The prognostic effect of mechanical, ultrastructural, and ECM signatures in glioblastoma core and rim.

Glioblastoma (GBM) is a highly invasive, aggressive brain cancer that carries a median survival of 15 months and is resistant to standard therapeutics. Recent studies have demonstrated that intratumoral heterogeneity plays a critical role in promoting resistance by mediating tumor adaptation through microenvironmental cues. GBM can be separated into two distinct regions-a core and a rim, which are thought to drive specific aspects of tumor evolution. These differences in tumor progression are regulated by the diverse biomolecular and biophysical signals in these regions, but the acellular biophysical characteristics remain poorly described. This study investigates the mechanical and ultrastructural characteristics of the tumor extracellular matrix (ECM) in patient-matched GBM core and rim tissues. Seven patient-matched tumor core and rim samples and one non-neoplastic control were analyzed using atomic force microscopy, scanning electron microscopy, and immunofluorescence imaging to quantify mechanical, ultrastructural, and ECM composition changes. The results reveal significant differences in biophysical parameters between GBM core, rim, and non-neoplastic tissues. The GBM core is stiffer, denser, and is rich in ECM proteins hyaluronic acid and tenascin-C when compared to tumor rim and non-neoplastic tissues. These alterations are intimately related and have prognostic effect with stiff, dense tissue correlating with longer progression-free survival. These findings reveal new insights into the spatial heterogeneity of biophysical parameters in the GBM tumor microenvironment and identify a set of characteristics that may correlate with patient prognosis. In the long term, these characteristics may aid in the development of strategies to combat therapeutic resistance.

Partial coil embolization before surgical clipping of ruptured intracranial aneurysms.

OBJECTIVE: Intraoperative rupture (IOR) is the most common adverse event encountered during surgical clip obliteration of ruptured intracranial aneurysms. Besides increasing surgeon experience and early proximal control, no methods exist to decrease IOR risk. Thus, our objective was to assess if partial endovascular coil embolization to protect the aneurysm before clipping decreases IOR. METHODS: We conducted a retrospective analysis of patients with ruptured intracranial aneurysms that were treated with surgical clipping at two tertiary academic centers. We compared patient characteristics and outcomes of those who underwent partial endovascular coil embolization to protect the aneurysm before clipping to those who did not. The primary outcome was IOR. Secondary outcomes were inpatient mortality and discharge destination. RESULTS: We analyzed 100 patients. Partial endovascular aneurysm protection was performed in 27 patients. Age, sex, subarachnoid hemorrhage severity, and aneurysm location were similar between the partially-embolized and non-embolized groups. The median size of the partially-embolized aneurysms was larger (7.0 mm [interquartile range 5.95-8.7] vs. 4.6 mm [3.3-6.0]; P < 0.001). During surgical clipping, IOR occurred less frequently in the partially-embolized aneurysms than non-embolized aneurysms (2/27, 7.4%, vs. 30/73, 41%; P = 0.001). Inpatient mortality was 14.8% (4/27) in patients with partially-embolized aneurysms and 28.8% (21/73) in patients without embolization (P = 0.20). Discharge to home or inpatient rehabilitation was 74.0% in patients with partially-embolized aneurysms and 56.2% in patients without embolization (P = 0.11). A complication from partial embolization occurred in 2/27 (7.4%) patients. CONCLUSIONS: Preoperative partial endovascular coil embolization of ruptured aneurysms is associated with a reduced frequency of IOR during definitive treatment with surgical clip obliteration. These results and the impact of preoperative partial endovascular coil embolization on functional outcomes should be confirmed with a randomized trial.

The ENCODE mouse postnatal developmental time course identifies regulatory programs of cell types and cell states.

Postnatal genomic regulation significantly influences tissue and organ maturation but is under-studied relative to existing genomic catalogs of adult tissues or prenatal development in mouse. The ENCODE4 consortium generated the first comprehensive single-nucleus resource of postnatal regulatory events across a diverse set of mouse tissues. The collection spans seven postnatal time points, mirroring human development from childhood to adulthood, and encompasses five core tissues. We identified 30 cell types, further subdivided into 69 subtypes and cell states across adrenal gland, left cerebral cortex, hippocampus, heart, and gastrocnemius muscle. Our annotations cover both known and novel cell differentiation dynamics ranging from early hippocampal neurogenesis to a new sex-specific adrenal gland population during puberty. We used an ensemble Latent Dirichlet Allocation strategy with a curated vocabulary of 2,701 regulatory genes to identify regulatory "topics," each of which is a gene vector, linked to cell type differentiation, subtype specialization, and transitions between cell states. We find recurrent regulatory topics in tissue-resident macrophages, neural cell types, endothelial cells across multiple tissues, and cycling cells of the adrenal gland and heart. Cell-type-specific topics are enriched in transcription factors and microRNA host genes, while chromatin regulators dominate mitosis topics. Corresponding chromatin accessibility data reveal dynamic and sex-specific regulatory elements, with enriched motifs matching transcription factors in regulatory topics. Together, these analyses identify both tissue-specific and common regulatory programs in postnatal development across multiple tissues through the lens of the factors regulating transcription.

Risk factors for stroke in penetrating cerebrovascular injuries.

OBJECTIVE: Penetrating cerebrovascular injuries (PCVI) are associated with a high incidence of mortality and neurological events. The optimal treatment strategy of PCVI, especially when damage control measures are required, remains controversial. The aim of this study was to describe the management of PCVI and patient outcomes at a level 1 trauma center where vascular injuries are managed predominantly by trauma surgeons. METHODS: An institutional trauma registry was queried for patients with PCVI from 2011 to 2021. Patients with common carotid artery (CCA), internal carotid artery (ICA), or vertebral artery injuries were included for analysis. The primary outcome was in-hospital stroke. The secondary outcomes were in-hospital mortality and in-hospital stroke or death. A subgroup analysis was completed of arterial repair (primary repair or interposition graft) vs ligation or embolization vs temporary intravascular shunting at the index procedure. RESULTS: We analyzed 54 patients with PCVI. Overall, the in-hospital stroke rate was 17% and in-hospital mortality was 26%. Twenty-one patients (39%) underwent arterial interventions for PCVI. Ten patients underwent arterial repair, six patients underwent ligation or embolization, and five patients underwent intravascular shunting as a damage control strategy with a plan for delayed repair. The rate of in-hospital stroke was 30% after arterial repair, 0% after arterial ligation or embolization, and 80% after temporary intravascular shunting. There was a significant difference in the stroke rate between the three subgroups (P = .015). Of the 32 patients who did not have an intervention to the CCA, ICA, or vertebral artery, 1 patient with ICA occlusion and 1 patient with CCA intimal injury developed in-hospital stroke. The mortality rate was 0% after arterial repair, 50% after ligation or embolization, and 60% after intravascular shunting. The rate of stroke or death was 30% in the arterial repair group, 50% in the ligation or embolization group, and 100% in the temporary intravascular shunting group. CONCLUSIONS: High rates of stroke and mortality were seen in patients requiring damage control after PCVI. In particular, temporary intravascular shunting was associated with a high incidence of in-hospital stroke and a 100% rate of stroke or death. Further investigation is needed into the factors related to these finding and whether the use of temporary intravascular shunting in PCVI is an advisable strategy.

Systematic assessment of long-read RNA-seq methods for transcript identification and quantification.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

Childhood adversity and mental health admission patterns prior to young person suicide (CHASE): a case-control 36 year linked hospital data study, Scotland UK 1981-2017.

BACKGROUND: Childhood adversity is associated with increased later mental health problems and suicidal behaviour. Opportunities for earlier healthcare identification and intervention are needed. AIM: To determine associations between hospital admissions for childhood adversity and mental health in children who later die by suicide. METHOD: Population-based longitudinal case-control study. Scottish in-patient general and psychiatric records were summarised for individuals born 1981 or later who died by suicide between 1991 and 2017 (cases), and matched controls (1:10), for childhood adversity and mental health (broadly defined as psychiatric diagnoses and general hospital admissions for self-harm and substance use). RESULTS: Records were extracted for 2477 'cases' and 24 777 'controls'; 2106 cases (85%) and 13 589 controls (55%) had lifespan hospitalisations. Mean age at death was 23.7; 75.9% were male. Maltreatment or violence-related childhood adversity codes were recorded for 7.6% cases aged 10-17 (160/2106) versus 2.7% controls (371/13 589), odds ratio = 2.9 (95% CI, 2.4-3.6); mental health-related admissions were recorded for 21.7% cases (458/2106), versus 4.1% controls (560/13 589), odds ratio = 6.5 (95% CI, 5.7-7.4); 80% of mental health admissions were in general hospitals. Using conditional logistic models, we found a dose-response effect of mental health admissions <18y, with highest adjusted odds ratio (aOR) for three or more mental health admissions: aORmale = 8.17 (95% CI, 5.02-13.29), aORfemale = 15.08 (95% CI, 8.07-28.17). We estimated that each type of childhood adversity multiplied odds of suicide by aORmale = 1.90 (95% CI, 1.64-2.21), aORfemale = 2.65 (95% CI, 1.94-3.62), and each mental health admission by aORmale = 2.06 (95% CI, 1.81-2.34), aORfemale = 1.78 (95% CI, 1.50-2.10). CONCLUSIONS: Our lifespan study found that experiencing childhood adversity (primarily maltreatment or violence-related admissions) or mental health admissions increased odds of young person suicide, with highest odds for those experiencing both. Healthcare practitioners should identify and flag potential 'at-risk' adolescents to prevent future suicidal acts, especially those in general hospitals.

Addressing Substance Use in the Adolescent Transplant Population.

Adolescence is a unique period of physical and psychosocial changes as youth transition, over many years, to adulthood. The psychosocial changes that accompany adolescence include emotional separation from parents, greater influence of peer groups, an interest in self-identification and autonomy, and increased risk-taking behaviors. Substance use is a common form of risk-taking behavior in the adolescent developmental stage. Alcohol, nicotine, and cannabis are the most common types of substances used in the United States. In the adolescent transplant population, rates of substance use appear to be at, or slightly below, their peer counterparts. Substance use can lead to deleterious health outcomes for adolescent transplant patients as a result of impaired decision-making, reduction in medication and clinic visit compliance, increases in mental health disorders, and risk for developing dependence and a substance use disorder. Given the close relationship that many pediatric transplant providers have with their patients and families, transplant care teams are in an excellent position to help their patients by addressing adolescent substance use. This narrative review describes how providers can use proactive standardized approaches to identify and intervene with substance use behavior.

Longitudinal evolution of the HIV effective reproduction number following sequential expansion of treatment as prevention and pre-exposure prophylaxis in British Columbia, Canada: a population-level programme evaluation.

BACKGROUND: Treatment as prevention and pre-exposure prophylaxis (PrEP) are key strategies in the control of HIV/AIDS. We aimed to characterise the longitudinal effects of antiretroviral therapy (ART), followed by treatment as prevention and the addition of PrEP, on the HIV effective reproduction number (Re) in British Columbia, Canada. METHODS: This population-level programme evaluation used data from the Drug Treatment Program of the British Columbia Centre for Excellence in HIV/AIDS (Vancouver, British Columbia, Canada). We also used estimates of HIV incidence and prevalence from the Public Health Agency of Canada, data on the number of new HIV diagnoses per year from the British Columbia Centre for Disease Control, and mortality data from the British Columbia Vital Statistics Agency. Data were obtained from 1985 until 2022, depending on the database source. Outcomes were the annual HIV prevalence, HIV incidence, number of new HIV diagnoses, number of people living with HIV on ART, HIV/AIDS-related and all-cause mortality rates, the HIV incidence-to-all-cause-mortality ratio, and Re. We calculated the modified effective reproduction number (Rme) using two thresholds of viral suppression and compared these values with Re. FINDINGS: We found a 95% decline in HIV/AIDS-related mortality and a 91% decrease in HIV incidence over the study period. The Re progressively declined from 1996 to 2022; however, from 1996 to 2017, Rme remained stable (>1) when calculated for people living with HIV with unsuppressed viraemia, suggesting that treatment as prevention reduces HIV incidence by decreasing the pool of individuals who are potentially able to transmit the virus. From 2018 to 2022, a decline in the estimated Re and Rme (<1) was observed regardless of whether we considered all people living with HIV or only those who were virologically unsuppressed. This finding suggests that PrEP decreases HIV incidence by reducing the number of susceptible individuals in the community, independently of viral suppression. INTERPRETATION: Our results show the synergy between generalised treatment as prevention and targeted PrEP in terms of decreasing HIV incidence. These findings support the incorporation of longitudinal monitoring of Re at a programmatic level to identify opportunities for the optimisation of treatment-as-prevention and PrEP programmes. FUNDING: British Columbia Ministry of Health, Health Canada, Public Health Agency of Canada, Vancouver Coastal Health, Vancouver General Hospital Foundation, Genome British Columbia, and the Canadian Institutes of Health Research.

Evaluating the oral delivery of GalNAc-conjugated siRNAs in rodents and non-human primates.

Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.

Pathologic Outcomes of Short-Course and Long-Course Radiotherapy for Locally Advanced Rectal Cancers Treated With Total Neoadjuvant Therapy.

INTRODUCTION: Total neoadjuvant therapy (TNT) for patients with locally advanced rectal cancer (LARC) is now the standard of care. Randomized trials suggest the use of short-course radiotherapy (SCRT) and long-course radiotherapy (LCRT) are oncologically equivalent. OBJECTIVE: To describe pathologic outcomes after surgical resections of patients receiving SCRT versus LCRT as part of TNT for LARC. PARTICIPANTS: All patients with LARC treated at a single tertiary hospital who underwent proctectomy after completing TNT were included. Patients were excluded if adequate details of TNT were not available in the electronic medical record. RESULTS: A total of 53 patients with LARC were included. Thirty-nine patients (73.5%) received LCRT and 14 (26.4%) received SCRT. Forty-nine patients (92.5%) were clinical stage III (cN1-2) prior to treatment. The average lymph node yield after proctectomy was 20.9 for SCRT and 17.0 for LCRT (P = .075). Of the 49 patients with clinically positive nodes before treatment, 76.9% of those who received SCRT and 72.2% of those who received LCRT achieved pN0 disease after TNT. Additionally, there were no significant differences in rates of pathologic complete response between patients who received SCRT and LCRT, 7.1% and 12.8%, respectively (P = .565). CONCLUSION: Pathologic outcomes of patients with LARC treated with SCRT or LCRT, as part of TNT, may be similar. Further prospective trials are needed to assess long-term clinical outcomes and to determine best treatment protocols.

Emergency management and sustainability: Understanding the link between disaster and citizen participation for sustainability efforts and climate change.

The goal of this study is to examine how disaster experience influences local government views on citizen participation in addressing issues of sustainability, such as climate change. This study considers concepts such as wicked problems, the social order, the environment, economic development, and citizen participation where sustainability can be considered a solution to help manage and solve the challenges of disaster, like climate change. The data are taken from a 2015 International City/County Management Association national survey that examines the link between disaster and sustainability. The results show that more than half of the respondents do not view public participation as having much of an impact on sustainability; however, we can expect public participation to increasingly impact sustainability efforts as communities experience more disaster. This suggests that emergency management needs to understand public pressures regarding wicked problems, such as climate change, to collectively address the global influence of environmental, economic, and social issues that have local effects on their communities.

Identification of robust cellular programs using reproducible LDA that impact sex-specific disease progression in different genotypes of a mouse model of AD.

The gene expression profiles of distinct cell types reflect complex genomic interactions among multiple simultaneous biological processes within each cell that can be altered by disease progression as well as genetic background. The identification of these active cellular programs is an open challenge in the analysis of single-cell RNA-seq data. Latent Dirichlet Allocation (LDA) is a generative method used to identify recurring patterns in counts data, commonly referred to as topics that can be used to interpret the state of each cell. However, LDA's interpretability is hindered by several key factors including the hyperparameter selection of the number of topics as well as the variability in topic definitions due to random initialization. We developed Topyfic, a Reproducible LDA (rLDA) package, to accurately infer the identity and activity of cellular programs in single-cell data, providing insights into the relative contributions of each program in individual cells. We apply Topyfic to brain single-cell and single-nucleus datasets of two 5xFAD mouse models of Alzheimer's disease crossed with C57BL6/J or CAST/EiJ mice to identify distinct cell types and states in different cell types such as microglia. We find that 8-month 5xFAD/Cast F1 males show higher level of microglial activation than matching 5xFAD/BL6 F1 males, whereas female mice show similar levels of microglial activation. We show that regulatory genes such as TFs, microRNA host genes, and chromatin regulatory genes alone capture cell types and cell states. Our study highlights how topic modeling with a limited vocabulary of regulatory genes can identify gene expression programs in single-cell data in order to quantify similar and divergent cell states in distinct genotypes.

Spatially Resolved Microglia/Macrophages in Recurrent Glioblastomas Overexpress Fatty Acid Metabolism and Phagocytic Genes.

BACKGROUND: Glioblastoma (GBM) tumors are rich in tumor-associated microglia/macrophages. Changes associated with treatment in this specific cell population are poorly understood. Therefore, we studied changes in gene expression of tumor-associated microglia/macrophages (Iba1+) cells in de novo versus recurrent GBMs. METHODS: NanoString GeoMx® Digital Spatial Transcriptomic Profiling of microglia/macrophages (Iba1+) and glial cells (Gfap+) cells identified on tumor sections was performed on paired de novo and recurrent samples obtained from three IDH-wildtype GBM patients. The impact of differentially expressed genes on patient survival was evaluated using publicly available data. RESULTS: Unsupervised analyses of the NanoString GeoMx® Digital Spatial Profiling data revealed clustering based on the transcriptomic data from Iba1+ and Gfap+ cells. As expected, conventional differential gene expression and enrichment analyses revealed upregulation of immune-function-related genes in Iba1+ cells compared to Gfap+ cells. A focused differential gene expression analysis revealed upregulation of phagocytosis and fatty acid/lipid metabolism genes in Iba1+ cells in recurrent GBM samples compared to de novo GBM samples. Importantly, of these genes, the lipid metabolism gene PLD3 consistently correlated with survival in multiple different publicly available datasets. CONCLUSION: Tumor-associated microglia/macrophages in recurrent GBM overexpress genes involved in fatty acid/lipid metabolism. Further investigation is needed to fully delineate the role of PLD phospholipases in GBM progression.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer's-like neurodegeneration.

Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/fibrillar pTau, however, appears to vary depending on the animal model used. Our prior work suggested that antigen-specific memory CD8 T (" hi T") cells act upstream of Aß/pTau after brain injury. Here we examine whether hi T cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hi T mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. Our work is the first to identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD. Significance Statement: This study changes our view of Alzheimer's Disease (AD) initiation and progression. Mutations promoting cerebral beta-amyloid (Aß) deposition guarantee rare genetic forms of AD. Thus, the prevailing hypothesis has been that Aß is central to initiation and progression of all AD, despite contrary animal and patient evidence. We show that age-related T cells generate neurodegeneration with compelling features of AD in mice, with distinct T cell functions required for pathological initiation and neurodegenerative progression. Knowledge from these mice was applied to successfully predict previously unknown features of human AD and generate novel tools for its clinical management.

Quantifying EHR and Policy Factors Associated with the Gender Productivity Gap in Ambulatory, General Internal Medicine.

BACKGROUND: The gender gap in physician compensation has persisted for decades. Little is known about how differences in use of the electronic health record (EHR) may contribute. OBJECTIVE: To characterize how time on clinical activities, time on the EHR, and clinical productivity vary by physician gender and to identify factors associated with physician productivity. DESIGN, SETTING, AND PARTICIPANTS: This longitudinal study included general internal medicine physicians employed by a large ambulatory practice network in the Northeastern United States from August 2018 to June 2021. MAIN MEASURES: Monthly data on physician work relative value units (wRVUs), physician and practice characteristics, metrics of EHR use and note content, and temporal trend variables. KEY RESULTS: The analysis included 3227 physician-months of data for 108 physicians (44% women). Compared with men physicians, women physicians generated 23.8% fewer wRVUs per month, completed 22.1% fewer visits per month, spent 4.0 more minutes/visit and 8.72 more minutes on the EHR per hour worked (all p < 0.001), and typed or dictated 36.4% more note characters per note (p = 0.006). With multivariable adjustment for physician age, practice characteristics, EHR use, and temporal trends, physician gender was no longer associated with productivity (men 4.20 vs. women 3.88 wRVUs/hour, p = 0.31). Typing/dictating fewer characters per note, relying on greater teamwork to manage orders, and spending less time on documentation were associated with higher wRVUs/hour. The 2021 E/M code change was associated with higher wRVUs/hour for all physicians: 10% higher for men physicians and 18% higher for women physicians (p < 0.001 and p = 0.009, respectively). CONCLUSIONS: Increased team support, briefer documentation, and the 2021 E/M code change were associated with higher physician productivity. The E/M code change may have preferentially benefited women physicians by incentivizing time-intensive activities such as medical decision-making, preventive care discussion, and patient counseling that women physicians have historically spent more time performing.

A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

REMAP Periop: a randomised, embedded, multifactorial adaptive platform trial protocol for perioperative medicine to determine the optimal enhanced recovery pathway components in complex abdominal surgery patients within a US healthcare system.

INTRODUCTION: Implementation of enhanced recovery pathways (ERPs) has resulted in improved patient-centred outcomes and decreased costs. However, there is a lack of high-level evidence for many ERP elements. We have designed a randomised, embedded, multifactorial, adaptive platform perioperative medicine (REMAP Periop) trial to evaluate the effectiveness of several perioperative therapies for patients undergoing complex abdominal surgery as part of an ERP. This trial will begin with two domains: postoperative nausea/vomiting (PONV) prophylaxis and regional/neuraxial analgesia. Patients enrolled in the trial will be randomised to arms within both domains, with the possibility of adding additional domains in the future. METHODS AND ANALYSIS: In the PONV domain, patients are randomised to optimal versus supraoptimal prophylactic regimens. In the regional/neuraxial domain, patients are randomised to one of five different single-injection techniques/combination of techniques. The primary study endpoint is hospital-free days at 30 days, with additional domain-specific secondary endpoints of PONV incidence and postoperative opioid consumption. The efficacy of an intervention arm within a given domain will be evaluated at regular interim analyses using Bayesian statistical analysis. At the beginning of the trial, participants will have an equal probability of being allocated to any given intervention within a domain (ie, simple 1:1 randomisation), with response adaptive randomisation guiding changes to allocation ratios after interim analyses when applicable based on prespecified statistical triggers. Triggers met at interim analysis may also result in intervention dropping. ETHICS AND DISSEMINATION: The core protocol and domain-specific appendices were approved by the University of Pittsburgh Institutional Review Board. A waiver of informed consent was obtained for this trial. Trial results will be announced to the public and healthcare providers once prespecified statistical triggers of interest are reached as described in the core protocol, and the most favourable interventions will then be implemented as a standardised institutional protocol. TRIAL REGISTRATION NUMBER: NCT04606264.

Understanding capacity for implementing new interventions: A qualitative study of speech and language therapy services for children with speech sound disorder.

BACKGROUND: Many speech sound disorder (SSD) interventions with a long-term evidence base are 'new' to clinical practice, and the role of services in supporting or constraining capacity for practice change is underexplored. Innovations from implementation science may offer solutions to this research-practice gap but have not previously been applied to SSD. AIM: To explain variation in speech and language therapy service capacity to implement new SSD interventions. METHODS & PROCEDURES: We conducted an intensive, case-based qualitative study with 42 speech and language therapists (SLTs) in three NHS services (n = 39) and private practice (n = 3) in Scotland. We explored therapists' diverse experiences of SSD practice change through individual interviews (n = 28) or self-generated paired (n = 2) or focus groups (n = 3). A theoretical framework (Normalization Process Theory) helped us understand how the service context contributed to the way therapists engaged with different practice changes. OUTCOMES & RESULTS: We identified six types ('cases') of practice change, two of which involved the new SSD interventions. We focus on these two cases ('Transforming' and 'Venturing') and use Normalization Process Theory's Cognitive participation construct to explain implementation (or not) of new SSD interventions in routine practice. Therapists were becoming aware of the new interventions through knowledge brokers, professional networks and an intervention database. In the Transforming case, new SSD interventions for selected children were becoming part of local routine practice. Transforming was the result of a favourable service structure, a sustained and supported 'push' that made implementation of the new interventions a service priority, and considerable collective time to think about doing it. 'Venturing' happened where the new SSD interventions were not a service priority. It involved individual or informal groups of therapists trying out or using one or more of the new interventions with selected children within the constraints of their service context. CONCLUSIONS & IMPLICATIONS: New, evidence-based SSD interventions may be challenging to implement in routine practice because they have in common a need for therapists who understand applied linguistics and can be flexible with service delivery. Appreciating what it really takes to do routine intervention differently is vital for managers and services who have to make decisions about priorities for implementation, along with realistic plans for resourcing and supporting it. WHAT THIS PAPER ADDS: What is already known on the subject Many SSD interventions have an evidence base but are not widely adopted into routine clinical practice. Addressing this is not just about individual therapists or education/training, as workplace pressures and service delivery models make it difficult to change practice. What this paper adds to the existing knowledge This paper applies innovations from implementation science to help explain how what is going on in services can support or constrain capacity for implementing evidence-based SSD interventions. What are the potential or actual clinical implications of this work? Service managers and therapists will have a clearer idea of the time and support they may realistically have to invest for new SSD interventions to be used routinely.

Risk of developing glioblastoma following non-CNS primary cancer: a SEER analysis between 2000 and 2018.

BACKGROUND: Patients with a prior malignancy are at elevated risk of developing subsequent primary malignancies (SPMs). However, the risk of developing subsequent primary glioblastoma (SPGBM) in patients with a prior cancer history is poorly understood. METHODS: We used the Surveillance, Epidemiology, and End Results (SEER) database and identified patients diagnosed with non-CNS malignancy between 2000 and 2018. We calculated a modified standardized incidence ratio (M-SIR), defined as the ratio of the incidence of SPGBM among patients with initial non-CNS malignancy to the incidence of GBM in the general population, stratified by sex latency, and initial tumor location. RESULTS: Of the 5,326,172 patients diagnosed with a primary non-CNS malignancy, 3559 patients developed SPGBM (0.07%). Among patients with SPGBM, 2312 (65.0%) were men, compared to 2,706,933 (50.8%) men in the total primary non-CNS malignancy cohort. The median age at diagnosis of SPGBM was 65 years. The mean latency between a prior non-CNS malignancy and developing a SPGBM was 67.3 months (interquartile range [IQR] 27-100). Overall, patients with a primary non-CNS malignancy had a significantly elevated M-SIR (1.13, 95% CI 1.09-1.16), with a 13% increased incidence of SPGBM when compared to the incidence of developing GBM in the age-matched general population. When stratified by non-CNS tumor location, patients diagnosed with primary melanoma, lymphoma, prostate, breast, renal, or endocrine malignancies had a higher M-SIR (M-SIR ranges: 1.09-2.15). Patients with lung cancers (M-SIR 0.82, 95% CI 0.68-0.99), or stomach cancers (M-SIR 0.47, 95% CI 0.24-0.82) demonstrated a lower M-SIR. CONCLUSION: Patients with a history of prior non-CNS malignancy are at an overall increased risk of developing SPGBM relative to the incidence of developing GBM in the general population. However, the incidence of SPGBM after prior non-CNS malignancy varies by primary tumor location, with some non-CNS malignancies demonstrating either increased or decreased predisposition for SPGBM depending on tumor origin. These findings merit future investigation into whether these relationships represent treatment effects or a previously unknown shared predisposition for glioblastoma and non-CNS malignancy.