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Abstract Objectives The objective of this study is to report the pilot phase of the Targeted Prostate Health Check programme that aims to identify men in the Surrey and Sussex region who have prostate cancer and who failed to be detected during the Covid era. Subjects and methods Men aged 50 to 70, or 45 to 70 if Black or with a family history of prostate cancer, were identified from participating general practitioner (GP) records. Short message service (SMS) texts invited men to visit www.talkprostate.co.uk for information on prostate cancer and give consent to prostate‐specific antigen (PSA) checks coordinated by a third‐party virtual healthcare provider. Elevated age‐related PSA levels, or levels below age‐related thresholds but at 3 ng/mL or more, triggered referral to a rapid access urology clinic. GPs were informed of the results. Results From 1842 text messages inviting 1549 people, 544 men consented to a PSA check. From 500 phlebotomy appointments, 485 (30% of invited men) took the PSA test of whom 68 (14%) were referred with an elevated PSA. After clinical review with multiparametric magnetic resonance imaging (mp‐MRI), 22 patients underwent transperineal biopsies, and prostate cancer was detected in 18 men of whom 17 (95%) had clinically significant cancer. Conclusion Our Targeted Prostate Cancer Health Check system identifies men at risk without burdening primary care. Awareness on prostate cancer risk was raised in 1549 invited men, half of whom were further educated via the registration website. One third of invited men were checked in whom clinically significant prostate cancer was found in 3.5%.

Abstract Infections caused by Gram-negative bacteria are leading causes of mortality worldwide. Due to the rise in antibiotic resistant strains, there is a desperate need for alternative strategies to control infections caused by these organisms. One such approach is the prevention of infection through vaccination. While live attenuated and heat-killed bacterial vaccines are effective, they can lead to adverse reactions. Newer vaccine technologies focus on utilizing polysaccharide or protein subunits for safer and more targeted vaccination approaches. One promising avenue in this regard is the use of proteins released by the Type 5 secretion system (T5SS). This system is the most prevalent secretion system in Gram-negative bacteria. These proteins are compelling vaccine candidates due to their demonstrated protective role in current licensed vaccines. Notably, Pertactin, FHA, and NadA are integral components of licensed vaccines designed to prevent infections caused by Bordetella pertussis or Neisseria meningitidis. In this review, we delve into the significance of incorporating T5SS proteins into licensed vaccines, their contributions to virulence, conserved structural motifs, and the protective immune responses elicited by these proteins.

ABSTRACT Salmonella enterica is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As S. enterica serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of Salmonella infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated Salmonella. Despite decades of research, many aspects of Salmonella infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated S. Typhimurium strains. We reveal differential essential pathways between strains in vitro and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCESalmonella enterica is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, S. enterica is used widely as a model to understand host responses to infection. Understanding how Salmonella survives in vivo is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, aroA, is thought to attenuate Salmonella by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of Salmonella to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of aroA in vaccine development.

Background. Knee and hip arthroplasty are two of the most frequently performed procedures in orthopaedic surgery. They are associated with positive patient-reported outcomes and significant improvements in quality of life for patients. Despite this, there may be room for further progress by quantifying functional improvements with gait analysis. Our study therefore aims to characterise the disease-specific gait pattern of participants with knee and hip osteoarthritis undergoing total joint replacement using a single chest-based wearable sensor. Methods. Twenty-nine participants awaiting total hip replacement and 28 participants awaiting total knee replacement underwent three-dimensional motion analysis with inertial wearable sensors. These gait metrics were then compared with 28 healthy controls of similar ages. Differences in gait metrics were evaluated using a T-test. The participants were recruited through a single centre to participate in this cross-sectional observational study. Participants with osteoarthritis severity sufficient to warrant surgical intervention were considered for inclusion in our study. The participants were instructed to walk 15–120 m in a hospital environment while fitted with a chest-based wearable sensor. Results. In total, three domains were evaluated, including spatiotemporal, variability and asymmetry parameters. There were marked variations in the gait asymmetry parameters and step length variation in both the hip and knee osteoarthritis patients compared with the healthy controls. The magnitude of gait deterioration in terms of step length asymmetry was greater on average in the hip osteoarthritis group than the knee group. The hip osteoarthritis (+180%, p < 0.001) and knee osteoarthritis (+129%, p = 0.001) groups demonstrated marked differences in step length asymmetry. Discussion. A single chest-based sensor was found to be capable of detecting pathological gait signatures in osteoarthritis patients when compared with age-matched controls. Future studies should compare pre- and postoperative changes to disease-specific gait impairments to validate the use of wearable sensors as a clinical adjunct.

Abstract Social networks are a mathematical representation of interactions among individuals which are prevalent across various animal species. Studies of human populations have shown the breadth of what can spread throughout a social network: obesity, smoking cessation, happiness, drug use and divorce. ‘Betweenness centrality’ is a key property of social networks that indicates an individual’s importance in facilitating communication and cohesion within the network. Heritability of betweenness centrality has been suggested in several species, however the genetic regulation of this property remains enigmatic. Here, we demonstrate that the gene CG14109, referred to as degrees of kevin bacon (dokb), influences betweenness centrality in Drosophila melanogaster. We identify strain-specific alleles of dokb with distinct amino acid sequences and when the dokb allele is exchanged between strains, flies exhibit the betweenness centrality pattern dictated by the donor allele. By inserting a GAL4 reporter into the dokb locus, we confirm that dokb is expressed in the central nervous system. These findings define a novel genetic entry point to study social network structure and thereby establish gene-to-social structure relationships. While dokb sequence homology is exclusive to Diptera, we anticipate that dokb-associated molecular pathways could unveil convergent neural mechanisms of social behaviour that apply in diverse animal species.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Abstract Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cryo-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Periostin is a matricellular protein encoded by the POSTN gene that is alternatively spliced to produce ten different periostin isoforms with molecular weights ranging from 78 to 91 kDa. It is known to promote fibrillogenesis, organize the extracellular matrix, and bind integrin-receptors to induce cell signaling. As well as being a key component of the wound healing process, it is also known to participate in the pathogenesis of different diseases including atopic dermatitis, asthma, and cancer. In both health and disease, the functions of the different periostin isoforms are largely unknown. The ability to precisely determine the isoform profile of a given human sample is fundamental for characterizing their functional significance. Identification of periostin isoforms is most often carried out at the transcriptional level using RT-PCR based approaches, but due to high sequence homogeneity, identification on the protein level has always been challenging. Top-down proteomics, where whole proteins are measured by mass spectrometry, offers a fast and reliable method for isoform identification. Here we present a fully developed top-down mass spectrometry assay for the characterization of periostin splice isoforms at the protein level.

Abstract Background This study aimed to test, in real-world clinical practice, the effectiveness of a Transitional Care Stroke Intervention (TCSI) compared to usual care on health outcomes, self-management, patient experience, and health and social service use costs in older adults (≥ 55 years) with stroke and multimorbidity (≥ 2 chronic conditions). Methods This pragmatic randomized controlled trial (RCT) included older adults discharged from hospital to community with stroke and multimorbidity using outpatient stroke rehabilitation services in two communities in Ontario, Canada. Participants were randomized 1:1 to usual care (control group) or usual care plus the 6-month TCSI (intervention group). The TCSI was delivered virtually by an interprofessional (IP) team, and included care coordination/system navigation support, phone/video visits, monthly IP team conferences, and an online resource to support system navigation. The primary outcome was risk of hospital readmission (all cause) after six-months. Secondary outcomes included physical and mental functioning, stroke self-management, patient experience, and health and social service use costs. The intention-to-treat principle was used to conduct the primary and secondary analyses. Results Ninety participants were enrolled (44 intervention, 46 control); 11 (12%) participants were lost to follow-up, leaving 79 (39 intervention, 40 control). No significant between-group differences were seen for baseline to six-month risk of hospital readmission. Differences favouring the intervention group were seen in the following secondary outcomes: physical functioning (SF-12 PCS mean difference: 5.10; 95% CI: 1.58–8.62, p = 0.005), stroke self-management (Southampton Stroke Self-Management Questionnaire mean difference: 6.00; 95% CI: 0.51—11.50, p = 0.03), and patient experience (Person-Centred Coordinated Care Experiences Questionnaire mean difference: 2.64, 95% CI: 0.81, 4.47, p = 0.005). No between-group differences were found in total healthcare costs or other secondary outcomes. Conclusions Although participation in the TCSI did not impact hospital readmissions, there were improvements in physical functioning, stroke self-management and patient experience in older adults with stroke and multimorbidity without increasing total healthcare costs. Challenges associated with the COVID-19 pandemic, including the shift from in-person to virtual delivery, and re-deployment of interventionists could have influenced the results. A larger pragmatic RCT is needed to determine intervention effectiveness in diverse geographic settings and ethno-cultural populations and examine intervention scalability. Trial registration ClinicalTrials.gov Identifier: NCT04278794 . Registered May 2, 2020.

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.

The Unimak and Shumagin segments of the Alaska Aleutian Subduction Zone show extensional deformation of the forearc since the Miocene. Using legacy seismic profiles and modern multichannel seismic data, we update the structural map of the area, focusing on the intersection between trench-parallel, landward-dipping normal faults rooting in the plate interface and trench-oblique to trench-perpendicular normal faults, all showing signs of recent activity. We investigate for the first time the origin of the trench-parallel extension and explain the horsetail geometry of the Central Sanak Basin as the termination of a slip-partitioning right-lateral strike slip fault. Re-analysis of subduction zone thrust earthquakes slip vectors indicates a possible onset of slip partitioning in the vicinity of the Central Sanak Basin. In the hypothesis of a continuum of deformation, finite deformation from normal fault offsets show a slow sliver motion of less than 1 mm/yr, which is below the resolution of GNSS measurements. Both trench oblique and trench parallel faults have been cited as reactivated terrane sutures, the presence of which may act as upper-plate weaknesses needed to allow slip partitioning in a context of low convergence obliquity. Weak landward-dipping normal faults rooting in the plate interface have also been linked to the tsunamigenic rupture of the shallow plate interface. We infer that the trench parallel Unimak Ridge, associated with the 1946 Mw 8.6 tsunami earthquake, is the last expression of terrane sutures reactivation before their westward vanishing in the more recent Aleutian arc.

Abstract Background Allopurinol is a xanthine oxidase inhibitor that lowers serum uric acid and is used to prevent acute gout flares in patients with gout. Observational and small interventional studies have suggested beneficial cardiovascular effects of allopurinol. Objective To determine whether allopurinol improves major cardiovascular outcomes in patients with ischaemic heart disease. Design Prospective, randomised, open-label, blinded endpoint multicentre clinical trial. Setting Four hundred and twenty-four UK primary care practices. Participants Aged 60 years and over with ischaemic heart disease but no gout. Interventions Participants were randomised (1 : 1) using a central web-based randomisation system to receive allopurinol up to 600 mg daily that was added to usual care or to continue usual care. Main outcome measures The primary outcome was the composite of non-fatal myocardial infarction, non-fatal stroke or cardiovascular death. Secondary outcomes were non-fatal myocardial infarction, non-fatal stroke, cardiovascular death, all-cause mortality, hospitalisation for heart failure, hospitalisation for acute coronary syndrome, coronary revascularisation, hospitalisation for acute coronary syndrome or coronary revascularisation, all cardiovascular hospitalisations, quality of life and cost-effectiveness. The hazard ratio (allopurinol vs. usual care) in a Cox proportional hazards model was assessed for superiority in a modified intention-to-treat analysis. Results From 7 February 2014 to 2 October 2017, 5937 participants were enrolled and randomised to the allopurinol arm (n = 2979) or the usual care arm (n = 2958). A total of 5721 randomised participants (2853 allopurinol; 2868 usual care) were included in the modified intention-to-treat analysis population (mean age 72.0 years; 75.5% male). There was no difference between the allopurinol and usual care arms in the primary endpoint, 314 (11.0%) participants in the allopurinol arm (2.47 events per 100 patient-years) and 325 (11.3%) in the usual care arm (2.37 events per 100 patient-years), hazard ratio 1.04 (95% confidence interval 0.89 to 1.21); p = 0.65. Two hundred and eighty-eight (10.1%) participants in the allopurinol arm and 303 (10.6%) participants in the usual care arm died, hazard ratio 1.02 (95% confidence interval 0.87 to 1.20); p = 0.77. The pre-specified health economic analysis plan was to perform a ‘within trial’ cost-utility analysis if there was no statistically significant difference in the primary endpoint, so NHS costs and quality-adjusted life-years were estimated over a 5-year period. The difference in costs between treatment arms was +£115 higher for allopurinol (95% confidence interval £17 to £210) with no difference in quality-adjusted life-years (95% confidence interval −0.061 to +0.060). We conclude that there is no evidence that allopurinol used in line with the study protocol is cost-effective. Limitations The results may not be generalisable to younger populations, other ethnic groups or patients with more acute ischaemic heart disease. One thousand six hundred and thirty-seven participants (57.4%) in the allopurinol arm withdrew from randomised treatment, but an on-treatment analysis gave similar results to the main analysis. Conclusions The ALL-HEART study showed that treatment with allopurinol 600 mg daily did not improve cardiovascular outcomes compared to usual care in patients with ischaemic heart disease. We conclude that allopurinol should not be recommended for the secondary prevention of cardiovascular events in patients with ischaemic heart disease but no gout. Future work The effects of allopurinol on cardiovascular outcomes in patients with ischaemic heart disease and co-existing hyperuricaemia or clinical gout could be explored in future studies. Trial registration This trial is registered as EU Clinical Trials Register (EudraCT 2013-003559-39) and ISRCTN (ISRCTN 32017426). Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 11/36/41) and is published in full in Health Technology Assessment; Vol. 28, No. 18. See the NIHR Funding and Awards website for further award information. Plain language summary What was the question? The purpose of the ALL-HEART study was to determine whether giving allopurinol to people with ischaemic heart disease (also commonly known as coronary heart disease) would reduce their risk of having a heart attack, stroke or of dying from cardiovascular disease. Allopurinol is a medication usually given to patients with gout to prevent acute gout flares. It is not currently used to treat ischaemic heart disease. What did we do? We randomly allocated people aged over 60 years with ischaemic heart disease to take up to 600 mg of allopurinol daily (in addition to their usual care) or to continue with their usual care. We then monitored participants for several years and recorded any major health events such as heart attacks, strokes and deaths. We obtained most of the follow-up data from centrally held electronic hospital admissions and death records, making the study easier for participants and more cost-efficient. We asked participants in both groups to complete questionnaires to assess their quality of life during the study. We also collected data to determine whether there was any economic benefit to the NHS of using allopurinol in patients with ischaemic heart disease. What did we find? There was no difference in the risk of heart attacks, strokes or death from cardiovascular disease between the participants given allopurinol and those in the group continuing their usual care. We also found no difference in the risks of other cardiovascular events, deaths from any cause or quality-of-life measurements between the allopurinol and usual care groups. What does this mean? The results of the ALL-HEART study suggest that we should not recommend that allopurinol be given to people with ischaemic heart disease to prevent further cardiovascular events or deaths. Scientific summary Background Allopurinol is a xanthine oxidase inhibitor that lowers serum uric acid (SUA) and is widely used in patients with gout to prevent acute gout flares. Xanthine oxidase promotes inflammation and atherosclerosis via the production of reactive oxygen species and xanthine oxidase levels are raised in several conditions including coronary artery disease. The role of SUA in cardiovascular (CV) disease is controversial, with some studies associating higher SUA levels with worse CV outcomes, but more recent genome-wide association studies suggest no major role of uric acid levels in determining CV outcomes. Some observational studies have suggested that allopurinol therapy may improve CV outcomes, while others have not found an association. Small interventional studies have shown that allopurinol therapy improves some CV parameters, including endothelial function, left ventricular hypertrophy, blood pressure, carotid intimal media thickness and arterial stiffness. Allopurinol therapy was also found to improve outcomes after acute coronary syndrome (ACS) in one study and to improve chest pain in patients with chronic stable angina with documented coronary artery disease in another. However, results have not been consistent across different studies. Before the ALL-HEART study, no large, randomised trial of the effects of allopurinol therapy on CV outcomes in patients with ischaemic heart disease (IHD) had been performed. Objectives Primary Does allopurinol therapy added to usual care improve major CV outcomes in patients aged over 60 years with IHD but no gout? Secondary Does allopurinol therapy added to usual care improve all-cause mortality or other CV outcomes in patients with IHD? What is the cost-effectiveness of adding allopurinol up to 600 mg daily to usual care in patients with IHD? Does allopurinol therapy added to usual care improve quality of life assessed by general health survey [EuroQol-5 Dimensions, five-level version (EQ-5D-5L)] or coronary heart disease-specific questionnaire (Seattle angina questionnaire)? Methods Design and participants The ALL-HEART study was a prospective, randomised, open-label, blinded endpoint (PROBE) multicentre trial undertaken in patients with IHD. Participants were primarily recruited from 424 primary care practices via 18 regional centres in the UK, with a small number also referred into the study from secondary care centres. Eligible patients were aged 60 years or over, with a history of IHD [myocardial infarction (MI), angina or other evidence of IHD]. Exclusion criteria were: history of gout; known severe renal impairment [estimated glomerular filtration rate (eGFR) < 30 ml/minute/1.73 m2]; moderate-to-severe heart failure (HF) [New York Heart Association (NYHA) III–IV]; significant hepatic disease [e.g. alanine transaminase (ALT) > 3 × upper limit of normal, cirrhosis, ascites] (investigator opinion); currently taking part in another interventional clinical trial of an investigational medicinal product or medical device (or taken part in one within the last 3 months); previous allergy to allopurinol; previous serious adverse cutaneous (skin) reaction to any drug (e.g. Stevens–Johnson syndrome, toxic epidermal necrolysis, hospitalisation due to skin reaction to drug) (investigator opinion); already taking urate-lowering therapy (including allopurinol, febuxostat, sulfinpyrazone, benzbromarone, probenecid, rasburicase); taking azathioprine, mercaptopurine, ciclosporin or theophylline; malignancy (except non-metastatic, non-melanoma skin cancers, cervical in situ carcinoma, breast ductal carcinoma in situ, or stage 1 prostate carcinoma) within the last 5 years (investigator opinion). The exclusion criterion relating to renal impairment was originally ‘known renal impairment eGFR < 60 ml/minute/1.73 m2’ for patients recruited from the start of the trial (7 February 2014) until 4 April 2016 when an updated version of the protocol was implemented at all study sites to allow the inclusion of patients with moderate renal impairment in the study with the purpose of making the study results more generalisable. Fifty-two per cent of the target number of patients had been randomised by this date. Randomisation At a single screening and randomisation visit usually held at the patient’s primary care practice by a research nurse, patients were consented, screened and randomised to receive allopurinol therapy or to continue their usual care. Baseline demographics, medical history, CV risk factors and concomitant medications were recorded. Blood pressure, height and weight were measured. Baseline blood tests were taken for urea, creatinine and electrolytes, full blood count and SUA. Participants were randomised before screening blood results were available. When the screening results were available, a nurse telephoned the patient to advise them to start taking randomised therapy. If the screening visit eGFR result was below the exclusion limit, the participant did not receive any allopurinol and was excluded from the modified intention-to-treat (mITT) analysis population, whichever arm of the study they had been randomised to. Randomisation was via a web-based randomisation facility located at the Robertson Centre for Biostatistics, University of Glasgow, accessed using a web-based application or an interactive voice response system. Randomisation was based on randomised permuted blocks of variable size, stratified by history of MI, history of stroke and primary care practice. Randomised intervention Allopurinol 100 or 300 mg tablets were prescribed to participants by their primary care physicians via the usual NHS prescription system. Participants with a screening eGFR result ≥ 60 ml/minute/1.73 m2 were prescribed allopurinol at 100 mg daily for 2 weeks, then 300 mg daily for 2 weeks then 600 mg daily (given as 300 mg twice daily) thereafter if tolerated for the duration of the study. After 4 April 2016, participants with a screening eGFR result 30–59 ml/minute/1.73 m2 were prescribed allopurinol at 100 mg daily for 2 weeks, then 300 mg daily thereafter if tolerated for the duration of the study. If there were tolerability issues, the dose could be decreased at the discretion of a physician. Participants who stopped randomised therapy were encouraged to continue with study follow-up. The comparison arm of the study was ‘usual care’; no placebo was given as this was a pragmatic open-label study. Randomised therapy was blinded to the endpoint adjudication committee, but not to participants, study staff or treating healthcare professionals. If allopurinol was started in participants randomised to the usual care group at any point in the study (e.g. for clinical reasons such as developing gout), this was recorded. Other study procedures At the screening visit, participants completed the EQ-5D-5L questionnaire to assess general health outcomes and the Seattle angina questionnaire to assess coronary artery disease-specific quality of life. Participants who had taken any allopurinol therapy attended a 6-week study visit. Blood samples were taken for urea, creatinine and electrolytes, full blood count and SUA. Participants were then followed up remotely by electronic record-linkage with centralised databases of hospitalisations, cancers and deaths held by Public Health Scotland (PHS) and NHS Digital and by annual questionnaires (online, by post or by telephone). Data on adverse events, skin rashes, gout flares and self-reported adherence to randomised therapy were collected at the 6-week visit, then by annual questionnaire (but could also be reported at any time by participants or health professionals). Participants completed the EQ-5D-5L and Seattle angina questionnaires again after 1 year and at the end of the trial. The trial recruitment period was extended once, and the trial follow-up period was extended twice. Recruitment exceeded the target of 5215 randomised participants to a final total of 5937 randomised participants due to an increase in recruitment rate in the last weeks of recruitment. The follow-up period of the trial ended on 30 September 2021. After this date, participants stopped randomised therapy and continued to receive their usual care. Study outcomes The primary and secondary outcomes were as follows. Primary outcome: Composite of non-fatal MI, non-fatal stroke or CV death. Secondary outcomes: Non-fatal MI. Non-fatal stroke. CV death. All-cause mortality. Hospitalisation for ACS. Coronary revascularisation. Hospitalisation for ACS or coronary revascularisation. Hospitalisation for HF. All CV hospitalisations. Quality of life. Cost-effectiveness. Adverse events Serious adverse events (SAEs) occurring during the study (until 30 September 2021) were recorded and any events ongoing at that time were followed up for a further 30 days, unless consent had been withdrawn. Treatment-related adverse events (adverse reactions), gout flares and rashes were also recorded. Allopurinol therapy was stopped if participants developed a rash that may have been due to allopurinol. For any SAEs that were potential study endpoints, detailed information was obtained from medical records and death certificates to support the production of an anonymised endpoint package that was adjudicated by an independent clinical endpoint adjudication committee, blinded to randomised therapy allocation. Statistical analysis The power calculation suggested that 5215 participants would need to be randomised 1 : 1 to give 80% power to detect a 20% reduction in the primary outcome for the intervention (allowing for a 4% dropout for withdrawal of consent and non-CV deaths). A primary event rate of 14% over an average of 4 years follow-up was estimated from other trials in similar patient groups. The study ended when 631 adjudicated first primary events had occurred. Baseline characteristics are presented by treatment group as means [standard deviation (SD)] and medians [interquartile range (IQR)] for continuous variables and as numbers (%) for categorical variables. Clinical outcomes were analysed on a time-to-first event basis using Cox proportional hazards models. Treatment effects (allopurinol vs. usual care) were estimated as hazard ratios (HR) and 95% confidence intervals (CIs) for the Cox models. Analyses were adjusted for the stratification variables history of MI and history of stroke and p-values were calculated from Wald statistics. The primary analysis was a mITT analysis. Health economic analysis A health economic analysis was planned to determine whether allopurinol was a cost-effective intervention in the context of the NHS setting in the UK in patients with IHD. The analysis plan was based around NHS costs and estimation of quality-adjusted life-years (QALYs) and was to include a ‘within trial’ cost–utility analysis if the primary outcome was not statistically different between randomised study arms. Trial approvals and committees The study was approved by an ethics committee, Medicines and Healthcare Products Regulatory Agency (MHRA) and Health Research Authority (HRA). An Independent Data Monitoring Committee oversaw trial safety. A Trial Steering Committee including independent and patient/lay members oversaw trial progress. Results From 7 February 2014 to 29 September 2017, 6134 patients consented to enrol in the trial and were assessed for eligibility. Also, 167 of the 6134 consented participants were not eligible and 30 others were not randomised. The final randomisation was completed on 2 October 2017. Of the 5937 randomised participants, 216 were excluded post randomisation from the mITT analysis population (184 did not meet the eGFR entry criteria once their screening blood results were available; 32 were later found to have not met all of the inclusion/exclusion criteria). Five thousand seven hundred and twenty-one participants (2853 in the allopurinol arm and 2868 in the usual care arm) were included in the mITT analysis population for the efficacy analysis. The population for safety analyses consisted of 2805 participants in the allopurinol arm (excluding the 48 participants in the allopurinol arm who never received any randomised therapy) and 2868 participants in the usual care arm. The mean duration of follow-up in the trial was 4.8 years. Two hundred and fifty-eight participants (9.0%) in the allopurinol group and 76 participants (2.6%) in the usual care group withdrew consent for all follow-up. One thousand six hundred and thirty-seven participants (57.4%) in the allopurinol arm withdrew from randomised treatment. Baseline characteristics were well balanced in the two groups. The mean age at study entry was 72.0 years (SD 6.8), 4321 participants (75.5%) were male, 5676 (99.2%) were white and 1241 (21.7%) had a history of diabetes mellitus. The median duration of IHD at study entry was 10.1 years (IQR 5.1–16.1). Three thousand four hundred and sixty-four (60.5%) participants were recruited in England and 2257 (39.5%) participants in Scotland. The most commonly taken dose of allopurinol was 600 mg daily. In the 2447 participants in the allopurinol arm with both a baseline and 6-week SUA result, SUA fell from a mean of 0.34 (SD 0.08) mmol/l at baseline to a mean of 0.18 (SD 0.09) mmol/l 6 weeks after randomisation. Forty-five participants in the usual care arm started allopurinol therapy during follow-up (for clinical reasons, mainly gout). Primary and secondary outcomes There was no significant difference between the randomised treatment groups in the rates of the primary outcome or any of the secondary time-to-event outcomes. Three hundred and fourteen (11.0%) participants in the allopurinol arm (2.47 events per 100 patient-years) and 325 (11.3%) in the usual care arm (2.37 events per 100 patient-years) experienced a primary outcome, HR 1.04 (95% CI 0.89 to 1.21); p = 0.65. Two hundred and eighty-eight (10.1%) participants in the allopurinol arm and 303 (10.6%) participants in the usual care arm died, HR 1.02 (95% CI 0.87 to 1.20); p = 0.77. Results for the primary outcome were consistent across all pre-specified subgroups. In a supporting on-treatment analysis, results for the time-to-event clinical outcomes were broadly similar to those in the mITT analysis. There was limited evidence of any effect of allopurinol on quality-of-life outcomes, with no differences in EQ-5D-5L outcomes or Seattle angina questionnaire outcomes at the end of the first year or at the final visit, except for a nominally significant but only slightly greater fall in the physical domain score of the Seattle angina questionnaire at the end of the first year [treatment difference = 1.219 (95% CI 0.027 to 2.410); p = 0.045] in the allopurinol arm. Health economic analysis There was strong evidence that allopurinol treatment was associated with incremental costs relative to usual care [incremental cost per patient £115.4, 95% CI (£17.0 to £210.2)], with little evidence of improvement in relation to incremental QALYs [incremental QALYs −0.000, 95% CI (−0.061 to 0.060)]. The cost-effectiveness acceptability curve asymptoted at a probability of 0.456, meaning that even with a willingness to pay of an infinite amount, the probability of cost-effectiveness was only 0.465. At a willingness to pay of £20,000 the probability of cost-effectiveness was 0.41. Adverse events There was no difference in SAE rates between treatment arms, except for the grouping of endocrine disorders where no events occurred in the allopurinol treatment group but 14 in the usual care group. However, these endocrine events included events with a spread of different types. Fifteen participants had SAEs that were considered potentially treatment related and none of these were fatal. There was no difference between treatment arms in the rates of incident cancers. Adjudicated causes of death were well balanced between the treatment groups, including deaths from COVID-19 and COVID-19 pneumonia. Conclusions The ALL-HEART study showed that treatment with allopurinol 600 mg daily did not improve CV outcomes compared to usual care in patients with IHD. There were also no benefits on quality of life. There was no evidence that allopurinol used in line with the study protocol is cost-effective within the NHS system. We conclude that allopurinol should not be recommended for the secondary prevention of CV events in patients with IHD but no gout. Recommendations for research Future research should explore other therapeutic options for the improvement of CV outcomes and quality of life in patients with IHD. Further exploration of the effects of allopurinol on CV outcomes in patients with co-existing IHD and clinical gout or hyperuricaemia could be considered (patients with gout were excluded from this study). Trial registration The ALL-HEART trial is registered with the EU Clinical Trials Register (EudraCT 2013-003559-39) and ISRCTN (ISRCTN 32017426). Funding This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: 11/36/41) and is published in full in Health Technology Assessment; Vol. 28, No. 18. See the NIHR Funding and Awards website for further award information.

Background Patients with peripheral artery disease are at increased risk for major adverse cardiac events, major adverse limb events, and all‐cause death. Developing tools capable of identifying those patients with peripheral artery disease at greatest risk for major adverse events is the first step for outcome prevention. This study aimed to determine whether computer‐assisted analysis of a resting Doppler waveform using deep neural networks can accurately identify patients with peripheral artery disease at greatest risk for adverse outcome events. Methods and Results Consecutive patients (April 1, 2015, to December 31, 2020) undergoing ankle–brachial index testing were included. Patients were randomly allocated to training, validation, and testing subsets (60%/20%/20%). Deep neural networks were trained on resting posterior tibial arterial Doppler waveforms to predict major adverse cardiac events, major adverse limb events, and all‐cause death at 5 years. Patients were then analyzed in groups based on the quartiles of each prediction score in the training set. Among 11 384 total patients, 10 437 patients met study inclusion criteria (mean age, 65.8±14.8 years; 40.6% women). The test subset included 2084 patients. During 5 years of follow‐up, there were 447 deaths, 585 major adverse cardiac events, and 161 MALE events. After adjusting for age, sex, and Charlson comorbidity index, deep neural network analysis of the posterior tibial artery waveform provided independent prediction of death (hazard ratio [HR], 2.44 [95% CI, 1.78–3.34]), major adverse cardiac events (HR, 1.97 [95% CI, 1.49–2.61]), and major adverse limb events (HR, 11.03 [95% CI, 5.43–22.39]) at 5 years. Conclusions An artificial intelligence–enabled analysis of Doppler arterial waveforms enables identification of major adverse outcomes among patients with peripheral artery disease, which may promote early adoption and adherence of risk factor modification.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Abstract Background In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. Results We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. Conclusions We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.