgesics in doses that risk respiratory depression to the point of death. Forty percent believe they should obtain legal counsel when considering stopping life-sustaining treatment. One half believe that PAS should be made explicitly legal by statute for terminally ill patients. Under current law, 13% would participate in PAS and 4% would carry out VE; if those procedures were legalized, 44% would participate in PAS and 28% in VE. Approximately one third believe that physicians have the same ethical duty to honor a terminally ill patient's request for PAS as they do to honor a such a patient's refusal of life-sustaining therapy. CONCLUSIONS: There is a gap between established medical, legal, and ethical guidelines for the care of dying patients and the beliefs and practices of many neurologists, suggesting a need for graduate and postgraduate education programs in the principles and practices of palliative care medicine. Many neurologists would participate in PAS and carry out VE if legalized.

Multiple sclerosis in Fife.

Patients in Fife with multiple sclerosis were identified from three sources: a postal questionnaire to all general practices in Fife, hospital discharge data, rehabilitation service database. A total of 508 patients were identified in a population of 354,273 giving a crude prevalence rate of 143/100,000. The Standardised Prevalence Rate was 178/100,000. The sex ratio was 2.43 females to males. The prevalence was higher than identified in southern parts of the United Kingdom and similar to northern Scotland. A total of 200 (40%) cases were identified as having relapsing-remitting disease and of these 151 were ambulant, making them eligible for treatment with interferon beta-lb under current licensing criteria. Only 90 (60%) of the 151 currently eligible for the drug were under the care of a specialist and almost half the practices felt that they would change their referring habits. These factors may have implications for the workload of specialist clinics.