Rapid Peptide Cyclization Inspired by the Modular Logic of Nonribosomal Peptide Synthetases.

Nonribosomal cyclic peptides (NRcPs) are structurally complex natural products and a vital pool of therapeutics, particularly antibiotics. Their structural diversity arises from the ability of the multidomain enzyme assembly lines, nonribosomal peptide synthetases (NRPSs), to utilize bespoke nonproteinogenic amino acids, modify the linear peptide during elongation, and catalyze an array of cyclization modes, e.g., head to tail, side chain to tail. The study and drug development of NRcPs are often limited by a lack of easy synthetic access to NRcPs and their analogues, with selective macrolactamization being a major bottleneck. Herein, we report a generally applicable chemical macrocyclization method of unprecedented speed and selectivity. Inspired by biosynthetic cyclization, it combines the deprotected linear biosynthetic precursor peptide sequence with a highly reactive C-terminus to produce NRcPs and analogues in minutes. The method was applied to several NRcPs of varying sequences, ring sizes, and cyclization modes including rufomycin, colistin, and gramicidin S with comparable success. We thus demonstrate that the linear order of modules in NRPS enzymes that determines peptide sequence encodes the key structural information to produce peptides conformationally biased toward macrocyclization. To fully exploit this conformational bias synthetically, a highly reactive C-terminal acyl azide is also required, alongside carefully balanced pH and solvent conditions. This allows for consistent, facile cyclization of exceptional speed, selectivity, and atom efficiency. This exciting macrolactamization method represents a new enabling technology for the biosynthetic study of NRcPs and their development as therapeutics.

Insights into E.†coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

Machine learning-enabled maternal risk assessment for women with pre-eclampsia (the PIERS-ML model): a modelling study.

BACKGROUND: Affecting 2-4% of pregnancies, pre-eclampsia is a leading cause of maternal death and morbidity worldwide. Using routinely available data, we aimed to develop and validate a novel machine learning-based and clinical setting-responsive time-of-disease model to rule out and rule in adverse maternal outcomes in women presenting with pre-eclampsia. METHODS: We used health system, demographic, and clinical data from the day of first assessment with pre-eclampsia to predict a Delphi-derived composite outcome of maternal mortality or severe morbidity within 2 days. Machine learning methods, multiple imputation, and ten-fold cross-validation were used to fit models on a development dataset (75% of combined published data of 8843 patients from 11 low-income, middle-income, and high-income countries). Validation was undertaken on the unseen 25%, and an additional external validation was performed in 2901 inpatient women admitted with pre-eclampsia to two hospitals in south-east England. Predictive risk accuracy was determined by area-under-the-receiver-operator characteristic (AUROC), and risk categories were data-driven and defined by negative (-LR) and positive (+LR) likelihood ratios. FINDINGS: Of 8843 participants, 590 (6·7%) developed the composite adverse maternal outcome within 2 days, 813 (9·2%) within 7 days, and 1083 (12·2%) at any time. An 18-variable random forest-based prediction model, PIERS-ML, was accurate (AUROC 0·80 [95% CI 0·76-0·84] vs the currently used logistic regression model, fullPIERS: AUROC 0·68 [0·63-0·74]) and categorised women into very low risk (-LR <0·1; eight [0·7%] of 1103 women), low risk (-LR 0·1 to 0·2; 321 [29·1%] women), moderate risk (-LR >0·2 and +LR <5·0; 676 [61·3%] women), high risk (+LR 5·0 to 10·0, 87 [7·9%] women), and very high risk (+LR >10·0; 11 [1·0%] women). Adverse maternal event rates were 0% for very low risk, 2% for low risk, 5% for moderate risk, 26% for high risk, and 91% for very high risk within 48 h. The 2901 women in the external validation dataset were accurately classified as being at very low risk (0% with outcomes), low risk (1%), moderate risk (4%), high risk (33%), or very high risk (67%). INTERPRETATION: The PIERS-ML model improves identification of women with pre-eclampsia who are at lowest and greatest risk of severe adverse maternal outcomes within 2 days of assessment, and can support provision of accurate guidance to women, their families, and their maternity care providers. FUNDING: University of Strathclyde Diversity in Data Linkage Centre for Doctoral Training, the Fetal Medicine Foundation, The Canadian Institutes of Health Research, and the Bill & Melinda Gates Foundation.

A realist synthesis of multicentre comparative audit implementation: exploring what works and in which healthcare contexts.

BACKGROUND: Multicentre comparative clinical audits have the potential to improve patient care, allow benchmarking and inform resource allocation. However, implementing effective and sustainable large-scale audit can be difficult within busy and resource-constrained contemporary healthcare settings. There are little data on what facilitates the successful implementation of multicentre audits. As healthcare environments are complex sociocultural organisational environments, implementing multicentre audits within them is likely to be highly context dependent. OBJECTIVE: We aimed to examine factors that were influential in the implementation process of multicentre comparative audits within healthcare contexts-what worked, why, how and for whom? METHODS: A realist review was conducted in accordance with the Realist and Meta-narrative Evidence Syntheses: Evolving Standards reporting standards. A preliminary programme theory informed two systematic literature searches of peer-reviewed and grey literature. The main context-mechanism-outcome (CMO) configurations underlying the implementation processes of multicentre audits were identified and formed a final programme theory. RESULTS: 69 original articles were included in the realist synthesis. Four discrete CMO configurations were deduced from this synthesis, which together made up the final programme theory. These were: (1) generating trustworthy data; (2) encouraging audit participation; (3) ensuring audit sustainability; and (4) facilitating audit cycle completion. CONCLUSIONS: This study elucidated contexts, mechanisms and outcomes influential to the implementation processes of multicentre or national comparative audits in healthcare. The relevance of these contextual factors and generative mechanisms were supported by established theories of behaviour and findings from previous empirical research. These findings highlight the importance of balancing reliability with pragmatism within complex adaptive systems, generating and protecting human capital, ensuring fair and credible leadership and prioritising change facilitation.

The activity-regulated cytoskeleton-associated protein, Arc, functions in the nucleus accumbens shell to limit multiple triggers of cocaine-seeking behaviour.

Use of addictive substances like cocaine produces enduring associations between the drug experience and cues in the drug-taking environment. In individuals with a substance use disorder (SUD) and attempting to remain abstinent, these powerful drug-cue associations can trigger a return to active drug use, but the molecular mechanisms regulating drug-cue associations remain poorly understood. The activity-regulated cytoskeleton-associated protein (Arc) is induced by cocaine in the nucleus accumbens (NAc), an important brain reward region, but Arc's NAc function in SUD-related behaviour remains unclear. We show here that cocaine self-administration (SA) in rats produced a significant upregulation of Arc protein in both the core and shell subregions of the NAc. Subregion-specific Arc reduction (shRNA) in the medial NAc Shell enhanced both context-associated and cue-reinstated cocaine seeking, but without altering the motivation to work for cocaine, the sensitivity to the reinforcing effects of cocaine or the ability of cocaine priming to reinstate drug seeking. In contrast, we observed no effects of Arc knockdown in the NAc core on any aspect of cocaine SA, extinction or reinstated cocaine seeking, suggesting that Arc functions within the medial NAc shell, but not NAc core, to limit the strength of drug-context and drug-cue associations that promote cocaine-seeking behaviour.

How International Health System Austerity Responses to the 2008 Financial Crisis Impacted Health System and Workforce Resilience - A Realist Review.

BACKGROUND: The Great Recession, following the 2008 financial crisis, led many governments to adopt programmes of austerity. This had a lasting impact on health system functionality, resources, staff (numbers, motivation and morale) and patient outcomes. This study aimed to understand how health system resilience was impacted and how this affects readiness for subsequent shocks. METHODS: A realist review identified legacies associated with austerity (proximal outcomes) and how these impact the distal outcome of health system resilience. EMBASE, CINAHL, MEDLINE, EconLit and Web of Science were searched (2007-May 2021), resulting in 1081 articles. Further theory-driven searches resulted in an additional 60 studies. Descriptive, inductive, deductive and retroductive realist analysis (utilising excel and Nvivo) aided the development of context-mechanism-outcome configurations (CMOCs), alongside stakeholder engagement to confirm or refute emerging results. Causal pathways, and the interplay between context and mechanisms that led to proximal and distal outcomes, were revealed. The refined CMOCs and policy recommendations focused primarily on workforce resilience. RESULTS: Five CMOCs demonstrated how austerity-driven policy decisions can impact health systems when driven by the priorities of external agents. This created a real or perceived shift away from the values and interests of health professionals, a distrust in decision-making processes and resistance to change. Their values were at odds with the realities of implementing such policy decisions within sustained restrictive working conditions (rationing of staff, consumables, treatment options). A diminished view of the profession and an inability to provide high-quality, equitable, and needs-led care, alongside stagnant or degraded working conditions, led to moral distress. This can forge legacies that may adversely impact resilience when faced with future shocks. CONCLUSION: This review reveals the importance of transparent, open communication, in addition to co-produced policies in order to avoid scenarios that can be detrimental to workforce and health system resilience.

Associations between significant head injury in male juveniles in prison in Scotland UK and cognitive function, disability and crime: A cross sectional study.

BACKGROUND: Although the prevalence of head injury is estimated to be high in juveniles in prison, the extent of persisting disability is unknown and relationships with offending uncertain. This limited understanding makes it difficult to develop effective management strategies and interventions to improve health or reduce recidivism. This study investigates effects of significant head injury (SHI) on cognitive function, disability and offending in juvenile prisoners, and considers relationships with common comorbidities. METHODS: This cross-sectional study recruited male juvenile prisoners in Scotland from Her Majesty's Young Offenders Institute (HMYOI) Polmont (detaining approximately 305 of 310 male juveniles in prison in Scotland). To be included juveniles had to be 16 years or older, fluent in English, able to participate in assessment, provide informed consent and not have a severe acute disorder of cognition or communication. Head injury, cognition, disability, history of abuse, mental health and problematic substance use were assessed by interview and questionnaire. RESULTS: We recruited 103 (34%) of 305 juvenile males in HMYOI Polmont. The sample was demographically representative of juvenile males in prisons for young offenders in Scotland. SHI was found in 82/103 (80%) and head injury repeated over long periods of time in 69/82 (85%). Disability was associated with SHI in 11/82 (13%) and was significantly associated with mental health problems, particularly anxiety. Group differences on cognitive tests were not found. However the SHI group reported poorer behavioural control on the Dysexecutive Questionnaire and were more often reported for incidents in prison than those without SHI. Characteristics of offending, including violence, did not differ between groups. CONCLUSIONS: Although SHI is highly prevalent in juvenile prisoners, associated disability was relatively uncommon. There was no evidence for differences in cognitive test performance or offending in juveniles with and without SHI. However, signs of poorer behavioural control and greater psychological distress in juveniles with SHI suggest that they may be at greater risk of recidivism and of potentially becoming lifelong offenders. This implies a need for remedial programmes for juvenile prisoners to take account of persisting effects of SHI on mental health and self-control and education and to improve their understanding of the effects of SHI reduce the likelihood of cumulative effects from further SHI.

Discovery and Rational Mutagenesis of Methionine Sulfoxide Reductase Biocatalysts To Expand the Substrate Scope of the Kinetic Resolution of Chiral Sulfoxides.

Methionine sulfoxide reductase A (MsrA) enzymes have recently found applications as nonoxidative biocatalysts in the enantioselective kinetic resolution of racemic sulfoxides. This work describes the identification of selective and robust MsrA biocatalysts able to catalyze the enantioselective reduction of a variety of aromatic and aliphatic chiral sulfoxides at 8-64 mM concentration with high yields and excellent ees (up to 99%). Moreover, with the aim to expand the substrate scope of MsrA biocatalysts, a library of mutant enzymes has been designed via rational mutagenesis utilizing in silico docking, molecular dynamics, and structural nuclear magnetic resonance (NMR) studies. The mutant enzyme MsrA33 was found to catalyze the kinetic resolution of bulky sulfoxide substrates bearing non-methyl substituents on the sulfur atom with ees up to 99%, overcoming a significant limitation of the currently available MsrA biocatalysts.

Production of copropophyrin III, biliverdin and bilirubin by the rufomycin producer, Streptomyces atratus.

Heme is best known for its role as a versatile prosthetic group in prokaryotic and eukaryotic proteins with diverse biological functions including gas and electron transport, as well as a wide array of redox chemistry. However, free heme and related tetrapyrroles also have important roles in the cell. In several bacterial strains, heme biosynthetic precursors and degradation products have been proposed to function as signaling molecules, ion chelators, antioxidants and photoprotectants. While the uptake and degradation of heme by bacterial pathogens is well studied, less is understood about the physiological role of these processes and their products in non-pathogenic bacteria. Streptomyces are slow growing soil bacteria known for their extraordinary capacity to produce complex secondary metabolites, particularly many clinically used antibiotics. Here we report the unambiguous identification of three tetrapyrrole metabolites from heme metabolism, coproporphyrin III, biliverdin and bilirubin, in culture extracts of the rufomycin antibiotic producing Streptomyces atratus DSM41673. We propose that biliverdin and bilirubin may combat oxidative stress induced by nitric oxide production during rufomycin biosynthesis, and indicate the genes involved in their production. This is, to our knowledge, the first report of the production of all three of these tetrapyrroles by a Streptomycete.

Epigenetic function during heroin self-administration controls future relapse-associated behavior in a cell type-specific manner.

Opioid use produces enduring associations between drug reinforcement/euphoria and discreet or diffuse cues in the drug-taking environment. These powerful associations can trigger relapse in individuals recovering from opioid use disorder (OUD). Here, we sought to determine whether the epigenetic enzyme, histone deacetylase 5 (HDAC5), regulates relapse-associated behavior in an animal model of OUD. We examined the effects of nucleus accumbens (NAc) HDAC5 on both heroin- and sucrose-seeking behaviors using operant self-administration paradigms. We utilized cre-dependent viral-mediated approaches to investigate the cell-type-specific effects of HDAC5 on heroin-seeking behavior, gene expression, and medium spiny neuron (MSN) cell and synaptic physiology. We found that NAc HDAC5 functions during the acquisition phase of heroin self-administration to limit future relapse-associated behavior. Moreover, overexpressing HDAC5 in the NAc suppressed context-associated and reinstated heroin-seeking behaviors, but it did not alter sucrose seeking. We also found that HDAC5 functions within dopamine D1 receptor-expressing MSNs to suppress cue-induced heroin seeking, and within dopamine D2 receptor-expressing MSNs to suppress drug-primed heroin seeking. Assessing cell-type-specific transcriptomics, we found that HDAC5 reduced expression of multiple ion transport genes in both D1- and D2-MSNs. Consistent with this observation, HDAC5 also produced firing rate depression in both MSN classes. These findings revealed roles for HDAC5 during active heroin use in both D1- and D2-MSNs to limit distinct triggers of drug-seeking behavior. Together, our results suggest that HDAC5 might limit relapse vulnerability through regulation of ion channel gene expression and suppression of MSN firing rates during active heroin use.

Reliability of Gallon-Jug Shelf-Transfer Power Equations in Older Women.

ABSTRACT: Ly, A, Strand, KL, Courtney, KJ, Barry, SS, Liscano, JA, Trebotich, TL, Martin-Diala, C, Martin, E, and Signorile, JF. Reliability of gallon-jug shelf-transfer test power equations in older women. J Strength Cond Res 37(5): 1124-1130, 2023-This study examined the test-retest reliability of the gallon-jug shelf-transfer (GJST) test as a measure of upper-body functional power in older women. Although the validity of the predictive equations for power during the GJST test has been established, for the test to be viable in either a laboratory or clinical environment, between-day and within-day reliability must be established. Thirty-four independently living older women (mean ± SD : 75.0 ± 6.4 years) performed 2 sets of 3 repetitions of the GJST test on 2 days separated by at least 48 hours. Using the established predictive equations, the values for peak power and average power were then computed. Statistical analyses to assess reliability included intraclass correlation coefficient, coefficient of variation (CV), SEM , minimal detectable change (MDC), and Cronbach's α values. Furthermore, Bland-Altman plots evaluated the agreement between the tests. Intraclass correlation coefficient (>0.91, p < 0 001), CV (<8.1%), SEM (<5.94 W), MDC (<14 W), and Cronbach's α (>0.95) indicated excellent reliability. The lines of equality for all Bland-Altman plots fell within the 95% confidence interval of the mean difference, implying that there were no significant differences between tests. Furthermore, bias values were small (<11.15 W), and the limits of agreement (LOA) were within an acceptable range. Based on our statistical analyses, the GJST test is a highly reliable assessment for determining object transfer power for healthy older women.

General and age-specific fertility rates in non-affective psychosis: population-based analysis of Scottish women.

PURPOSE: Women diagnosed with non-affective psychosis have a lower general fertility rate (GFR) and age-specific fertility rate (ASFR) than women in the general population. Contemporary data on GFR in this group remain limited, despite substantive changes in prescribing and management. We calculated contemporary estimates of the GFR and ASFR for women diagnosed with non-affective psychosis compared with the general population of women without this diagnosis. METHODS: A population-based design combined routinely collected historical maternity and psychiatric data from two representative areas of Scotland. Women were included from the NHS Grampian or Greater Glasgow and Clyde areas and were aged 15-44 between 2005 and 2013 inclusive. The 'exposed' group had a diagnosis of non-affective psychosis (ICD-10 F20-F29) and was compared to the general population of 'unexposed' women in the same geographical areas. RESULTS: Annual GFR between 2005 and 2013 for women with non-affective psychosis varied from 9.6 to 21.3 live births/1000 women per year in the exposed cohort and 52.7 to 57.8 live births/1000 women per year in the unexposed cohort, a rate ratio (RR) of 0.28 [p < 0.001; 95% CI (0.24, 0.32)]. ASFR for all 5-year age groups was lower in the exposed cohort than amongst unexposed women. CONCLUSION: We highlight continued low fertility rates in women with a diagnosis of non-affective psychosis, despite widespread availability of prolactin-sparing atypical antipsychotics. Accurate estimation of fertility rates remains crucial in developing needs-matched perinatal care for these women. Methodological improvements using routine datasets to investigate perinatal mental health are also urgently needed.

Validity and Reliability of the Seated Medicine Ball Throw as a Measure of Upper Body Power in Older Women.

ABSTRACT: Strand, KL, Ly, AS, Barry, SS, Liscano, JA, Trebotich, TL, Martin-Diala, C, Martin, E, and Signorile, JF. Validity and reliability of the seated medicine ball throw as a measure of upper body power in older women. J Strength Cond Res 37(4): 902-908, 2023-In women, aging is associated with diminishing upper body power, which may increase the risk of falls and fall-related injury; however, the validity and reliability of clinical tests to evaluate upper body power need to be confirmed. The seated medicine ball throw (SMBT) is an upper body performance test used to monitor muscle function among older individuals. The purpose of this study was to evaluate the validity and test-retest reliability of the SMBT in older women. Thirty-five women (age = 75.15 ± 6.39 years) participated in this study. Subjects performed SMBT trials using common ball masses (SMBT 4lb and SMBT 3kg ) over 3 sessions. Familiarization with the SMBT and chest press 1 repetition maximum (CP 1RM ) was provided on the first day. On day 2, subjects repeated the tests, but data were recorded. On day 3, SMBT was retested followed by an evaluation of chest press peak power (CP PP ) values at 30-80% of CP 1RM . Significant correlations ( p ≤ 0.05) were found between the CP PP and SMBT 4lb ( r = 0.775, p < 0.001) and SMBT 3kg ( r = 0.734, p < 0.001), and SMBT distance showed expected declines with age ( r = -0.724 to -0.626, p < 0.001), demonstrating its validity. High reliability between testing days was found, and Bland-Altman plots showed few points that fell outside the limits of agreement. In conclusion, the SMBT is a valid and highly reliable tool that can be used by health professionals to monitor deficits in upper body muscular power to improve treatment protocols in older women.

E. coli bacteraemia and antimicrobial resistance following antimicrobial prescribing for urinary tract infection in the community.

BACKGROUND: Urinary tract infections are one of the most common infections in primary and secondary care, with the majority of antimicrobial therapy initiated empirically before culture results are available. In some cases, however, over 40% of the bacteria that cause UTIs are resistant to some of the antimicrobials used, yet we do not know how the patient outcome is affected in terms of relapse, treatment failure, progression to more serious illness (bacteraemia) requiring hospitalization, and ultimately death. This study analyzed the current patterns of antimicrobial use for UTI in the community in Scotland, and factors for poor outcomes. OBJECTIVES: To explore antimicrobial use for UTI in the community in Scotland, and the relationship with patient characteristics and antimicrobial resistance in E. coli bloodstream infections and subsequent mortality. METHODS: We included all adult patients in Scotland with a positive blood culture with E. coli growth, receiving at least one UTI-related antimicrobial (amoxicillin, amoxicillin/clavulanic acid, ciprofloxacin, trimethoprim, and nitrofurantoin) between 1st January 2009 and 31st December 2012. Univariate and multivariate logistic regression analysis was performed to understand the impact of age, gender, socioeconomic status, previous community antimicrobial exposure (including long-term use), prior treatment failure, and multi-morbidity, on the occurrence of E. coli bacteraemia, trimethoprim and nitrofurantoin resistance, and mortality. RESULTS: There were 1,093,227 patients aged 16 to 100 years old identified as receiving at least one prescription for the 5 UTI-related antimicrobials during the study period. Antimicrobial use was particularly prevalent in the female elderly population, and 10% study population was on long-term antimicrobials. The greatest predictor for trimethoprim resistance in E. coli bacteraemia was increasing age (OR 7.18, 95% CI 5.70 to 9.04 for the 65 years old and over group), followed by multi-morbidity (OR 5.42, 95% CI 4.82 to 6.09 for Charlson Index 3+). Prior antimicrobial use, along with prior treatment failure, male gender, and higher deprivation were also associated with a greater likelihood of a resistant E. coli bacteraemia. Mortality was significantly associated with both having an E. coli bloodstream infection, and those with resistant growth. CONCLUSION: Increasing age, increasing co-morbidity, lower socioeconomic status, and prior community antibiotic exposure were significantly associated with a resistant E. coli bacteraemia, which leads to increased mortality.

Mapping Geographic Trends in Early Childhood Social, Emotional, and Behavioural Difficulties in Glasgow: 2010-2017.

Measuring variation in childhood mental health supports the development of local early intervention strategies. The methodological approach used to investigate mental health trends (often determined by the availability of individual level data) can affect decision making. We apply two approaches to identify geographic trends in childhood social, emotional, and behavioural difficulties using the Strengths and Difficulties Questionnaire (SDQ). SDQ forms were analysed for 35,171 children aged 4-6 years old across 180 preschools in Glasgow, UK, between 2010 and 2017 as part of routine monitoring. The number of children in each electoral ward and year with a high SDQ total difficulties score (≥15), indicating a high risk of psychopathology, was modelled using a disease mapping model. The total difficulties score for an individual child nested in their preschool and electoral ward was modelled using a multilevel model. For each approach, linear time trends and unstructured spatial random effects were estimated. The disease mapping model estimated a yearly rise in the relative rate (RR) of high scores of 1.5-5.0%. The multilevel model estimated an RR increase of 0.3-1.2% in average total scores across the years, with higher variation between preschools than between electoral wards. Rising temporal trends may indicate worsening social, emotional, and behavioural difficulties over time, with a faster rate for the proportion with high scores than for the average total scores. Preschool and ward variation, although minimal, highlight potential priority areas for local service provision. Both methodological approaches have utility in estimating and predicting children's difficulties and local areas requiring greater intervention.

Redesigning Enzymes for Biocatalysis: Exploiting Structural Understanding for Improved Selectivity.

The discovery of new enzymes, alongside the push to make chemical processes more sustainable, has resulted in increased industrial interest in the use of biocatalytic processes to produce high-value and chiral precursor chemicals. Huge strides in protein engineering methodology and in silico tools have facilitated significant progress in the discovery and production of enzymes for biocatalytic processes. However, there are significant gaps in our knowledge of the relationship between enzyme structure and function. This has demonstrated the need for improved computational methods to model mechanisms and understand structure dynamics. Here, we explore efforts to rationally modify enzymes toward changing aspects of their catalyzed chemistry. We highlight examples of enzymes where links between enzyme function and structure have been made, thus enabling rational changes to the enzyme structure to give predictable chemical outcomes. We look at future directions the field could take and the technologies that will enable it.

Evaluations of heat action plans for reducing the health impacts of extreme heat: methodological developments (2012-2021) and remaining challenges.

The recent report of the Intergovernmental Panel on Climate Change is stark in its warnings about the changing climate, including future increases in the frequency and intensity of extremely hot weather. The well-established impacts of extreme heat on human health have led to widespread implementation of national and city-wide heat plans for mitigating such impacts. Evaluations of the effectiveness of some heat plans have been published, with previous reviews highlighting key methodological challenges. This article reviews methods used since and that address those challenges, so helping to set an agenda for improving evaluations of heat plans in terms of their effectiveness in reducing heat-health impacts. We examined the reviews that identified the methodological challenges and systematically searched the literature to find evaluations that had since been conducted. We found 11 evaluations. Their methods help address the key challenge of identifying study control groups and address other challenges to a limited extent. For future evaluations, we recommend: utilising recent evaluation methodologies, such as difference-in-differences quasi-experimental designs where appropriate; cross-agency working to utilise data on morbidity and confounders; adoption of a proposed universal heat index; and greater publication of evaluations. More evaluations should assess morbidity outcomes and be conducted in low- and middle-income countries. Evaluations of heat plans globally should employ robust methodologies, as demonstrated in existing studies and potentially transferrable from other fields. Publication of such evaluations will advance the field and thus help address some of the health challenges resulting from our changing climate.

A Subset of Nucleus Accumbens Neurons Receiving Dense and Functional Prelimbic Cortical Input Are Required for Cocaine Seeking.

Background: Prelimbic cortical projections to the nucleus accumbens core are critical for cue-induced cocaine seeking, but the identity of the accumbens neuron(s) targeted by this projection, and the transient neuroadaptations contributing to relapse within these cells, remain unknown. Methods: Male Sprague-Dawley rats underwent cocaine or sucrose self-administration, extinction, and cue-induced reinstatement. Pathway-specific chemogenetics, patch-clamp electrophysiology, in vivo electrochemistry, and high-resolution confocal microscopy were used to identify and characterize a small population of nucleus accumbens core neurons that receive dense prelimbic cortical input to determine their role in regulating cue-induced cocaine and natural reward seeking. Results: Chemogenetic inhibition of prelimbic cortical projections to the nucleus accumbens core suppressed cue-induced cocaine relapse and normalized real-time cue-evoked increases in accumbens glutamate release to that of sucrose seeking animals. Furthermore, chemogenetic inhibition of the population of nucleus accumbens core neurons receiving the densest prelimbic cortical input suppressed cocaine, but not sucrose seeking. These neurons also underwent morphological plasticity during the peak of cocaine seeking in the form of dendritic spine expansion and increased ensheathment by astroglial processes at large spines. Conclusion: We identified and characterized a unique subpopulation of nucleus accumbens neurons that receive dense prelimbic cortical input. The functional specificity of this subpopulation is underscored by their ability to mediate cue-induced cocaine relapse, but not sucrose seeking. This subset of cells represents a novel target for addiction therapeutics revealed by anterograde targeting to interrogate functional circuits imbedded within a known network.

A comparison of 2020 health policy responses to the COVID-19 pandemic in Canada, Ireland, the United Kingdom and the United States of America.

This paper compares health policy responses to COVID-19 in Canada, Ireland, the United Kingdom and United States of America (US) from January to November 2020, with the aim of facilitating cross-country learning. Evidence is taken from the COVID-19 Health System Response Monitor, a joint initiative of the European Observatory on Health Systems and Policies, the WHO Regional Office for Europe, and the European Commission, which has documented country responses to COVID-19 using a structured template completed by country experts. We show all countries faced common challenges during the pandemic, including difficulties in scaling-up testing capacity, implementing timely and appropriate containment measures amid much uncertainty and overcoming shortages of health and social care workers, personal protective equipment and other medical technologies. Country responses to address these issues were similar in many ways, but dissimilar in others, reflecting differences in health system organization and financing, political leadership and governance structures. In the US, lack of universal health coverage have created barriers to accessing care, while political pushback against scientific leadership has likely undermined the crisis response. Our findings highlight the importance of consistent messaging and alignment between health experts and political leadership to increase the level of compliance with public health measures, alongside the need to invest in health infrastructure and training and retaining an adequate domestic health workforce. Building on innovations in care delivery seen during the pandemic, including increased use of digital technology, can also help inform development of more resilient health systems longer-term.