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The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau). Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduces tau levels. However, the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to be useful tools for studying tau homeostasis.

The membrane permeability of nucleotide-based drugs, such as sofosbuvir (Sovaldi), requires installation of phosphate-caging groups. One strategy, termed "ProTide", masks the anionic phosphate through an N-linked amino ester and an O-linked aromatic phospho-ester, such that release of the active drug requires consecutive enzymatic liberation by an esterase and then a phosphoramidase, such as Hint1. Because Hint1 is known to be selective for nucleotides, it was not clear if the ProTide approach could be deployed for non-nucleotides. Here, we demonstrate that caging of a phosphate-containing inhibitor of the prolyl isomerase Pin1 increases its permeability. Moreover, this compound was processed by both esterase and phosphoramidase activity, releasing the active molecule to bind and inhibit Pin1 in cells. Thus, Hint1 appears to recognize a broader set of substrates than previously appreciated. It seems possible that other potent, but impermeable, phosphate-containing inhibitors might likewise benefit from this approach.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Correlated motions of proteins are critical to function, but these features are difficult to resolve using traditional structure determination techniques. Time-resolved X-ray methods hold promise for addressing this challenge, but have relied on the exploitation of exotic protein photoactivity, and are therefore not generalizable. Temperature jumps, through thermal excitation of the solvent, have been utilized to study protein dynamics using spectroscopic techniques, but their implementation in X-ray scattering experiments has been limited. Here, we perform temperature-jump small- and wide-angle X-ray scattering measurements on a dynamic enzyme, cyclophilin A, demonstrating that these experiments are able to capture functional intramolecular protein dynamics on the microsecond timescale. We show that cyclophilin A displays rich dynamics following a temperature jump, and use the resulting time-resolved signal to assess the kinetics of conformational changes. Two relaxation processes are resolved: a fast process is related to surface loop motions, and a slower process is related to motions in the core of the protein that are critical for catalytic turnover.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Abstract Background Healthcare policy implemented during the COVID-19 pandemic may have impacted the health of patients with heart failure. Australian data provide a unique opportunity to examine service disruption independent of significant COVID-19 burden. This study aimed to examine heart failure care during the pandemic in New South Wales (NSW). Methods Analysis of hospital utilisation among patients aged ≥ 18 years with a primary diagnosis of heart failure conducted using linked administrative health records from hospital admission, emergency department, non-admitted services, and mortality data collections. Health service utilisation and outcomes were compared “Pre-pandemic” (PP): 16th March 2018 – 28th August 2019 and “During pandemic” (DP): 16th March 2020 – 28th August 2021. Mortality data were available until December 2021. Results Heart failure-related ED presentations and hospital admissions were similar between the periods (PP = 15,324 vs DP = 15,023 ED presentations, PP = 24,072 vs DP = 23,145 hospital admissions), though rates of admission from ED were lower DP (PP = 12,783/15,324 (83.4% [95% CI 82.8-84.0]) vs DP = 12,230/15,023 (81.4% [95% CI 80.8-82.0%]). There was no difference according to age, sex, rurality, or socioeconomic status. Outpatient volume reduced DP (PP = 44,447 vs DP = 35,801 occasions of service), but telehealth visits increased nearly threefold (PP = 5,978/44,447 (13.4% [95% CI 13.1-13.8%]) vs DP = 15,901/35,801 (44.4% [95% CI 43.9-44.9%]) with highest uptake among the wealthy and those in major cities. Time to heart failure-related ED presentation, hospitalisation or all-cause mortality following index admission was longer DP (PP = 273 [IQR 259, 290] days, DP = 323 [IQR 300, 342] days, HR 0.91 [95% CI 0.88, 0.95]). Conclusions Policies implemented DP had minimal impact on volumes of inpatient heart failure care in NSW hospitals, but there were fewer admissions from ED and reduced volumes of publicly funded outpatient care. A rapid shift from patient-facing to remotely delivered care enabled compliance with restrictions and was associated with increased time to heart failure-related adverse events, but access was not afforded equally across the socio-demographic spectrum.

Abstract Recent research highlights the significant impact of the gut microbiota on health and disease. Thalassemia, a hereditary blood disorder, requires regular blood transfusions, leading to an accumulation of iron in the body. Such changes could potentially alter the intestinal microbiota, thereby increasing the susceptibility of thalassemic patients to infection. In this study, we analyzed the fecal microbiota of 70 non-transfusion-dependent (NTDT) β-thalassemia/HbE patients and 30 healthy controls. Our findings indicate that iron chelation intervention had no detectable effect on the microbiome profile of thalassemic patients. However, the cross-sectional analysis revealed that the bacterial diversity and community structure in patients were significantly less diverse and distinct compared to those of healthy subjects. Using reference frames, we were also able to demonstrate that bacterial taxa that are known to produce short chain fatty acids, from the genera Alistipes, Coprococcus, and Oscillospira, and those from the family Ruminococcaceae, were less prevalent in the patients. In contrast, bacterial taxa associated with an unhealthy gut, including the genus Clostridium and those from the families Fusobacteriaceae, Enterobacteriaceae, and Peptostrptococcaceae, were more prevalent in patients and found to be correlated with higher levels of ferritin. Collectively, these changes in the microbiota could be regarded as markers of raised ferritin levels, and therefore, awareness should be exercised as they could interfere, albeit indirectly, with the treatment of the co-morbidities of thalassemia.

Critical Infrastructures (CIs), which serve as the foundation of our modern society, are facing increasing risks from cyber threats, physical attacks, and natural disasters. Additionally, the interdependencies between CIs throughout their operational lifespan can also significantly impact their integrity and safety. As a result, enhancing the resilience of CIs has emerged as a top priority for many countries, including the European Union. This involves not only understanding the threats/attacks themselves but also gaining knowledge about the areas and infrastructures that could potentially be affected. A European Union-funded project named PRECINCT (Preparedness and Resilience Enforcement for Critical INfrastructure Cascading Cyber-Physical Threats), under the Horizon 2020 program, tries to connect private and public stakeholders of CIs in a specific geographical area. The key objective of this project is to establish a common cyber-physical security management approach that will ensure the protection of both citizens and infrastructures, creating a secure territory. This paper presents the components of PRECINCT, including a directory of PRECINCT Critical Infrastructure Protection (CIP) blueprints. These blueprints support CI communities in designing integrated ecosystems, operating and replicating PRECINCT components (or toolkits). The integration enables coordinated security and resilience management, incorporating improved 'installation-specific' security solutions. Additionally, Serious Games (SG), and Digital Twins (DT) are a significant part of this project, serving as a novel vulnerability evaluation method for analysing complicated multi-system cascading effects in the PRECINCT Living Labs (LLs). The use of SG supports the concentrated advancement of innovative resilience enhancement services.

Abstract The use of observed wearable sensor data (e.g., photoplethysmograms [PPG]) to infer health measures (e.g., glucose level or blood pressure) is a very active area of research. Such technology can have a significant impact on health screening, chronic disease management and remote monitoring. A common approach is to collect sensor data and corresponding labels from a clinical grade device (e.g., blood pressure cuff) and train deep learning models to map one to the other. Although well intentioned, this approach often ignores a principled analysis of whether the input sensor data have enough information to predict the desired metric. We analyze the task of predicting blood pressure from PPG pulse wave analysis. Our review of the prior work reveals that many papers fall prey to data leakage and unrealistic constraints on the task and preprocessing steps. We propose a set of tools to help determine if the input signal in question (e.g., PPG) is indeed a good predictor of the desired label (e.g., blood pressure). Using our proposed tools, we found that blood pressure prediction using PPG has a high multi-valued mapping factor of 33.2% and low mutual information of 9.8%. In comparison, heart rate prediction using PPG, a well-established task, has a very low multi-valued mapping factor of 0.75% and high mutual information of 87.7%. We argue that these results provide a more realistic representation of the current progress toward the goal of wearable blood pressure measurement via PPG pulse wave analysis. For code, see our project page: https://github.com/lirus7/PPG-BP-Analysis

Abstract Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017–2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies. Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395 .

Abstract Large national-level electronic health record (EHR) datasets offer new opportunities for disentangling the role of genes and environment through deep phenotype information and approximate pedigree structures. Here we use the approximate geographical locations of patients as a proxy for spatially correlated community-level environmental risk factors. We develop a spatial mixed linear effect (SMILE) model that incorporates both genetics and environmental contribution. We extract EHR and geographical locations from 257,620 nuclear families and compile 1083 disease outcome measurements from the MarketScan dataset. We augment the EHR with publicly available environmental data, including levels of particulate matter 2.5 (PM2.5), nitrogen dioxide (NO2), climate, and sociodemographic data. We refine the estimates of genetic heritability and quantify community-level environmental contributions. We also use wind speed and direction as instrumental variables to assess the causal effects of air pollution. In total, we find PM2.5 or NO2 have statistically significant causal effects on 135 diseases, including respiratory, musculoskeletal, digestive, metabolic, and sleep disorders, where PM2.5 and NO2 tend to affect biologically distinct disease categories. These analyses showcase several robust strategies for jointly modeling genetic and environmental effects on disease risk using large EHR datasets and will benefit upcoming biobank studies in the era of precision medicine.

Abstract Background Evidence links lifestyle factors with Alzheimer’s disease (AD). We report the first randomized, controlled clinical trial to determine if intensive lifestyle changes may beneficially affect the progression of mild cognitive impairment (MCI) or early dementia due to AD. Methods A 1:1 multicenter randomized controlled phase 2 trial, ages 45-90 with MCI or early dementia due to AD and a Montreal Cognitive Assessment (MoCA) score of 18 or higher. The primary outcome measures were changes in cognition and function tests: Clinical Global Impression of Change (CGIC), Alzheimer’s Disease Assessment Scale (ADAS-Cog), Clinical Dementia Rating–Sum of Boxes (CDR-SB), and Clinical Dementia Rating Global (CDR-G) after 20 weeks of an intensive multidomain lifestyle intervention compared to a wait-list usual care control group. ADAS-Cog, CDR-SB, and CDR-Global scales were compared using a Mann-Whitney-Wilcoxon rank-sum test, and CGIC was compared using Fisher’s exact test. Secondary outcomes included plasma Aβ42/40 ratio, other biomarkers, and correlating lifestyle with the degree of change in these measures. Results Fifty-one AD patients enrolled, mean age 73.5. No significant differences in any measures at baseline. Only two patients withdrew. All patients had plasma Aβ42/40 ratios

ABSTRACT Large-scale studies are essential to answer questions about complex microbial communities that can be extremely dynamic across hosts, environments, and time points. However, managing acquisition, processing, and analysis of large numbers of samples poses many challenges, with cross-contamination being the biggest obstacle. Contamination complicates analysis and results in sample loss, leading to higher costs and constraints on mixed sample type study designs. While many researchers opt for 96-well plates for their workflows, these plates present a significant issue: the shared seal and weak separation between wells leads to well-to-well contamination. To address this concern, we propose an innovative high-throughput approach, termed as the Matrix method, which employs barcoded Matrix Tubes for sample acquisition. This method is complemented by a paired nucleic acid and metabolite extraction, utilizing 95% (vol/vol) ethanol to stabilize microbial communities and as a solvent for extracting metabolites. Comparative analysis between conventional 96-well plate extractions and the Matrix method, measuring 16S rRNA gene levels via quantitative polymerase chain reaction, demonstrates a notable decrease in well-to-well contamination with the Matrix method. Metagenomics, 16S rRNA gene amplicon sequencing (16S), and untargeted metabolomics analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the Matrix method recovers reproducible microbial and metabolite compositions that can distinguish between subjects. This advancement is critical for large-scale study design as it minimizes well-to-well contamination and technical variation, shortens processing times, and integrates with automated infrastructure for enhancing sample randomization and metadata generation.IMPORTANCEUnderstanding dynamic microbial communities typically requires large-scale studies. However, handling large numbers of samples introduces many challenges, with cross-contamination being a major issue. It not only complicates analysis but also leads to sample loss and increased costs and restricts diverse study designs. The prevalent use of 96-well plates for nucleic acid and metabolite extractions exacerbates this problem due to their wells having little separation and being connected by a single plate seal. To address this, we propose a new strategy using barcoded Matrix Tubes, showing a significant reduction in cross-contamination compared to conventional plate-based approaches. Additionally, this method facilitates the extraction of both nucleic acids and metabolites from a single tubed sample, eliminating the need to collect separate aliquots for each extraction. This innovation improves large-scale study design by shortening processing times, simplifying analysis, facilitating metadata curation, and producing more reliable results.

Objectives Generation Scotland (GS) is a family-based genetic epidemiology study. Initial recruitment between 2006-11 recruited ~24,000 adults from ~7000 families across Scotland with consent for medical record linkage and re-contact. In 2022 we began recruiting another 20,000, with consent extended to administrative records, with age range now 12+. Methods Original volunteers completed a baseline questionnaire, provided biological samples and underwent clinical assessment. The samples, phenotype and genotype (including methylation) data are linked to routine NHS hospital, maternity, lab test, prescriptions, dentistry, mortality, imaging, cancer screening, GP data records, Covid-19 testing and vaccinations, and follow-up questionnaires. The new wave of recruitment is all online and can be done on a smartphone, with DNA from saliva collected by post. Teenagers aged 12-15 can join with parental consent. Results Researchers can find prevalent and incident disease cases and controls, to test research hypotheses on a stratified population. GS is a vanguard cohort testing emerging linkage opportunities in Scotland – including neonatal bloodspots and medical imaging reports. GS has established and validated E-HR linkage with the NHS Scotland CHI Register, overcoming technical and governance issues in the process. We contribute to major international consortia, with collaborators from institutions worldwide, both academic and commercial. Conclusion We plan to extend the linkage process to include other administrative data from national datasets as and when approvals are obtained. New types of data can also be collected by online questionnaires. The Research Tissue Bank resources are available to academic and commercial researchers through a managed access process.

This paper investigates the robustness of a single 3D volumetric corner-supported module made of square hollow-section (SHS) columns. Typically, the moment–rotation (M-θ) behaviour of connections within the module (intra-module) is assumed to be fully rigid rather than semi-rigid, resulting in inaccurate assessment (i.e., overestimated vertical stiffness) during extreme loading events, such as progressive collapse. The intra-module connections are not capable of rigidly transferring the moment from the beams to the SHS columns. In this paper, a computationally intensive shell element model (SEM) of the module frame is created. The M-θ relationship of the intra-module connections in the SEM is firstly validated against test results by others and then replicated in a new simplified phenomenological beam element model (BEM), using nonlinear spring elements to capture the M-θ relationship. Comparing the structural behaviour of the SEM and BEM, under notional support removal, shows that the proposed BEM with semi-rigid connections (SR-BEM) agrees well with the validated SEM and requires substantially lower modelling time (98.7% lower) and computational effort (97.4% less RAM). When compared to a BEM with the typically modelled fully rigid intra-module connections (FR-BEM), the vertical displacement in the SR-BEM is at least 16% higher. The results demonstrate the importance of an accurate assessment of framing rotational stiffness and the benefits of a computationally efficient model.

Abstract In this study, we aimed to understand the potential role of the gut microbiome in the development of Alzheimer's disease (AD). We took a multi-faceted approach to investigate this relationship. Urine metabolomics were examined in individuals with AD and controls, revealing decreased formate and fumarate concentrations in AD. Additionally, we utilised whole-genome sequencing (WGS) data obtained from a separate group of individuals with AD and controls. This information allowed us to create and investigate host-microbiome personalised whole-body metabolic models. Notably, AD individuals displayed diminished formate microbial secretion in these models. Additionally, we identified specific reactions responsible for the production of formate in the host, and interestingly, these reactions were linked to genes that have correlations with AD. This study suggests formate as a possible early AD marker and highlights genetic and microbiome contributions to its production. The reduced formate secretion and its genetic associations point to a complex connection between gut microbiota and AD. This holistic understanding might pave the way for novel diagnostic and therapeutic avenues in AD management.

ABSTRACTWhile coaching has been employed as a success strategy in many areas such as athletics and business for decades, its use is relatively new in the medical field despite evidence of its benefits. Implementation and engagement regarding coaching in graduate medical education (GME) for residents and fellows is particularly scarce. We report our three-year experience of a GME success coaching program that aims to help trainees reach their full potential by addressing various areas of medical knowledge, clinical skills, efficiency, interpersonal skills and communication, professionalism, and mental health and well-being. The majority of participants (87%) were identified by themselves, their program director, and/or the GME coaches to have more than one area of need. The majority (79%) of referrals were identified by the coaches to have additional needs to the reasons for referral. We provide a framework for implementation of a GME coaching program and propose that coaching in GME may provide an additional safe environment for learners to reveal areas of concerns or difficulty that otherwise would not be disclosed and/or addressed.

Objectives: Most prior advance care planning (ACP) interventions lack integration of the social context of patients' ACP process, which patients indicate is critically important. The current study developed the Planning Advance Care Together (PACT) website to foster inclusion of loved ones in the ACP process. Methods: To provide feedback about the PACT website, patients with advanced cancer (N = 11), their caregivers (N = 11), and experts (N = 10) participated in semi-structured interviews. Patients and caregivers also completed standardized ratings of acceptability and usability. Results: Overall, patient (n = 11) and caregiver (n = 11) ratings of acceptability and usability of the website exceeded benchmark cut-offs (≥24 on the Acceptability E-Scale and ≥ 68 on the System Usability Scale). Patients, caregivers, and experts liked the topic of ACP but felt that it could be emotionally challenging. They recommended focusing more on planning and less on end of life. They appreciated being able to include loved ones and recommended adding resources for caregivers. Conclusions: Study findings support the preliminary usability and acceptability of the PACT website. Findings will be used to inform a modified prototype of the PACT website that is interactive and ready for field testing with patients with advanced cancer and their loved ones. Innovation: We utilized a novel application of the shared mind framework to support patients with advanced cancer in engaging their loved ones in the ACP process.

This paper offers a reflective account of delivering art workshops in women’s prisons in England, as one method in a larger, qualitative study around the role of food in prisons. We present reflections around the role of art workshops in building relationships and community between the researcher and the participants, and the participants themselves. We found that building connection through art workshops, in a hostile and challenging environment like prison, enabled us to gather richer and deeper data during the qualitive interviews that succeeded the art workshops. We propose that attention from researchers to the ‘unintended consequences’ can contribute to research projects which are impactful for both the participants and researchers. This paper addresses gaps in literature around the use of visual art as method in women’s prisons and the role of arts-based methods in larger qualitative research projects in secure settings.

Background Australia does not have a national strategy for Aboriginal and Torres Strait Islander adolescent health and as a result, policy and programming actions are fragmented and may not be responsive to needs. Efforts to date have also rarely engaged Aboriginal and Torres Strait Islander people in co-designing solutions. The Roadmap Project aims to work in partnership with young people to define priority areas of health and well-being need and establish the corresponding developmentally appropriate, evidence-based actions.Methods and analysis All aspects of this project are governed by a group of Aboriginal and Torres Strait Islander young people. Needs, determinants and corresponding responses will be explored with Aboriginal and Torres Strait Islander adolescents (aged 10–24 years) across Australia through an online qualitative survey, interviews and focus group discussions. Parents, service providers and policy makers (stakeholders) will share their perspectives on needs and support required through interviews. Data generated will be co-analysed with the governance group and integrated with population health data, policy frameworks and evidence of effective programmes (established through reviews) to define responsive and effective actions for Aboriginal and Torres Strait Islander adolescent health and well-being.Ethics and dissemination Ethical approval for this study has been obtained from the Aboriginal Health Council of South Australia (Ref: 04-21-956), the Aboriginal Health and Medical Research Council of New South Wales (Ref: 1918/22), the Western Australian Aboriginal Health Ethics Committee (Ref: HREC1147), the Northern Territory Health and Menzies School of Health Research (Ref: 2022–4371), ACT Health Human Research Ethics Committee (Ref: 2022.ETH.00133), the St. Vincent’s Hospital, Victoria (Ref: HREC 129/22), University of Tasmania (Ref: 28020), Far North Queensland Human Research Ethics Committee (Ref: HREC/2023/QCH/89911) and Griffith University (Ref: 2023/135). Prospective adolescent participants will provide their own consent for the online survey (aged 13–24 years) and, interviews or focus group discussions (aged 15–24 years); with parental consent and adolescent assent required for younger adolescents (aged 10–14 years) participating in interviews.Study findings (priority needs and evidence-based responses) will be presented at a series of co-design workshops with adolescents and stakeholders from relevant sectors. We will also communicate findings through reports, multimedia clips and peer-reviewed publications as directed by the governance group.

Abstract We utilize ocean 10‐m wind speed (U10m) from the microwave Multi‐sensor Advanced Climatology data set to examine the coupling between convective cloud and precipitation processes, synoptic state, and U10m and to evaluate the representation of U10m in global climate models (GCMs). We find that midlatitude U10m is underestimated by GCMs relative to observations. We examine two potential mechanisms to explain this model behavior: cold pool formation in cold air outbreaks (CAOs) associated with downdrafts that enhance U10m and sea surface temperature (SST) gradients affecting U10m through thermally forced surface winds at regional scales. When the effects of the CAO index (M) and SST gradients on U10m are accounted for, a relationship between GCM horizontal resolution and U10m appears. The strongest correlation between resolution and U10m is over the western boundary currents characterized by frequent CAOs atop strong SST gradients which drives the strongest surface fluxes on Earth.

Objectives Long-term sickness absence from employment has negative consequences for the economy and can lead to widened health inequalities. Sick notes (also called ‘fit notes’) are issued by general practitioners when a person cannot work for health reasons for more than 7 days. We quantified the sick note rate in people with evidence of COVID-19 in 2020, 2021 and 2022, as an indication of the burden for people recovering from COVID-19.Design Cohort study.Setting With National Health Service (NHS) England approval, we used routine clinical data (primary care, hospital and COVID-19 testing records) within the OpenSAFELY-TPP database.Participants People 18–64 years with a recorded positive test or diagnosis of COVID-19 in 2020 (n=365 421), 2021 (n=1 206 555) or 2022 (n=1 321 313); general population matched in age, sex and region in 2019 (n=3 140 326), 2020 (n=3 439 534), 2021 (n=4 571 469) and 2022 (n=4 818 870); people hospitalised with pneumonia in 2019 (n=29 673).Primary outcome measure Receipt of a sick note in primary care.Results Among people with a positive SARS-CoV-2 test or COVID-19 diagnosis, the sick note rate was 4.88 per 100 person-months (95% CI 4.83 to 4.93) in 2020, 2.66 (95% CI 2.64 to 2.67) in 2021 and 1.73 (95% CI 1.72 to 1.73) in 2022. Compared with the age, sex and region-matched general population, the adjusted HR for receipt of a sick note over the entire follow-up period (up to 10 months) was 4.07 (95% CI 4.02 to 4.12) in 2020 decreasing to 1.57 (95% CI 1.56 to 1.58) in 2022. The HR was highest in the first 30 days postdiagnosis in all years. Among people hospitalised with COVID-19, after adjustment, the sick note rate was lower than in people hospitalised with pneumonia.Conclusions Given the under-recording of postacute COVID-19-related symptoms, these findings contribute a valuable perspective on the long-term effects of COVID-19. Despite likely underestimation of the sick note rate, sick notes were issued more frequently to people with COVID-19 compared with those without, even in an era when most people are vaccinated. Most sick notes occurred in the first 30 days postdiagnosis, but the increased risk several months postdiagnosis may provide further evidence of the long-term impact.

Polyphenolic compounds are a class of phytonutrients that play important roles in plants and contribute to human health when incorporated into our diet through fruit consumption. A large proportion occur as glycoconjugates but the enzymes responsible for their glycosylation are poorly characterized. Here, we report the biochemical and structural characterization of two glycosyltransferases from sweet cherry named PaUGT1 and PaUGT2. Both are promiscuous glucosyltransferases active on diverse anthocyanidins and flavonols, as well as phenolic acids in the case of PaUGT1. They also exhibit weaker galactosyltransferase activity. The expression of the gene encoding PaUGT1, the most active of the two proteins, follows anthocyanin accumulation during fruit ripening, suggesting that this enzyme is the primary glycosyltransferase involved in flavonoid glycosylation in sweet cherry. It can potentially be used to synthesize diverse glycoconjugates of flavonoids for integration into bioactive formulations, and for generating new fruit cultivars with enhanced health-promoting properties using breeding methods.

Purpose Generation Scotland (GS) is a large family-based cohort study established as a longitudinal resource for research into the genetic, lifestyle and environmental determinants of physical and mental health. It comprises extensive genetic, sociodemographic and clinical data from volunteers in Scotland.Participants A total of 24 084 adult participants, including 5501 families, were recruited between 2006 and 2011. Within the cohort, 59% (approximately 14 209) are women, with an average age at recruitment of 49 years. Participants completed a health questionnaire and attended an in-person clinic visit, where detailed baseline data were collected on lifestyle information, cognitive function, personality traits and mental and physical health. Genotype array data are available for 20 026 (83%) participants, and blood-based DNA methylation (DNAm) data for 18 869 (78%) participants. Linkage to routine National Health Service datasets has been possible for 93% (n=22 402) of the cohort, creating a longitudinal resource that includes primary care, hospital attendance, prescription and mortality records. Multimodal brain imaging is available in 1069 individuals.Findings to date GS has been widely used by researchers across the world to study the genetic and environmental basis of common complex diseases. Over 350 peer-reviewed papers have been published using GS data, contributing to research areas such as ageing, cancer, cardiovascular disease and mental health. Recontact studies have built on the GS cohort to collect additional prospective data to study chronic pain, major depressive disorder and COVID-19.Future plans To create a larger, richer, longitudinal resource, ‘Next Generation Scotland’ launched in May 2022 to expand the existing cohort by a target of 20 000 additional volunteers, now including anyone aged 12+ years. New participants complete online consent and questionnaires and provide postal saliva samples, from which genotype and salivary DNAm array data will be generated. The latest cohort information and how to access data can be found on the GS website (www.generationscotland.org).

Aneurysmal bone cysts (ABCs) are rare benign bone lesions with a predilection for the metaphysis of long bones. They are often cystic, expansive, and osteolytic and may result in bony deformity. In general, there remains debate about optimal treatment for ABCs; however, the mainstay typically consists of a combination of curettage, bone grafting, and considering the need for internal fixation and osteotomies. The goals of treatment include preserving bony anatomy while eliminating the lesion. There is sparse literature regarding the treatment of osteoarthritis adjacent to benign bony tumors. If total hip arthroplasty is chosen as a treatment option, diligent preoperative planning is required, and the surgeon must assess the patient’s bone stock, account for bony deformity, and utilize specific implants and techniques based on the patient’s characteristics. We present a case of an adult patient with proximal femur ABC and symptomatic adjacent hip osteoarthritis who underwent treatment with total hip arthroplasty.

Abstract Biochar production from woody biomass generated during forest management (slash) offers significant benefits for soil health and carbon emissions, yet its adoption remains limited in the western United States (U.S.). To address this challenge, the U.S. Department of Agriculture (U.S.D.A.) Forest Service Rocky Mountain Research Station organized two workshops focused on forest management-centric biochar production. These workshops convened a diverse group of stakeholders, including investors, land management practitioners, industry professionals, and research scientists, each with unique roles in slash-based biochar production. This article presents a synthesis of the insights and perspectives gathered from these workshops, aiming to identify barriers and propose viable pathways for overcoming them. The barriers encompass governance issues such as policy and permitting, economic challenges related to costs, funding, and market stability, technological hurdles concerning methods and equipment, and a need for further research and improved science dissemination. In response to these challenges, workshop attendees collaboratively outlined specific strategies to reduce these barriers. These strategies emphasize the expansion of operational initiatives, the development of proactive policies, the stabilization of biochar markets, and the generation of additional case studies showcasing the effects of biochar amendments across various soils and environments. Collectively, the insights gleaned from this workshop series provide a comprehensive roadmap outlining both the struggles and the necessary actions and investments required to enhance the scale of slash-based biochar production and application in the western U.S. Graphical Abstract

Abstract Mortality and morbidity of Acute Respiratory Distress Syndrome (ARDS) are largely unaltered. A possible new approach to treatment of ARDS is offered by the discovery of inflammatory subphenotypes. In an ovine model of ARDS phenotypes, matching key features of the human subphenotypes, we provide an imaging characterization using computer tomography (CT). Nine animals were randomized into (a) OA (oleic acid, hypoinflammatory; n = 5) and (b) OA-LPS (oleic acid and lipopolysaccharides, hyperinflammatory; n = 4). 48 h after ARDS induction and anti-inflammatory treatment, CT scans were performed at high (H) and then low (L) airway pressure. After CT, the animals were euthanized and lung tissue was collected. OA-LPS showed a higher air fraction and OA a higher tissue fraction, resulting in more normally aerated lungs in OA-LPS in contrast to more non-aerated lung in OA. The change in lung and air volume between H and L was more accentuated in OA-LPS, indicating a higher recruitment potential. Strain was higher in OA, indicating a higher level of lung damage, while the amount of lung edema and histological lung injury were largely comparable. Anti-inflammatory treatment might be beneficial in terms of overall ventilated lung portion and recruitment potential, especially in the OA-LPS group.

Abstract Biologic therapies targeting the IL-23/IL-17 axis have transformed the treatment of psoriasis. However, the early mechanisms of action of these drugs remain poorly understood. Here, we perform longitudinal single-cell RNA-sequencing in affected individuals receiving IL-23 inhibitor therapy. By profiling skin at baseline, day 3 and day 14 of treatment, we demonstrate that IL-23 blockade causes marked gene expression shifts, with fibroblast and myeloid populations displaying the most extensive changes at day 3. We also identify a transient WNT5A+/IL24+ fibroblast state, which is only detectable in lesional skin. In-silico and in-vitro studies indicate that signals stemming from these WNT5A+/IL24+ fibroblasts upregulate multiple inflammatory genes in keratinocytes. Importantly, the abundance of WNT5A+/IL24+ fibroblasts is significantly reduced after treatment. This observation is validated in-silico, by deconvolution of multiple transcriptomic datasets, and experimentally, by RNA in-situ hybridization. These findings demonstrate that the evolution of inflammatory fibroblast states is a key feature of resolving psoriasis skin.

Background: Treatment inertia, non-adherence and non-persistence to medical treatment contribute to poor blood pressure (BP) control worldwide. Fixed dose combination (FDC) antihypertensive medicines simplify prescribing patterns and improve adherence. The aim of this study was to identify factors associated with prescribing FDC antihypertensive medicines and to understand if these factors differ among doctors worldwide. Methods: A cross-sectional survey was conducted online from June 2023 to January 2024 to recruit doctors. We collaborated with an international network of researchers and clinicians identified through institutional connections. A passive snowballing recruitment strategy was employed, where network members forwarded the survey link to their clinical colleagues. The survey instrument, developed through a literature review, interviews with academic and clinical researchers, and pilot testing, assessed participants perspectives on prescribing FDC antihypertensive medicines for hypertension. Participants rated their level of agreement (5-point Likert scale) with statements representing six barriers and four facilitators to FDC use. Findings: Data from 191 surveys were available for analysis. 25% (n = 47) of participants worked in high-income countries, 38% (n = 73) in upper-middle income, 25% (n = 48) in lower-middle income, 6% (n = 10) in low-income countries. Forty percent (n = 70) of participants were between 36–45 years of age; two thirds were male. Cost was reported as a barrier to prescribing FDC antihypertensive medicines [51% (n = 87) agreeing or strongly agreeing], followed by doctors’ confidence in BP measured in clinic [40%, (n = 70)], access [37%, (n = 67)], appointment duration [35%, (n = 61)], concerns about side-effects [(21%, n = 37)], and non-adherence [12%, (n = 21)]. Facilitators to FDC antihypertensive polypills prescribing were clinician facing, such as access to educational supports [79%, (n = 143)], more BP measurement data [67%, (n = 120)], a clinical nudge in health records [61%, (n = 109)] and patient-facing including improved patient health literacy [49%, (n = 88)]. The levels of agreement and strong agreement across all barriers and facilitators were similar for participants working in higher or lower income countries. Across all countries, participants rated FDC antihypertensive medications highly valuable for managing patients with non-adherence, (82% reported high or very high value), for patients with high pill burden (80%). Interpretation: Cost and access were the most common barriers to prescribing FDCs across high- and low-income countries. While greater educational support for clinicians was perceived as the leading potential facilitator of FDC use, this seems unlikely to be effective without addressing access.

Advancing imaging technologies are drastically increasing the rate of marine video and image data collection. Often these datasets are not analysed to their full potential as extracting information for multiple species is incredibly time-consuming. This study demonstrates the capability of the open-source interactive machine learning tool, RootPainter, to analyse large marine image datasets quickly and accurately. The ability of RootPainter to extract the presence and surface area of the cold-water coral reef associate sponge species, Mycale lingua, was tested in two datasets: 18 346 time-lapse images and 1420 remotely operated vehicle video frames. New corrective annotation metrics integrated with RootPainter allow objective assessment of when to stop model training and reduce the need for manual model validation. Three highly accurate M. lingua models were created using RootPainter, with an average dice score of 0.94 ± 0.06. Transfer learning aided the production of two of the models, increasing analysis efficiency from 6 to 16 times faster than manual annotation for time-lapse images. Surface area measurements were extracted from both datasets allowing future investigation of sponge behaviours and distributions. Moving forward, interactive machine learning tools and model sharing could dramatically increase image analysis speeds, collaborative research and our understanding of spatiotemporal patterns in biodiversity.

Background Generation Scotland (GS) is large family-based cohort study established as a resource for research into the genetic and environmental determinants of physical and mental health. Participants completed a baseline questionnaire and clinic visit in 2006-2011. 24,084 Participants 5,501 Families • NHS data linkage 22,402 (93%) • Genotype array data 20,026 (83%) • DNA methylation data 18,876 (78%) In 2019 funding was obtained to expand the existing GS cohort. By 2025 NextGenScot aims to: • Recruit 20,000 new participants online • Expand eligibility to 12-17 year olds • Collect saliva samples by post for genotyping • Linkage to NHS and other administrative data Details on how to join at www.gen.scot Participants consented to linkage to NHS Scotland medical records which offer longitudinal phenotype information. Linkage is conducted using the Community Health Index (CHI). Introduction GS receives 30-40 requests for access to data and/or samples every year, and has been included in over 380 publications. Research proposals are subject to review by the GS Access Committee. Details of the application process terms and conditions for access are available on the GS website, and now on the new research access website. The previous GS access process was based on an application form in Word, downloaded from the main GS website. We are replacing this with a new online research access system, adhering to transparency standards1, helped by HDR Alliance Transparency funding. After researchers publish their results, a link to the publication and a lay summary is currently posted on the GS website, with a spreadsheet listing papers. We will now make this information available and searchable for researchers, participants and the public on the new research access website. Methods We are developing a website, using in-house staff, with HDR Alliance Transparency contributing to their salaries. The system coding uses Django (a Python tool) linked to a MySQL database, a platform widely used for such projects, enabling faster development in a secure environment. Transparent access to GS data for registered users • View summary of data available • Query the summary data to see if it fits research requirements • Select data requested from data dictionaries • Get a detailed quote for access charges • Complete application form showing the project complies with the Five Safes2 • Upload supporting documentation (e.g. protocol, DPIA, ethics, etc) • Track progress of their application review, approval and delivery • Researchers will be required to submit a lay summary of their application GS participants, researchers and the public can view: • List of approved applications with lay summaries • Summaries of ongoing projects which will link to eventual publications • Searchable list of publications Result The new system is undergoing internal testing, using data imported from previous and ongoing access requests (May 2024). We will then pilot it with selected external researchers and GS participants (June 2024), with a plan to launch it for public access in July/August 2024. The GS Access Committee and admin team will be able to streamline the review and approval process, giving an improved turnaround time. Conclusions We were already planning to develop a new online application system, but the Transparency grant funding has enabled us to extend the functionality beyond a simple form-filling system. The new access website will give researchers more information about the application process, the data and samples available, see what other research has been approved in their area of interest, and track the progress of their project from enquiry through to publication. By adding a participant/public access area, with lay summaries of projects and publications, GS participants will be able to see how their data is being used for public benefit, as can potential new volunteers or other interested parties. Notes The URL for the new website is www.gsaccess.org The main GS website is at www.generationscotland.org References • Transparency Standards https://zenodo.org/record/8262453 • Five Safes https://ukdataservice.ac.uk/help/secure-lab/what-is-the-five-safes-framework/

Introduction Improving the health of Indigenous adolescents is central to addressing the health inequities faced by Indigenous peoples. To achieve this, it is critical to understand what is needed from the perspectives of Indigenous adolescents themselves. There have been many qualitative studies that capture the perspectives of Indigenous young people, but synthesis of these has been limited to date.Methods and analysis This scoping review seeks to understand the specific health needs and priorities of Indigenous adolescents aged 10–24 years captured via qualitative studies conducted across Australia, Aotearoa New Zealand, Canada, the USA, Greenland and Sami populations (Norway and Sweden). A team of Indigenous and non-Indigenous researchers from these nations will systematically search PubMed (including the MEDLINE, PubMed Central and Bookshelf databases), CINAHL, Embase, Scopus, the Informit Indigenous and Health Collections, Google Scholar, Arctic Health, the Circumpolar Health Bibliographic Database, Native Health Database, iPortal and NZresearch.org, as well as specific websites and clearinghouses within each nation for qualitative studies. We will limit our search to articles published in any language during the preceding 5 years given that needs may have changed significantly over time. Two independent reviewers will identify relevant articles using a two-step process, with disagreements resolved by a third reviewer and the wider research group. Data will then be extracted from included articles using a standardised form, with descriptive synthesis focussing on key needs and priorities. This scoping review will be conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines.Ethics and dissemination Ethics approval was not required for this review. Findings will be disseminated via a peer-reviewed journal article and will inform a broader international collaboration for Indigenous adolescent health to develop evidence-based actions and solutions.

BackgroundThe impact of digital device use on health and well-being is a pressing question. However, the scientific literature on this topic, to date, is marred by small and unrepresentative samples, poor measurement of core constructs, and a limited ability to address the psychological and behavioral mechanisms that may underlie the relationships between device use and well-being. Recent authoritative reviews have made urgent calls for future research projects to address these limitations. The critical role of research is to identify which patterns of use are associated with benefits versus risks and who is more vulnerable to harmful versus beneficial outcomes, so that we can pursue evidence-based product design, education, and regulation aimed at maximizing benefits and minimizing the risks of smartphones and other digital devices. ObjectiveThe objective of this study is to provide normative data on objective patterns of smartphone use. We aim to (1) identify how patterns of smartphone use impact well-being and identify groups of individuals who show similar patterns of covariation between smartphone use and well-being measures across time; (2) examine sociodemographic and personality or mental health predictors and which patterns of smartphone use and well-being are associated with pre-post changes in mental health and functioning; (3) discover which nondevice behavior patterns mediate the association between device use and well-being; (4) identify and explore recruitment strategies to increase and improve the representation of traditionally underrepresented populations; and (5) provide a real-world baseline of observed stress, mood, insomnia, physical activity, and sleep across a representative population. MethodsThis is a prospective, nonrandomized study to investigate the patterns and relationships among digital device use, sensor-based measures (including both behavioral and physiological signals), and self-reported measures of mental health and well-being. The study duration is 4 weeks per participant and includes passive sensing based on smartphone sensors, and optionally a wearable (Fitbit), for the complete study period. The smartphone device will provide activity, location, phone unlocks and app usage, and battery status information. ResultsAt the time of submission, the study infrastructure and app have been designed and built, the institutional review board of the University of Oregon has approved the study protocol, and data collection is underway. Data from 4182 enrolled and consented participants have been collected as of March 27, 2023. We have made many efforts to sample a study population that matches the general population, and the demographic breakdown we have been able to achieve, to date, is not a perfect match. ConclusionsThe impact of digital devices on mental health and well-being raises important questions. The Digital Well-Being Study is designed to help answer questions about the association between patterns of smartphone use and well-being. International Registered Report Identifier (IRRID)DERR1-10.2196/49189

Background Patient safety learning systems play a critical role in supporting safety culture in healthcare organisations. A lack of explicit standards leads to inconsistent implementation across organisations, causing uncertainty about their roles and impact. Organisations can address inconsistent implementation by using a self-assessment tool based on agreed-on best practices. Therefore, we aimed to create a survey instrument to assess an organisation’s approach to learning from safety events.Methods The foundation for this work was a recent systematic review that defined features associated with the performance of a safety learning system. We organised features into themes and rephrased them into questions (items). Face validity was checked, which included independent pre-testing to ensure comprehensibility and parsimony. It also included clinical sensibility testing in which a representative sample of leaders in quality at a large teaching hospital (The Ottawa Hospital) answered two questions to judge each item for clarity and necessity. If more than 20% of respondents judged a question unclear or unnecessary, we modified or removed that question accordingly. Finally, we checked the internal consistency of the questionnaire using Cronbach’s alpha.Results We initially developed a 47-item questionnaire based on a prior systematic review. Pre-testing resulted in the modification of 15 of the questions, 2 were removed and 2 questions were added to ensure comprehensiveness and relevance. Face validity was assessed through yes/no responses, with over 80% of respondents confirming the clarity and 85% the necessity of each question, leading to the retention of all 47 questions. Data collected from the five-point responses (strongly disagree to strongly agree) for each question were used to assess the questionnaire’s internal consistency. The Cronbach’s alpha was 0.94, indicating a high internal consistency.Conclusion This self-assessment questionnaire is evidence-based and on preliminary testing is deemed valid, comprehensible and reliable. Future work should assess the range of survey responses in a large sample of respondents from different hospitals.

Abstract Within oxygen minimum zones, anaerobic processes transform bioavailable nitrogen (N) into the gases dinitrogen (N2) and nitrous oxide (N2O), a potent greenhouse gas. Mesoscale eddies in these regions create heterogeneity in dissolved N tracers and O2 concentrations, influencing nonlinear N cycle reactions that depend on them. Here, we use an eddy‐resolving model of the Eastern Tropical South Pacific to show that eddies enhance N2 production by between 43% and 64% at the expense of reducing N2O production by between 94% and 104% due to both the steep increase of progressive denitrification steps at vanishing oxygen, and the more effective inhibition of N2O consumption relative to production. Our findings reveal the critical role of eddies in shaping the N cycle of oxygen minimum zones, which is not currently represented by coarse models used for climate studies.

Abstract Background In January 2018, the Government of Ontario, Canada, initiated a universal pharmacare program (OHIP+) for all individuals aged 24 years and younger. In April 2019, the program was amended to cover only children and youth without private insurance. Because benzodiazepines are commonly prescribed to children and youth despite their potential hazards, we examined whether changes in publicly-funded drug coverage influenced benzodiazepine dispensing trends in this demographic. Methods We conducted a population-based natural experiment study of benzodiazepine dispensing to children and youth in Ontario between January 2013 and March 2020. We used interventional autoregressive integrated moving average models to estimate the impact of OHIP + and its subsequent modification on these trends. Results The implementation of OHIP + was associated with an immediate increase in the monthly rate of benzodiazepine dispensing of 12.9 individuals per 100,000 population (95% confidence interval [CI]; 7.5 to 18.3 per 100,000). Benzodiazepine dispensing rates rose from 214.2 to 241.5 per 100,000 from December 2017 to March 2019, a 12.8% (95% CI 9.6–16.0%) increase. In stratified analyses, increases were most pronounced among females, children and youth living in the lowest income neighbourhoods and individuals aged 20 to 24. The April 2019 modification to OHIP + was not associated with changes in monthly benzodiazepine dispensing trends (0.39 individuals per 100,000; 95% CI -1.3 to 2.1 per 100,000). However, rates remained elevated relative to the period preceding OHIP + implementation. Conclusions Implementation of a publicly-funded pharmacare program resulted in more children and youth being prescribed benzodiazepines.

Abstract Background Sepsis is characterized by highly heterogeneous immune responses associated with a spectrum of disease severity. Methods that rapidly and sensitively profile these immune responses can potentially personalize immune-adjuvant therapies for sepsis. We hypothesized that the ELLA microfluidic approach to measure cytokine production from the whole blood of septic and critically ill patients would deliver faster, more precise results than the existing optic-driven ELISpot quantification. We tested our hypothesis by measuring ex vivo-stimulated production of TNF and IFNγ in critically ill and septic patients (n = 22), critically ill and non-septic patients (n = 10), and healthy volunteers (n = 10) through both ELLA and ELISpot immunoassays. Blood samples were subjected to one of three stimulants for 4 h or 18 h durations during days 1, 7–10, and 14 of critical illness. Stimulants for lymphocytes included anti-CD3/anti-CD28 and phorbol 12-myristate 13-acetate (PMA), whereas LPS was used for monocytes. Stimulated TNF and IFNγ concentrations were then associated with 30-day mortality. Results Both ELISpot and ELLA immunoassays showed substantial agreement in TNF concentrations post 4 h and 18 h LPS stimulation, with concordance correlation coefficients at 0.62 and 0.60, respectively. ELLA had a broad dynamic measurement range and provided accurate TNF and IFNγ readings at both minimal and elevated cytokine concentrations (with mean coefficients of variation between triplicate readings at 2.1 ± 1.4% and 4.9 ± 7.2%, respectively). However, there was no association between the ELLA-determined cytokine concentrations on the first day of critical illness and 30-day mortality rate. In contrast, using the ELISpot for cytokine quantification revealed that non-survivors had reduced baseline TNF levels at 18 h, decreased LPS-induced TNF levels at 18 h, and diminished TNF levels post 4 h/18 h anti-CD3/28 stimulation. Conclusions Our study affirms the feasibility of obtaining dependable immune phenotyping data within 6 h of blood collection from critically ill patients, both septic and non-septic, using the ELLA immunoassay. Both ELLA and ELISpot can offer valuable insights into prognosis, therapeutic strategies, and the underlying mechanisms of sepsis development.

Abstract Background In February 2018, Canada’s National Advisory Committee on Immunization (NACI) recommended antenatal tetanus–diphtheria–acellular pertussis (Tdap) immunization in every pregnancy regardless of previous Tdap immunization history. We examined the impact of the NACI recommendation on rates of infant pertussis in Ontario, Canada. Methods We conducted a population-based time-series study of all live births in Ontario between August 1, 2011 and February 28, 2020. We used interventional autoregressive integrated moving average models to examine the impact of the NACI recommendation on monthly rates of pertussis among infants ≤ 3 months of age. Results We observed 675 incident cases of pertussis among 1,368,024 infants 3 months of age or less between August 2011 and February 2020. The average monthly percent change in infant pertussis during the period up to and including publication of the NACI guidance and the period following publication were 0.0% (95% CI: -0.4–0.3%) and − 0.8% (95% CI -2.3% to -0.1%), respectively. Following interventional ARIMA modelling, publication of the NACI guidance was not associated with a statistically significant decrease in the monthly pertussis incidence trend (-0.67 cases per 100,000 infants; p = 0.73). Conclusion Publication of national recommendations for antenatal Tdap immunization in every pregnancy did not significantly reduce infant pertussis rates. This may reflect the persistently low rate of antenatal vaccination following publication of the recommendations. Expanding the scope of practice of allied health care providers to include antenatal Tdap immunization and patient education regarding antenatal pertussis immunization should be considered to further optimize uptake of vaccination.

Abstract The gut microbiome is important for human health, yet modulation requires more insight into inter-individual variation. Here, we explored latent structures of the human gut microbiome across the human lifespan, applying partitioning, pseudotime, and ordination approaches to >35,000 samples. Specifically, three major gut microbiome branches were identified, within which multiple partitions were observed in adulthood, with differential abundances of species along branches. Different compositions and metabolic functions characterized the branches’ tips, reflecting ecological differences. An unsupervised network analysis from longitudinal data from 745 individuals showed that partitions exhibited connected gut microbiome states rather than over-partitioning. Stability in the Bacteroides-enriched branch was associated with specific ratios of Faecalibacterium:Bacteroides. We also showed that associations with factors (intrinsic and extrinsic) could be generic, branch- or partition-specific. Our ecological framework for cross-sectional and longitudinal data allows a better understanding of overall variation in the human gut microbiome and disentangles factors associated with specific configurations.

Abstract Background Mercaptopurine is an important component of acute lymphoblastic leukemia (ALL) and lymphoma (LLy) maintenance therapy. The 6‐thioguanine nucleosides (6‐TGN) are believed to be the primary contributor to myelosuppression and immunosuppressive effects, while 6‐methylmercaptopurine (6‐MMPN) is believed to be responsible for several toxicities including hepatotoxicity, pancreatitis, and hypoglycemia. Previous reports suggest the addition of allopurinol may reduce these toxicities. Aims To assess the use of allopurinol to improve both safety and efficacy of mercaptopurine in pediatric patients with ALL and LLy during maintenance therapy. Secondary objectives included evaluating patient tolerability and skewed metabolism. In addition, we also analyzed mercaptopurine daily dose reduction upon allopurinol initiation. Methods and Results The primary endpoint was time within goal ANC prior to and after initiation of allopurinol. Secondary endpoints included; improvement in selective toxicities (hepatotoxicity, pancreatitis, and hypoglycemia) and 6‐MMPN to 6‐TGN ratio prior to and after allopurinol initiation. In addition, an exploratory endpoint assessing mercaptopurine daily dose reduction prior to and after allopurinol initiation was included. Sixteen patients met inclusion criteria and 15 (94%) of which were included in this study. Median percent of maintenance days within goal ANC prior to and after initiation of allopurinol was 27.8 (IQR 22.6–44.9) and 41.6 (IQR 20.2–58.2) respectively. All patients experienced selective toxicities; 15 (100%) hepatotoxicity, 1 (7%) pancreatitis, and 3 (20%) hypoglycemia. Improvement of toxicities was seen in 13/15 (87%), 1/1 (100%), and 2/3 (67%) respectively. Average 6‐MMPN:6‐TGN ratio prior to allopurinol initiation was 304:1 and after, allopurinol initiation improved to 15:1, resulting in a 95% reduction. Average mercaptopurine dose prior to and after allopurinol initiation decreased by about 56% (63 to 28 mg/m2/day). Conclusion Results suggest that the use of allopurinol in pediatric patients with ALL and LLy receiving mercaptopurine during maintenance therapy is both safe and effective.