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Internal carotid artery terminus aneurysms can be complex entities that present a challenge for both surgical and endovascular treatment. 1 , 2 Definitive microsurgical treatment can be difficult due to anatomic features such as a wide neck, high density of perforators originating at this location, and proximity of vital arteries (e.g., the anterior choroidal artery) originating from the distal internal carotid artery. Conversely, endovascular treatment may be ineffective due to low complete aneurysm occlusion rates and high risk of recurrence. Hybrid surgical and endovascular treatment of complex aneurysms not readily amenable to treatment with a single modality has been described. 3 In Video 1, we present a case of a 55-year-old woman found to have a large internal carotid artery terminus aneurysm, which was managed with a hybrid approach. The aneurysm was large, broad-based, and mostly saccular, but had a fusiform component at the base containing the origin of the anterior choroidal artery. Definitive microsurgical clipping was attempted, but ultimately was not possible as the anterior choroidal artery origin could not be preserved. The aneurysm was subsequently partially clipped, facilitating coil embolization of the saccular component with clip assistance., (Copyright © 2025 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transcription factor MYT1L supports proper neuronal differentiation and maturation during brain development. MYT1L haploinsufficiency results in a neurodevelopmental disorder characterized by intellectual disability, developmental delay, autism, behavioral disruptions, aggression, obesity and epilepsy. While MYT1L is expressed throughout the brain, how it supports proper neuronal function in distinct regions has not been assessed. Some features of MYT1L Neurodevelopmental Syndrome suggest disruption of hypothalamic function, such as obesity and endocrine issues, and previous research showed changes in hypothalamic neuropeptide expression following knockdown in zebrafish. Here, we leveraged our heterozygous Myt1l mutant, previously shown to recapitulate aspects of the human syndrome such as hyperactivity, social challenges, and obesity, to examine the impact of MYT1L loss on hypothalamic function. Examining the molecular profile of the MYT1L haploinsufficient hypothalamus revealed a similar scale of disruption to previously studied brain regions, yet with region-specific roles for MYT1L, including regulation of neuropeptide systems. Alterations in oxytocin and arginine vasopressin cell numbers were also found. Behaviors studied included maternal care, social group hierarchies, and aggression, all of which were unchanged. Feeding and metabolic markers were also largely unchanged in MYT1L haploinsufficient mice, yet an interaction was observed between diet and MYT1L genotype on weight gain. Our findings here suggest that gross endocrine function was not altered by MYT1L haploinsufficiency, and that key sex-specific behaviors related to proper hypothalamic function remain intact. Further study is needed to understand the functional impact of the altered hypothalamic molecular profile and changes in neuropeptide cell numbers that result from MYT1L haploinsufficiency., Competing Interests: Declaration of Generative AI and AI-assisted technologies in the writing process: None

Background: Anterior communicating artery (ACOM) aneurysms are among the most common aneurysms associated with aneurysmal subarachnoid hemorrhage (International Study of Unruptured Intracranial Aneurysms I (N Engl J Med 339:1725-1733, 1998), Wiebers (Lancet 362:103-110, 2003)). Surgical clipping of posterior-superiorly projecting ACOM aneurysms can be challenging, as the ipsilateral A2 can interfere with clip trajectory and ACOM perforating vessels obstructed from view. Intraluminal coils can further increase the difficulty of the procedure., Method: The relevant surgical anatomy with illustration is presented. A video detailing our technique on an illustrative case is provided., Conclusion: Surgical clipping of posterior-superiorly projecting ACOM aneurysms can require complex clip configurations. We describe the key steps of posterior-superiorly projecting ACOM aneurysm clipping through a lateral supraorbital craniotomy and fenestrated tandem clipping., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Compared to men, women often develop COPD at an earlier age with worse respiratory symptoms despite lower smoking exposure. However, most preventive, and therapeutic strategies ignore biological sex differences in COPD. Our goal was to better understand sex-specific gene regulatory processes in lung tissue and the molecular basis for sex differences in COPD onset and severity. We analyzed lung tissue gene expression and DNA methylation data from 747 individuals in the Lung Tissue Research Consortium (LTRC), and 85 individuals in an independent dataset. We identified sex differences in COPD-associated gene regulation using gene regulatory networks. We used linear regression to test for sex-biased associations of methylation with lung function, emphysema, smoking, and age. Analyzing gene regulatory networks in the control group, we identified that genes involved in the extracellular matrix (ECM) have higher transcriptional factor targeting in females than in males. However, this pattern is reversed in COPD, with males showing stronger regulatory targeting of ECM-related genes than females. Smoking exposure, age, lung function, and emphysema were all associated with sex-specific differential methylation of ECM-related genes. We identified sex-based gene regulatory patterns of ECM-related genes associated with lung function and emphysema. Multiple factors including epigenetics, smoking, aging, and cell heterogeneity influence sex-specific gene regulation in COPD. Our findings underscore the importance of considering sex as a key factor in disease susceptibility and severity.

Health workers, especially auxiliary nurse midwives (ANMs), are among the most critical resources in improving the quality of immunization services and reducing vaccine hesitancy under the Universal Immunization Programme (UIP) in India. To improve health worker immunization skills, UIP trainings in India are primarily conducted through instructor-led classroom, cascade trainings. However, a 2018 capacity-building need assessment revealed several challenges involved in traditional classroom training, such as a single-time exposure to new guidelines, complicated logistics arrangements, a lack of refresher training, and varying quality of training. These complexities make it difficult to meet the timely knowledge and skill needs of every health worker effectively and uniformly in a rapidly changing scenario of UIP. To meet health worker capacity-building needs and address these challenges, Rapid Immunization Skill Enhancement (RISE), a learning management system (LMS) application, was conceptualized. The RISE LMS application was developed as a human-centered, interactive, continuous, and adaptable knowledge and skill-building platform for health workers engaged in the UIP. RISE complements existing classroom-based cascade training for health workers by leveraging digital technologies for faster, easier, and more effective knowledge transfer to accommodate the fast-changing needs of a dynamic health program like UIP. In this article, we share the challenges and strategic solutions to digital training applications, lessons learned, sustainability of the application, and the impact RISE has made in India, all of which stemmed from leadership, coordinated efforts from a team of skilled professionals, government acceptance, detailed planning, and continued stakeholder engagement., (© Chase et al.)

Rationale: While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort., Objectives: Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples., Methods: Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses., Measurements and Main Results: In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations., Conclusions: This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

Background: Sex differences in the brain may play an important role in sex-differential prevalence of neuropsychiatric conditions., Methods: In order to understand the transcriptional basis of sex differences, we analyzed multiple, large-scale, human postmortem brain RNA-Seq datasets using both within-region and pan-regional frameworks., Results: We find evidence of sex-biased transcription in many autosomal genes, some of which provide evidence for pathways and cell population differences between chromosomally male and female individuals. These analyses also highlight regional differences in the extent of sex-differential gene expression. We observe an increase in specific neuronal transcripts in male brains and an increase in immune and glial function-related transcripts in female brains. Integration with single-nucleus data suggests this corresponds to sex differences in cellular states rather than cell abundance. Integration with case-control gene expression studies suggests a female molecular predisposition towards Alzheimer's disease, a female-biased disease. Autism, a male-biased diagnosis, does not exhibit a male predisposition pattern in our analysis., Conclusion: Overall, these analyses highlight mechanisms by which sex differences may interact with sex-biased conditions in the brain. Furthermore, we provide region-specific analyses of sex differences in brain gene expression to enable additional studies at the interface of gene expression and diagnostic differences., (© 2024. The Author(s).)

Demands to manage the risks of artificial intelligence (AI) are growing. These demands and the government standards arising from them both call for trustworthy AI. In response, we adopt a convergent approach to review, evaluate, and synthesize research on the trust and trustworthiness of AI in the environmental sciences and propose a research agenda. Evidential and conceptual histories of research on trust and trustworthiness reveal persisting ambiguities and measurement shortcomings related to inconsistent attention to the contextual and social dependencies and dynamics of trust. Potentially underappreciated in the development of trustworthy AI for environmental sciences is the importance of engaging AI users and other stakeholders, which human-AI teaming perspectives on AI development similarly underscore. Co-development strategies may also help reconcile efforts to develop performance-based trustworthiness standards with dynamic and contextual notions of trust. We illustrate the importance of these themes with applied examples and show how insights from research on trust and the communication of risk and uncertainty can help advance the understanding of trust and trustworthiness of AI in the environmental sciences., (© 2023 The Authors. Risk Analysis published by Wiley Periodicals LLC on behalf of Society for Risk Analysis.)

Vitamin D possesses immunomodulatory functions and vitamin D deficiency has been associated with the rise in chronic inflammatory diseases, including asthma (Litonjua and Weiss, 2007). Vitamin D supplementation studies do not provide insight into the molecular genetic mechanisms of vitamin D-mediated immunoregulation. Here, we provide evidence for vitamin D regulation of two human chromosomal loci, Chr17q12-21.1 and Chr17q21.2, reliably associated with autoimmune and chronic inflammatory diseases. We demonstrate increased vitamin D receptor ( Vdr ) expression in mouse lung CD4+ Th2 cells, differential expression of Chr17q12-21.1 and Chr17q21.2 genes in Th2 cells based on vitamin D status and identify the IL-2/Stat5 pathway as a target of vitamin D signaling. Vitamin D deficiency caused severe lung inflammation after allergen challenge in mice that was prevented by long-term prenatal vitamin D supplementation. Mechanistically, vitamin D induced the expression of the Ikzf3 -encoded protein Aiolos to suppress IL-2 signaling and ameliorate cytokine production in Th2 cells. These translational findings demonstrate mechanisms for the immune protective effect of vitamin D in allergic lung inflammation with a strong molecular genetic link to the regulation of both Chr17q12-21.1 and Chr17q21.2 genes and suggest further functional studies and interventional strategies for long-term prevention of asthma and other autoimmune disorders., Competing Interests: AK, AH, MD, EM, MD, TB, JR, RC, HM, AS, JK, TN, HP, NK, RA, KG, AL, MD, HR, BL No competing interests declared, SW receives royalties from UpToDate and is an investor in Histolix, (© 2023, Kilic, Halu et al.)

Introduction: B-cells are essential components of the immune system that neutralize infectious agents through the generation of antigen-specific antibodies and through the phagocytic functions of naïve and memory B-cells. However, the B-cell response can become compromised by a variety of conditions that alter the overall inflammatory milieu, be that due to substantial, acute insults as seen in sepsis, or due to those that produce low-level, smoldering background inflammation such as diabetes, obesity, or advanced age. This B-cell dysfunction, mediated by the inflammatory cytokines Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α), increases the susceptibility of late-stage sepsis patients to nosocomial infections and increases the incidence or severity of recurrent infections, such as SARS-CoV-2, in those with chronic conditions. We propose that modeling B-cell dynamics can aid the investigation of their responses to different levels and patterns of systemic inflammation., Methods: The B-cell Immunity Agent-based Model (BCIABM) was developed by integrating knowledge regarding naïve B-cells, short-lived plasma cells, long-lived plasma cells, memory B-cells, and regulatory B-cells, along with their various differentiation pathways and cytokines/mediators. The BCIABM was calibrated to reflect physiologic behaviors in response to: 1) mild antigen stimuli expected to result in immune sensitization through the generation of effective immune memory, and 2) severe antigen challenges representing the acute substantial inflammation seen during sepsis, previously documented in studies on B-cell behavior in septic patients. Once calibrated, the BCIABM was used to simulate the B-cell response to repeat antigen stimuli during states of low, chronic background inflammation, implemented as low background levels of IL-6 and TNF-α often seen in patients with conditions such as diabetes, obesity, or advanced age. The levels of immune responsiveness were evaluated and validated by comparing to a Veteran's Administration (VA) patient cohort with COVID-19 infection known to have a higher incidence of such comorbidities., Results: The BCIABM was successfully able to reproduce the expected appropriate development of immune memory to mild antigen exposure, as well as the immunoparalysis seen in septic patients. Simulation experiments then revealed significantly decreased B-cell responsiveness as levels of background chronic inflammation increased, reproducing the different COVID-19 infection data seen in a VA population., Conclusion: The BCIABM proved useful in dynamically representing known mechanisms of B-cell function and reproduced immune memory responses across a range of different antigen exposures and inflammatory statuses. These results elucidate previous studies demonstrating a similar negative correlation between the B-cell response and background inflammation by positing an established and conserved mechanism that explains B-cell dysfunction across a wide range of phenotypic presentations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: Timely data on drug overdose deaths can help identify community needs, evaluate the effectiveness of interventions, and allocate resources. We identified variations in death investigation and reporting systems within and between states that affect the timeliness and accuracy of death certificate information., Methods: The HEALing Communities Study (HCS) is a community-engaged, data-driven approach to combating the opioid crisis in 67 communities in 4 states: Kentucky, Massachusetts, New York, and Ohio. HCS conducted a survey of coroners and medical examiners to understand variability in drug overdose death data. We compared survey results in Massachusetts, New York, and Ohio with national data to investigate the completeness of provisional death counts by type of death investigation system., Results: Communities in each HCS state had different ways of collecting and reporting mortality data. Completion of death certificates for drug overdoses ranged from <2 weeks in 23% (7 of 31) of those surveyed to more than 3 months in 10% (3 of 31) of those surveyed. Variabilities in the timeliness of reporting drug overdose deaths were not associated with type of coroner or medical examiner office in each state, urban versus rural setting, or specificity of drug information on the death certificate., Conclusion: Having specific drug information on the death certificate may increase death certificate quality, comparability, and accuracy. We recommend the following: (1) all coroners and medical examiners should be trained on conducting death investigations, interpreting toxicology reports, and completing death certificates; (2) 1 office in each state should oversee all coroners and medical examiners to increase data consistency; and (3) communities should identify and address barriers to timely death certification., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

De novo mutations cause a variety of neurodevelopmental disorders including autism. Recent whole genome sequencing from individuals with autism has shown that many de novo mutations also occur in untranslated regions (UTRs) of genes, but it is difficult to predict from sequence alone which mutations are functional, let alone causal. Therefore, we developed a high throughput assay to screen the transcriptional and translational effects of 997 variants from 5'UTR patient mutations. This assay successfully enriched for elements that alter reporter translation, identifying over 100 potentially functional mutations from probands. Studies in patient-derived cell lines further confirmed that these mutations can alter protein production in individuals with autism, and some variants fall in genes known to cause syndromic forms of autism, suggesting a diagnosis for these individual patients. Since UTR function varies by cell type, we further optimized this high throughput assay to enable assessment of mutations in neurons in vivo . First, comparing in cellulo to in vivo results, we demonstrate neurons have different principles of regulation by 5'UTRs, consistent with a more robust mechanism for reducing the impact of RNA secondary structure. Finally, we discovered patient mutations specifically altering the translational activity of additional known syndromic genes LRRC4 and ZNF644 in neurons of the brain. Overall our results highlight a new approach for assessing the impact of 5'UTR mutations across cell types and suggest that some cases of neurodevelopmental disorder may be caused by such variants.

Introduction: The clinical characterization of the functional status of active wounds in terms of their driving cellular and molecular biology remains a considerable challenge that currently requires excision via a tissue biopsy. In this pilot study, we use convolutional Siamese neural network (SNN) architecture to predict the functional state of a wound using digital photographs of wounds in a canine model of volumetric muscle loss (VML)., Methods: Digital images of VML injuries and tissue biopsies were obtained in a standardized fashion from an established canine model of VML. Gene expression profiles for each biopsy site were obtained using RNA sequencing. These profiles were converted to functional profiles by a manual review of validated gene ontology databases in which we determined a hierarchical representation of gene functions based on functional specificity. An SNN was trained to regress functional profile expression values, informed by an image segment showing the surface of a small tissue biopsy., Results: The SNN was able to predict the functional expression of a range of functions based with error ranging from ∼5% to ∼30%, with functions that are most closely associated with the early state of wound healing to be those best-predicted., Conclusions: These initial results suggest promise for further research regarding this novel use of machine learning regression on medical images. The regression of functional profiles, as opposed to specific genes, both addresses the challenge of genetic redundancy and gives a deeper insight into the mechanistic configuration of a region of tissue in wounds., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: There is increasing recognition of extensive crosstalk between programmed cell death pathways (PCDPs), such as apoptosis, pyroptosis, and necroptosis, resulting in a highly redundant system responsive to a breadth of potential pathogens. However, because pyroptosis and necroptosis propagate inflammation, these redundancies also present challenges for therapeutic control of dysregulated hyperinflammation seen in cytokine storm (CS) generated organ dysfunction. Hypothesis: We hypothesize that the conversion of existing knowledge regarding apoptosis, pyroptosis, and necroptosis into a computational model can enhance our understanding of the crosstalk between PCDPs via simulation experiments of microbe interactions and experimental interventions. Materials and Methods: Literature regarding apoptosis, pyroptosis, and necroptosis was reviewed and transposed into an agent-based model, the programmed cell death agent-based model (PCDABM). Computational experiments were performed to simulate the activation of various PCDPs as seen by differing microbes, specifically: influenza A virus (IAV), enteropathic Escherichia coli (EPEC), and Salmonella enterica (SE). The potential protective value of PCDP crosstalk was evaluated by silencing either pyroptosis, necroptosis, or both. Computational experiments were also performed simulating the effect of potential therapies blocking tumor necrosis factor (TNF) and interleukin (IL)-1. Results: The PCDABM was implemented in the agent-based modeling toolkit NetLogo. Computational experiments of infection with IAV, EPEC, and SE reproduced cross-activation of PCDPs with effective microbial clearance. Simulations of anti-TNF and anti-IL-1 did not reduce the aggregated amount of inflammation-generated system damage, the surrogate for CS-generated tissue damage. Conclusions: Redundancies have evolved in host PCDPs to maintain protection against a wide range of pathogens. However, these redundancies also challenge attempts at dampening the pathogenic hyperinflammatory state of CS using therapeutic immunomodulation. Integrative simulation models such as the PCDABM can aid in identifying potentially targetable inflection points to mitigate CS while maintaining effective host defense.

Background: Though governed by the same underlying biology, the differential physiology of children causes the temporal evolution from health to a septic/diseased state to follow trajectories that are distinct from adult cases. As pediatric sepsis data sets are less readily available than for adult sepsis, we aim to leverage this shared underlying biology by normalizing pediatric physiological data such that it would be directly comparable to adult data, and then develop machine-learning (ML) based classifiers to predict the onset of sepsis in the pediatric population. We then externally validated the classifiers in an independent adult dataset., Methods: Vital signs and laboratory observables were obtained from the Pediatric Intensive Care (PIC) database. These data elements were normalized for age and placed on a continuous scale, termed the Continuous Age-Normalized SOFA (CAN-SOFA) score. The XGBoost algorithm was used to classify pediatric patients that are septic. We tested the trained model using adult data from the MIMIC-IV database., Results: On the pediatric population, the sepsis classifier has an accuracy of 0.84 and an F1-Score of 0.867. On the adult population, the sepsis classifier has an accuracy of 0.80 and an F1-score of 0.88; when tested on the adult population, the model showed similar performance degradation ("data drift") as in the pediatric population., Conclusions: In this work, we demonstrate that, using a straightforward age-normalization method, EHR's can be generalizable compared (at least in the context of sepsis) between the pediatric and adult populations., Competing Interests: The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article., (© 2023 The Authors.)

Objective: Inferior petrosal sinus (IPS) sampling (IPSS) is a diagnostic procedure used to guide diagnostic localization of imaging-negative adrenocorticotropic hormone (ACTH)-secreting pituitary microadenomas. However, the efficacy of IPSS has been suboptimal at accurately lateralizing the adenoma, reducing surgical cure rates and leading to unintended pituitary dysfunction due to the added exploration. One rationale for the occasional imprecision is the existence of additional petrosal sinus collateral channels that connect the IPS bilaterally, which may lead to false localization results during sampling. The aim of this study was to explore a potential connection between normal anatomical variation in the angioarchitecture of the IPSs and the ACTH results obtained in subsequent IPSS tests., Methods: A retrospective review was performed on all cases between 1998 and 2013 involving patients at a single institution who underwent IPSS for radiographically equivocal pituitary microadenomas. Cases were reviewed for tumor laterality noted on either operative or pathology reports, as well as the presence of angiographic evidence of cross-filling between the sinuses. In addition, ACTH levels from the right and left IPSs were documented at baseline and at 2, 5, and 10 minutes after corticotropin-releasing hormone (CRH) administration. A ratio of the change in ACTH levels measured at the time of maximal response (10 minutes) versus the levels measured at the initial response (2 minutes) was computed for each patient and compared between patients by their angiographic cross-filling status., Results: There were 41 patients with a histopathologically confirmed right- or left-sided ACTH-secreting pituitary microadenoma who underwent preoperative IPSS. Among these patients, 28 (68%) showed angiographic evidence of cross-filling between the IPSs, and 13 showed no cross-filling. On average, ACTH levels increased by a factor of 3.91 ± 0.77 in the contralateral IPS in patients with angiographic cross-filling, compared with a factor increase of only 1.80 ± 0.27 in patients without cross-filling (p = 0.014). In comparison, ACTH levels increased by a factor of 2.01 ± 0.57 in the ipsilateral IPS in patients with cross-filling, and by 8.78 ± 7.30 in those without cross-filling (p = 0.373)., Conclusions: The presence of angiographic cross-filling, suggestive of a greater degree of vascular channel networking between the right and left IPS, is a significant factor influencing the measured rates of change of ACTH in IPSS and may impact the specificity of this test to accurately determine microadenoma laterality in the preoperative setting.

Rationale: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPDs) are associated with a significant disease burden. Blood immune phenotyping may improve our understanding of a COPD endotype at increased risk of exacerbations. Objective: To determine the relationship between the transcriptome of circulating leukocytes and COPD exacerbations. Methods: Blood RNA sequencing data ( n  = 3,618) from the COPDGene (Genetic Epidemiology of COPD) study were analyzed. Blood microarray data ( n  = 646) from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study were used for validation. We tested the association between blood gene expression and AE-COPDs. We imputed the abundance of leukocyte subtypes and tested their association with prospective AE-COPDs. Flow cytometry was performed on blood in SPIROMICS (Subpopulations and Intermediate Outcomes in COPD Study) ( n  = 127), and activation markers for T cells were tested for association with prospective AE-COPDs. Measurements and Main Results: Exacerbations were reported 4,030 and 2,368 times during follow-up in COPDGene (5.3 ± 1.7 yr) and ECLIPSE (3 yr), respectively. We identified 890, 675, and 3,217 genes associated with a history of AE-COPDs, persistent exacerbations (at least one exacerbation per year), and prospective exacerbation rate, respectively. In COPDGene, the number of prospective exacerbations in patients with COPD (Global Initiative for Chronic Obstructive Lung Disease stage ⩾2) was negatively associated with circulating CD8 + T cells, CD4 + T cells, and resting natural killer cells. The negative association with naive CD4 + T cells was replicated in ECLIPSE. In the flow-cytometry study, an increase in CTLA4 on CD4 + T cells was positively associated with AE-COPDs. Conclusions: Individuals with COPD with lower circulating lymphocyte counts, particularly decreased CD4 + T cells, are more susceptible to AE-COPDs, including persistent exacerbations.

RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of ∼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR. SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption., (Copyright © 2023 the authors.)

Background: As trachoma is eliminated, skilled field graders become less adept at correctly identifying active disease (trachomatous inflammation-follicular [TF]). Deciding if trachoma has been eliminated from a district or if treatment strategies need to be continued or reinstated is of critical public health importance. Telemedicine solutions require both connectivity, which can be poor in the resource-limited regions of the world in which trachoma occurs, and accurate grading of the images., Objective: Our purpose was to develop and validate a cloud-based "virtual reading center" (VRC) model using crowdsourcing for image interpretation., Methods: The Amazon Mechanical Turk (AMT) platform was used to recruit lay graders to interpret 2299 gradable images from a prior field trial of a smartphone-based camera system. Each image received 7 grades for US $0.05 per grade in this VRC. The resultant data set was divided into training and test sets to internally validate the VRC. In the training set, crowdsourcing scores were summed, and the optimal raw score cutoff was chosen to optimize kappa agreement and the resulting prevalence of TF. The best method was then applied to the test set, and the sensitivity, specificity, kappa, and TF prevalence were calculated., Results: In this trial, over 16,000 grades were rendered in just over 60 minutes for US $1098 including AMT fees. After choosing an AMT raw score cut point to optimize kappa near the World Health Organization (WHO)-endorsed level of 0.7 (with a simulated 40% prevalence TF), crowdsourcing was 95% sensitive and 87% specific for TF in the training set with a kappa of 0.797. All 196 crowdsourced-positive images received a skilled overread to mimic a tiered reading center and specificity improved to 99%, while sensitivity remained above 78%. Kappa for the entire sample improved from 0.162 to 0.685 with overreads, and the skilled grader burden was reduced by over 80%. This tiered VRC model was then applied to the test set and produced a sensitivity of 99% and a specificity of 76% with a kappa of 0.775 in the entire set. The prevalence estimated by the VRC was 2.70% (95% CI 1.84%-3.80%) compared to the ground truth prevalence of 2.87% (95% CI 1.98%-4.01%)., Conclusions: A VRC model using crowdsourcing as a first pass with skilled grading of positive images was able to identify TF rapidly and accurately in a low prevalence setting. The findings from this study support further validation of a VRC and crowdsourcing for image grading and estimation of trachoma prevalence from field-acquired images, although further prospective field testing is required to determine if diagnostic characteristics are acceptable in real-world surveys with a low prevalence of the disease., (©Christopher J Brady, R Chase Cockrell, Lindsay R Aldrich, Meraf A Wolle, Sheila K West. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 06.04.2023.)

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair., (© 2022 S. Karger AG, Basel.)

Peripheral nerve injury results in loss of motor and sensory function distal to the nerve injury and is often permanent in nerve gaps longer than 5 cm. Autologous nerve grafts (nerve autografts) utilize patients' own nerve tissue from another part of their body to repair the defect and are the gold standard in care. However, there is a limited autologous tissue supply, size mismatch between donor nerve and injured nerve, and morbidity at the site of nerve donation. Decellularized cadaveric nerve tissue alleviates some of these limitations and has demonstrated success clinically. We previously developed an alternative apoptosis-assisted decellularization process for nerve tissue. This new process may result in an ideal scaffold for peripheral nerve regeneration by gently removing cells and antigens while preserving delicate topographical cues. In addition, the apoptosis-assisted process requires less active processing time and is inexpensive. This study examines the utility of apoptosis-decellularized peripheral nerve scaffolds compared to detergent-decellularized peripheral nerve scaffolds and isograft controls in a rat nerve gap model. Results indicate that, at 8 weeks post-injury, apoptosis-decellularized peripheral nerve scaffolds perform similarly to detergent-decellularized and isograft controls in both functional (muscle weight recovery, gait analysis) and histological measures (neurofilament staining, macrophage infiltration). These new apoptosis-decellularized scaffolds hold great promise to provide a less expensive scaffold for nerve injury repair, with the potential to improve nerve regeneration and functional outcomes compared to current detergent-decellularized scaffolds., (© 2022 S. Karger AG, Basel.)

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor. SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators., (Copyright © 2022 the authors.)

Background: Though significant progress in disease elimination has been made over the past decades, trachoma is the leading infectious cause of blindness globally. Further efforts in trachoma elimination are paradoxically being limited by the relative rarity of the disease, which makes clinical training for monitoring surveys difficult. In this work, we evaluate the plausibility of an Artificial Intelligence model to augment or replace human image graders in the evaluation/diagnosis of trachomatous inflammation-follicular (TF)., Methods: We utilized a dataset consisting of 2300 images with a 5% positivity rate for TF. We developed classifiers by implementing two state-of-the-art Convolutional Neural Network architectures, ResNet101 and VGG16, and applying a suite of data augmentation/oversampling techniques to the positive images. We then augmented our data set with additional images from independent research groups and evaluated performance., Results: Models performed well in minimizing the number of false negatives, given the constraint of the low numbers of images in which TF was present. The best performing models achieved a sensitivity of 95% and positive predictive value of 50-70% while reducing the number images requiring skilled grading by 66-75%. Basic oversampling and data augmentation techniques were most successful at improving model performance, while techniques that are grounded in clinical experience, such as highlighting follicles, were less successful., Discussion: The developed models perform well and significantly reduce the burden on graders by minimizing the number of false negative identifications. Further improvements in model skill will benefit from data sets with more TF as well as a range in image quality and image capture techniques used. While these models approach/meet the community-accepted standard for skilled field graders (i.e., Cohen's Kappa >0.7), they are insufficient to be deployed independently/clinically at this time; rather, they can be utilized to significantly reduce the burden on skilled image graders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Socia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cancer-associated fibroblasts (CAFs) are integral to the solid tumor microenvironment. CAFs were once thought to be a relatively uniform population of matrix-producing cells, but single-cell RNA sequencing has revealed diverse CAF phenotypes. Here, we further probed CAF heterogeneity with a comprehensive multiomics approach. Using paired, same-cell chromatin accessibility and transcriptome analysis, we provided an integrated analysis of CAF subpopulations over a complex spatial transcriptomic and proteomic landscape to identify three superclusters: steady state-like (SSL), mechanoresponsive (MR), and immunomodulatory (IM) CAFs. These superclusters are recapitulated across multiple tissue types and species. Selective disruption of underlying mechanical force or immune checkpoint inhibition therapy results in shifts in CAF subpopulation distributions and affected tumor growth. As such, the balance among CAF superclusters may have considerable translational implications. Collectively, this research expands our understanding of CAF biology, identifying regulatory pathways in CAF differentiation and elucidating therapeutic targets in a species- and tumor-agnostic manner., Competing Interests: Declaration of interests D.S.F., M.J., D.D., H.Y.C., J.A.N., and M.T.L. have applied for a provisional patent through Stanford University related to the research presented in this manuscript. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and Cartography Biosciences, and an advisor of 10X Genomics, Arsenal Biosciences, and Spring Discovery. K.E.Y. is a consultant for Cartography Biosciences., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The development of novel pain therapeutics hinges on the identification and rigorous validation of potential targets. Model organisms provide a means to test the involvement of specific genes and regulatory elements in pain. Here we provide a list of genes linked to pain-associated behaviors. We capitalize on results spanning over three decades to identify a set of 242 genes. They support a remarkable diversity of functions spanning action potential propagation, immune response, GPCR signaling, enzymatic catalysis, nucleic acid regulation, and intercellular signaling. Making use of existing tissue and single-cell high-throughput RNA sequencing datasets, we examine their patterns of expression. For each gene class, we discuss archetypal members, with an emphasis on opportunities for additional experimentation. Finally, we discuss how powerful and increasingly ubiquitous forward genetic screening approaches could be used to improve our ability to identify pain genes. This article is categorized under: Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Molecular and Cellular Physiology., (© 2022 The Authors. WIREs Mechanisms of Disease published by Wiley Periodicals LLC.)

Introduction: Chronic obstructive pulmonary disease (COPD) can progress across several domains, complicating the identification of the determinants of disease progression. In our previous work, we applied k-means clustering to spirometric and chest radiological measures to identify four COPD-related subtypes: 'relatively resistant smokers (RRS)', 'mild upper lobe-predominant emphysema (ULE)', 'airway-predominant disease (AD)' and 'severe emphysema (SE)'. In the current study, we examined the associations of these subtypes to longitudinal COPD-related health measures as well as blood transcriptomic and plasma proteomic biomarkers., Methods: We included 8266 non-Hispanic white and African-American smokers from the COPDGene study. We used linear regression to investigate cluster associations to 5-year prospective changes in spirometric and radiological measures and to gene expression and protein levels. We used Cox-proportional hazard test to test for cluster associations to prospective exacerbations, comorbidities and mortality., Results: The RRS, ULE, AD and SE clusters represented 39%, 15%, 26% and 20% of the studied cohort at baseline, respectively. The SE cluster had the greatest 5-year FEV 1 (forced expiratory volume in 1 s) and emphysema progression, and the highest risks of exacerbations, cardiovascular disease and mortality. The AD cluster had the highest diabetes risk. After adjustments, only the SE cluster had an elevated respiratory mortality risk, while the ULE, AD and SE clusters had elevated all-cause mortality risks. These clusters also demonstrated differential protein and gene expression biomarker associations, mostly related to inflammatory and immune processes., Conclusion: COPD k-means subtypes demonstrate varying rates of disease progression, prospective comorbidities, mortality and associations to transcriptomic and proteomic biomarkers. These findings emphasise the clinical and biological relevance of these subtypes, which call for more study for translation into clinical practice., Trail Registration Number: NCT00608764., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males., (© 2022 Federation of American Societies for Experimental Biology.)

Background: The molecular basis of airway remodelling in chronic obstructive pulmonary disease (COPD) remains poorly understood. We identified gene expression signatures associated with chest computed tomography (CT) scan airway measures to understand molecular pathways associated with airway disease., Methods: In 2396 subjects in the COPDGene Study, we examined the relationship between quantitative CT airway phenotypes and blood transcriptomes to identify airway disease-specific genes and to define an airway wall thickness (AWT) gene set score. Multivariable regression analyses were performed to identify associations of the AWT score with clinical phenotypes, bronchial gene expression and genetic variants., Results: Type 1 interferon (IFN)-induced genes were consistently associated with AWT, square root wall area of a hypothetical airway with 10 mm internal perimeter (Pi10) and wall area percentage, with the strongest enrichment in AWT. A score derived from 18 genes whose expression was associated with AWT was associated with COPD-related phenotypes including reduced lung function (forced expiratory volume in 1 s percentage predicted β= -3.4; p<0.05) and increased exacerbations (incidence rate ratio 1.7; p<0.05). The AWT score was reproducibly associated with AWT in bronchial samples from 23 subjects (β=3.22; p<0.05). The blood AWT score was associated with genetic variant rs876039, an expression quantitative trait locus for IKZF1 , a gene that regulates IFN signalling and is associated with inflammatory diseases., Conclusions: A gene expression signature with IFN-stimulated genes from peripheral blood and bronchial brushings is associated with CT AWT, lung function and exacerbations. Shared genes and genetic associations suggest viral responses and/or autoimmune dysregulation as potential underlying mechanisms of airway disease in COPD., Competing Interests: Conflict of interest: J.H. Yun reports grants from the National Institutes of Health, during the conduct of the study; personal fees from Bridge Biotherapeutics, outside the submitted work. Conflict of interest: J. Morrow reports grants from the National Institutes of Health, during the conduct of the study. Conflict of interest: M. Cho reports personal fees from Genetech, AstraZeneca and Illumina, grants from Bayer and GlaxoSmithKline, outside the submitted work. Conflict of interest: P. Castaldi reports personal fees from GlaxoSmithKline and Novartis, grants from GlaxoSmithKline, outside the submitted work. Conflict of interest: C.P. Hersh reports grants from the National Heart, Lung, and Blood Institute, during the conduct of the study; grants from Bayer, Boehringer Ingelheim, Novartis and Vertex, and personal fees from Takeda, outside the submitted work. The remaining authors have nothing to disclose., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

There is a large assessment and treatment gap in child and adolescent mental health services, prominently so in low- and middle-income countries, where 90% of the world's children live. There is an urgent need to find evidence-based interventions that can be implemented successfully in these low-resource contexts. This pre-pilot study aimed to explore the barriers and facilitators to implementation as well as overall feasibility of Parent-Child Interaction Therapy (PCIT) in South Africa. A reflective and consensus building workshop was used to gather South African PCIT therapist (N = 4) perspectives on barriers, facilitators, and next steps to implementation in that country. Caregiver participants (N = 7) receiving the intervention in South Africa for the first time were also recruited to gather information on overall feasibility. Facilitators for implementation, including its strong evidence base, manualisation, and training model were described. Barriers relating to sustainability and scalability were highlighted. Largely positive views on acceptability from caregiver participants also indicated the promise of PCIT as an intervention in South Africa. Pilot data on the efficacy of the treatment for participating families are a next step. These initial results are positive, though research on how implementation factors contribute to the longer-term successful dissemination of PCIT in complex, heterogeneous low-resource settings is required.

Chemotherapy has been used to inhibit cancer growth for decades, but emerging evidence shows it can affect the tumor stroma, unintentionally promoting cancer malignancy. After treatment of primary tumors, remaining drugs drain via lymphatics. Though all drugs interact with the lymphatics, we know little of their impact on them. Here, we show a previously unknown effect of platinums, a widely used class of chemotherapeutics, to directly induce systemic lymphangiogenesis and activation. These changes are dose-dependent, long-lasting, and occur in healthy and cancerous tissue in multiple mouse models of breast cancer. We found similar effects in human ovarian and breast cancer patients whose treatment regimens included platinums. Carboplatin treatment of healthy mice prior to mammary tumor inoculation increased cancer metastasis as compared to no pre-treatment. These platinum-induced phenomena could be blocked by VEGFR3 inhibition. These findings have implications for cancer patients receiving platinums and may support the inclusion of anti-VEGFR3 therapy into treatment regimens or differential design of treatment regimens to alter these potential effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Harris, Esparza, Azimi, Cornelison, Azar, Llaneza, Belanger, Mathew, Tkachenko, Perez, Rosean, Bostic, Cornelison, Tate, Peirce-Cottler, Paquette, Mills, Landen, Saucerman, Dillon, Pompano, Rutkowski and Munson.)

Background: The clinical characterization of the biological status of complex wounds remains a considerable challenge. Digital photography provides a non-invasive means of obtaining wound information and is currently employed to assess wounds qualitatively. Advances in machine learning (ML) image processing provide a means of identifying "hidden" features in pictures. This pilot study trains a convolutional neural network (CNN) to predict gene expression based on digital photographs of wounds in a canine model of volumetric muscle loss (VML)., Materials and Methods: Images of volumetric muscle loss injuries and tissue biopsies were obtained in a canine model of VML. A CNN was trained to regress gene expression values as a function of the extracted image segment (color and spatial distribution). Performance of the CNN was assessed in a held-back test set of images using Mean Absolute Percentage Error (MAPE)., Results: The CNN was able to predict the gene expression of certain genes based on digital images, with a MAPE ranging from ∼10% to ∼30%, indicating the presence and identification of distinct, and identifiable patterns in gene expression throughout the wound., Conclusions: These initial results suggest promise for further research regarding this novel use of ML regression on medical images. Specifically, the use of CNNs to determine the mechanistic biological state of a VML wound could aid both the design of future mechanistic interventions and the design of trials to test those therapies. Future work will expand the CNN training and/or test set, with potential expansion to predicting functional gene modules., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: COPD can be assessed using multidimensional grading systems with components from three domains: pulmonary function tests, symptoms and systemic features. Clinically, measures may be used interchangeably, though it is not known if they share similar pathobiology., Objective: To use RNA sequencing (RNA-seq) to determine if there is an overlap in the underlying biological mechanisms and consequences driving different components of the multidimensional grading systems., Methods: Whole blood was collected for RNA-seq from current and former smokers in the Genetic Epidemiology of COPD study. We tested the overlap in gene expression and biological pathways associated with case-control status and quantitative COPD phenotypes within and between the three domains., Results: In 2647 subjects, there were 3030 genes differentially expressed in any of the three domains or case-control status. There were five genes that overlapped between the three domains and case-control status, including G protein-coupled receptor 15 ( GPR15 ), sestrin 1 ( SESN1 ) and interferon-induced guanylate-binding protein 1 ( GBP1 ), which were associated with longitudinal decline in FEV 1 . The overlap between the three domains was enriched for pathways related to cellular components., Conclusions: We identified gene sets and pathways that overlap between 12 COPD-related phenotypes and case-control status. There were no pathways represented in the overlap between the three domains and case-control status, but we identified multiple genes that demonstrated a consistent pattern of expression across several of the phenotypes. Patterns of gene expression correlation were generally similar to the correlation of clinical phenotypes in the PFT and symptom domains but not the systemic features., Competing Interests: Competing interests: Dr Castaldi reports grants from GSK, personal fees from GSK, personal fees from Novartis, outside the submitted work. Dr Hersh reports grants from NHLBI, during the conduct of the study; grants from Bayer, grants from Boehringer-Ingelheim, grants from Novartis, grants from Vertex, outside the submitted work., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background and Purpose: Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain-associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear., Experimental Approach: We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss-Webster mice, after treatment with nerve growth factor and IL-6. Expression dynamics of c-Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c-Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c-Fos (T-5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss-Webster mice using the hyperalgesic priming model., Key Results: c-Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c-Fos translation. The mTOR effector S6K1 is essential for c-Fos biosynthesis. Inhibition of S6K1 or c-Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity., Conclusion and Implications: Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c-Fos being one key downstream target. Targeting the S6 kinase pathway or c-Fos is an exciting new avenue for pain-modulating compounds., (© 2021 The British Pharmacological Society.)

Cigarette smoking induces a profound transcriptomic and systemic inflammatory response. Previous studies have focused on gene level differential expression of smoking, but the genome-wide effects of smoking on alternative isoform regulation have not yet been described. We conducted RNA sequencing in whole-blood samples of 454 current and 767 former smokers in the COPDGene Study, and we analyzed the effects of smoking on differential usage of isoforms and exons. At 10% FDR, we detected 3167 differentially expressed genes, 945 differentially used isoforms and 160 differentially used exons. Isoform switch analysis revealed widespread 3' UTR lengthening associated with cigarette smoking. The lengthening of these 3' UTRs was consistent with alternative usage of distal polyadenylation sites, and these extended 3' UTR regions were significantly enriched with functional sequence elements including microRNA and RNA-protein binding sites. These findings warrant further studies on alternative polyadenylation events as potential biomarkers and novel therapeutic targets for smoking-related diseases., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Disease states are being characterized at finer and finer levels of resolution via biomarker or gene expression profiles, while at the same time. Machine learning (ML) is increasingly used to analyze and potentially classify or predict the behavior of biological systems based on such characterization. As ML applications are extremely data-intensive, given the relative sparsity of biomedical data sets ML training of artificial neural networks (ANNs) often require the use of synthetic training data. Agent-based models (ABMs) that incorporate known biological mechanisms and their associated stochastic properties are a potential means of generating synthetic data. Herein we present an example of ML used to train an artificial neural network (ANN) as a surrogate system used to predict the time evolution of an ABM focusing on the clinical condition of sepsis. Methods: The disease trajectories for clinical sepsis, in terms of temporal cytokine and phenotypic dynamics, can be interpreted as a random dynamical system. The Innate Immune Response Agent-based Model (IIRABM) is a well-established model that utilizes known cellular and molecular rules to simulate disease trajectories corresponding to clinical sepsis. We have utilized two distinct neural network architectures, Long Short-Term Memory and Multi-Layer Perceptron, to take a time sequence of five measurements of eleven IIRABM simulated serum cytokine concentrations as input and to return both the future cytokine trajectories as well as an aggregate metric representing the patient's state of health. Results: The ANNs predicted model trajectories with the expected amount of error, due to stochasticity in the simulation, and recognizing that the mapping from a specific cytokine profile to a state-of-health is not unique. The Multi-Layer Perceptron neural network, generated predictions with a more accurate forecasted trajectory cone. Discussion: This work serves as a proof-of-concept for the use of ANNs to predict disease progression in sepsis as represented by an ABM. The findings demonstrate that multicellular systems with intrinsic stochasticity can be approximated with an ANN, but that forecasting a specific trajectory of the system requires sequential updating of the system state to provide a rolling forecast horizon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Larie, An and Cockrell.)

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive, in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to skin injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to reveal potential mechanisms controlling fibroblast fate during migration, proliferation, and differentiation following skin injury, and thereby reexamine the canonical phases of wound healing. These findings have broad implications for the study of tissue repair in complex organ systems., Competing Interests: Competing interest statement: H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, and an advisor of 10× Genomics, Arsenal Biosciences, and Spring Discovery. The entities had no role in the design, execution, or interpretation of this research. They had no role in the funding of this research. The paper reports basic research and has no financial implications for the listed companies. Two authors have co-published perspective or review articles with one reviewer over the last 4 y., (Copyright © 2021 the Author(s). Published by PNAS.)

There are over 1,000 described neurological and neurodegenerative disorders affecting nearly 100 million Americans - roughly one third of the U.S. population. Collectively, treatment of neurological conditions is estimated to cost $800 billion every year. Lowering this societal burden will require developing better model systems in which to study these diverse disorders. Microphysiological systems are promising tools for modeling healthy and diseased neural tissues to study mechanisms and treatment of neuropathology. One major benefit of microphysiological systems is the ability to incorporate biophysical forces, namely the forces derived from biological fluid flow. Fluid flow in the central nervous system (CNS) is a complex but important element of physiology, and pathologies as diverse as traumatic or ischemic injury, cancer, neurodegenerative disease, and natural aging have all been found to alter flow pathways. In this review, we summarize recent advances in three-dimensional microphysiological systems for studying the biology and therapy of CNS disorders and highlight the ability and growing need to incorporate biological fluid flow in these miniaturized model systems., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Epithelial tissue can transition from a jammed, solid-like, quiescent phase to an unjammed, fluid-like, migratory phase, but the underlying molecular events of the unjamming transition (UJT) remain largely unexplored. Using primary human bronchial epithelial cells (HBECs) and one well-defined trigger of the UJT, compression mimicking the mechanical effects of bronchoconstriction, here, we combine RNA sequencing data with protein-protein interaction networks to provide the first genome-wide analysis of the UJT. Our results show that compression induces an early transcriptional activation of the membrane and actomyosin network and a delayed activation of the extracellular matrix (ECM) and cell-matrix networks. This response is associated with a signaling cascade that promotes actin polymerization and cellular motility through the coordinated interplay of downstream pathways including ERK, JNK, integrin signaling, and energy metabolism. Moreover, in nonasthmatic versus asthmatic HBECs, common genomic patterns associated with ECM remodeling suggest a molecular connection between airway remodeling, bronchoconstriction, and the UJT., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

When sequencing small RNA libraries derived from whole blood, the most abundant microRNAs (miRs) detected are often miR-486-5p, miR-451a, and miR-92a-3p. These highly expressed erythropoietic miRs are released into the sample from red blood cell hemolysis. Next-generation sequencing of these unwanted miRs leads to a waste in sequencing cost and diminished detection of lowly expressed miRNAs, including many potential miRNA biomarkers. Previous work has developed a method to reduce targeted miRNAs using oligonucleotides that bind their target miRNA and prevent its ligation during library construction, although the extent to which oligonucleotides can be multiplexed and their effect on larger cohorts has not been thoroughly explored. We present a method for suppressing detection of three highly abundant heme miRs in a single multiplexed blocking oligonucleotide reaction. In a small paired-sample pilot (n = 8) and a large cohort of samples (n = 901), multiplexed oligos reduced detection of their target miRNAs by approximately 70%, allowing for an approximately 10-fold increase in reads mapping to nonheme miRs and increased detection of very lowly expressed miRs, with minimal off-target effects. By removing all three highly expressed erythropoietic miRNAs from next-generational sequencing libraries, this commercially available multiplexed blocking oligonucleotide method allows for greater detection of lowly expressed biomarkers, improving the efficacy, cost-efficiency, and sensitivity of biomarker studies and diagnostic tests., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Despite nearly 50 years of research there currently remains no mediator-directed therapy approved for the treatment of sepsis. The failure to effectively translate the copious mechanistic knowledge regarding systemic inflammation to effective therapies is a dramatic example of the translational dilemma. Dynamic computational modeling has been proposed as a vital means of integrating community-wide knowledge into an investigatory framework that allows the application of engineering-like principles to the problem of sepsis. Agent-based modeling is a computational modeling method that has been used to address some of the fundamental issues facing the sepsis research community. This chapter will introduce the rationale to augment traditional research practices with agent-based modeling, describe the basic steps in the construction and use of agent-based models, and provide examples of how the use of agent-based modeling can provide an investigatory pathway to solving the challenge of sepsis.

Background: Achieving functional repair after peripheral nerve injury (PNI) remains problematic despite considerable advances in surgical technique. Therein, questions lie regarding the variable capacity of peripheral nerves to regenerate based on environmental influence. In-depth analyses of multiple therapeutic strategies have ensued to overcome these natural obstacles. Of these candidate therapies, electrical stimulation has emerged a frontrunner. Extensive animal studies have reported the ability of brief intraoperative electrical stimulation (BES) to enhance functional regeneration after PNI. Despite these reports, the exact mechanisms by which BES enhances regeneration and its effects on long nerve lesions are largely unknown. Indeed, clinical translation of this seemingly simple therapeutic has not been so simple, but a few studies performed in humans have yielded highly encouraging results., Objective: We aimed to help bridge this translational gap by presenting the latest clinical trials on electrical stimulation for PNIs in combination with relevant etiologies, treatments and nonclinical findings., Methods: To do so, a systematic search was performed on PubMed, IEEE, and Web of Science databases up to February 2020 using keywords significant to our study. References of each manuscript were screened for additional manuscripts of relevance to our study., Results: We found multiple BES clinical studies reporting enhanced functional recovery or increased nerve regeneration. Although improved outcomes were reported, high variability after BES is seen between and within species likely due to injury severity, location and timeline along with other factors., Conclusion: Further clinical studies and introduction of novel delivery platforms are vital to uncover the true regenerative potential of electrical stimulationtherapy.

Introduction: Indigenous peoples in Canada have endured and continue to experience the impact of colonization by European settlers. The deleterious manifestations of intergenerational historic trauma (HT) are evidenced in the high HIV/AIDS epidemic-related premature mortality rates among Indigenous men, despite the availability of novel highly active antiretroviral therapies (HAARTs). Aim: The aims of this study were to explore the impact of historic trauma (HT) on treatment adherence and health promoting practices among Indigenous men living with HIV, and how resilience was both expressed and mediated by survivor status. Methods: This interpretive description study incorporated a cultural safety lens. Through partnership with the Vancouver Native Health Society, 36 male HT survivors were recruited using purposive and theoretical sampling. They told their lived experiences and health promoting practices with respect to HAART adherence through interviews and a focus group. Results: Two broad categories (findings) emerged: (1) resilience as facilitator of HAART adherence; and (2) differential views on HT's impact. Resilience was expressed through nine concepts. Conclusion: Most Indigenous men in this study demonstrate health promoting behavior, stay on HAART and have better health and well-being even if the environments they live in are marginalized or heavily stigmatizing. This study shows that areas of strength and adaptation, including factors promoting resilience can be harnessed to foster HAART adherence. With a consideration of these areas of strength and adaptation, this study offers implications for research and recommendations to improve treatment-adherent behavior, fostering healing from HT, and reducing HIV/AIDS-related deaths., (Copyright © 2020 Chongo, Lavoie, Mignone, Caron, Harder and Chase.)

Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, and may cause bowel obstruction, chronic pain, or infertility. Our understanding of adhesion biology is limited, which explains the paucity of anti-adhesion treatments. Here we present a systematic analysis of mouse and human adhesion tissues. First, we show that adhesions derive primarily from the visceral peritoneum, consistent with our clinical experience that adhesions form primarily following laparotomy rather than laparoscopy. Second, adhesions are formed by poly-clonal proliferating tissue-resident fibroblasts. Third, using single cell RNA-sequencing, we identify heterogeneity among adhesion fibroblasts, which is more pronounced at early timepoints. Fourth, JUN promotes adhesion formation and results in upregulation of PDGFRA expression. With JUN suppression, adhesion formation is diminished. Our findings support JUN as a therapeutic target to prevent adhesions. An anti-JUN therapy that could be applied intra-operatively to prevent adhesion formation could dramatically improve the lives of surgical patients.

Competing Interests: Conflict of interest: S.G. Chu has nothing to disclose. Conflict of interest: S. Poli De Frias has nothing to disclose. Conflict of interest: Y. Sakairi reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: R.S. Kelly reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: R. Chase reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: K. Konishi reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: A. Blau reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: E. Tsai reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: K. Tsoyi has nothing to disclose. Conflict of interest: R.F. Padera reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: L.M. Sholl reports grants from National Institutes of Health (P01HL114501), during the conduct of the study; personal fees for consultancyfrom Foghorn Therapeutics, outside the submitted work. Conflict of interest: H.J. Goldberg reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: H.R. Mallidi reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: P.C. Camp reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: S.Y. El-Chemaly has nothing to disclose. Conflict of interest: M.A. Perrella reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: A.M.K. Choi reports grants from National Institutes of Health (P01HL114501, R01HL132198, P01HL108801, R01HL055330, R01HL133801), during the conduct of the study. Conflict of interest: G.R. Washko reports grants from NIH and BTG Interventional Medicine, grants from and consultancy/advisory board work for Boehringer Ingelheim, consultancy for Genentech, Regeneron and GlaxoSmithKline, consultancy/data monitoring committee work for PulmonX, advisory board work for ModoSpira and Toshiba, grants from and consultancy for Janssen Pharmaceuticals, outside the submitted work; is founder and co-owner of Quantitative Imaging Solutions; and G.R Washko's spouse works for Biogen, which is focused on developing therapies for fibrotic lung disease. Conflict of interest: B.A. Raby reports grants from National Institutes of Health (P01HL114501), during the conduct of the study. Conflict of interest: I.O. Rosas reports grants from National Institutes of Health (P01HL114501), during the conduct of the study.

Fluorescent proteins are used extensively in transgenic animal models to label and study specific cell and tissue types. Expression of these proteins can be imaged and analyzed using fluorescent and confocal microscopy. Conventional confocal microscopes cannot penetrate through tissue more than 4-6 μm thick. Tissue clearing procedures overcome this challenge by rendering thick specimens into translucent tissue. However, most tissue clearing techniques do not satisfactorily preserve expression of endogenous fluorophores. Using simple adjustments to the BABB (Benzoic Acid Benzyl Benzoate) clearing methodology, preservation of fluorophore expression can be maintained. Modified BABB tissue clearing is a reliable technique to clear skin and soft tissue specimens for the study of dermal biology, wound healing and fibrotic pathologies.

Background: Hydralazine, labetalol, and nifedipine are the recommended first-line treatments for severe hypertension in pregnancy. While all three are effective, there is a lack of sufficient evidence regarding their comparative safety and efficacy., Objective: To determine the comparative safety and efficacy of the first-line treatment options for severe hypertension in pregnancy., Methods: A systematic search of Medline, Embase, and Cochrane Central Register of Controlled Trials up to May 31, 2018 was conducted. RCTs in pregnancy comparing a first-line antihypertensive agent to another first-line agent for the treatment of severe hypertension in pregnancy. Screening, data abstraction, and quality assessment were done by two independent reviewers. To estimate relative effects from all available evidence, a Bayesian network meta-analysis with vague priors was conducted., Main Results: Of the 1330 publications identified, 17 RCTs comprised of a total of 1591 women met our selection criteria. For successful treatment of severe hypertension, nifedipine was found to be superior to hydralazine (OR 4.13 [95% CrI 1.01-20.75]) but not labetalol (OR 3.43 [95% CrI 0.94-19.95]). This was not associated with an increased risk for caesarean delivery or maternal side effects. There was no significant difference between labetalol and hydralazine., Conclusions: Given the results of this systematic review and network meta-analysis, maternity care providers should feel comfortable initiating management of severe hypertension in pregnancy using oral nifedipine., (Copyright © 2019 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Hepatitis C virus (HCV) genotype (GT) 2 represents approximately 9% of all viral infections globally. While treatment outcomes for GT2-infected patients have improved substantially with direct-acting antiviral agents (DAAs) compared to interferon-α, the presence of polymorphisms in NS5A can impact efficacy of NS5A inhibitor-containing regimens. Thus, pathways of NS5A resistance were explored in GT2 subtypes using elbasvir, an NS5A inhibitor with broad genotype activity. Resistance selection studies, resistance analysis in NS5A-inhibitor treated virologic failures, antiviral activities in replicons bearing a panel of GT2 subtype sequences and amino acid substitutions introduced by site-directed mutagenesis were performed to define determinants of inhibitor susceptibility. Elbasvir showed differential antiviral activity in replicons bearing GT2 sequences. The EC 50 values for replicons bearing reference NS5A sequences for GT2a and GT2b were 0.003 and 3.4 nanomolar (nM) respectively. Studies with recombinant replicons demonstrated crosstalk between amino acid positions 28 and 31. The combination of phenylalanine and methionine at positions 28 and 31 respectively, conferred the highest potency reduction for elbasvir in GT2a and GT2b. This combination was observed in failures from the C-SCAPE trial. Addition of grazoprevir, an NS3/4A protease inhibitor, to elbasvir more effectively suppressed the emergence of resistance in GT2 at modest inhibitor concentrations (3X EC 90 ). Ruzasvir, a potent, pan-genotype NS5A inhibitor successfully inhibited replicons bearing GT2 resistance-associated substitutions (RASs) at positions 28 and 31. The studies demonstrate crosstalk between amino acids at positions 28 and 31 in NS5A modulate inhibitor potency and may impact treatment outcomes in some HCV GT2-infected patients., (Copyright © 2019 American Society for Microbiology.)

Bioengineers have built models of the tumour microenvironment (TME) in which to study cell-cell interactions, mechanisms of cancer growth and metastasis, and to test new therapies. These models allow researchers to culture cells in conditions that include features of the in vivo TME implicated in regulating cancer progression, such as extracellular matrix (ECM) stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a three-dimensional geometry more representative of the in vivo TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical models and clinical trials. However, it can be challenging to compare drug response reports across different biomaterial platforms in the current literature. This is, in part, a result of inconsistent reporting and improper use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial designs. This study attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to measure the growth rate of cells in the absence of drug, and follow our 'decision tree' when reporting drug response metrics. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.

Murine studies have linked TGF-β signaling to emphysema, and human genome-wide association studies (GWAS) studies of lung function and COPD have identified associated regions near genes in the TGF-β superfamily. However, the functional regulatory mechanisms at these loci have not been identified. We performed the largest GWAS of emphysema patterns to date, identifying 10 GWAS loci including an association peak spanning a 200 kb region downstream from TGFB2 . Integrative analysis of publicly available eQTL, DNaseI, and chromatin conformation data identified a putative functional variant, rs1690789, that may regulate TGFB2 expression in human fibroblasts. Using chromatin conformation capture, we confirmed that the region containing rs1690789 contacts the TGFB2 promoter in fibroblasts, and CRISPR/Cas-9 targeted deletion of a ~ 100 bp region containing rs1690789 resulted in decreased TGFB2 expression in primary human lung fibroblasts. These data provide novel mechanistic evidence linking genetic variation affecting the TGF-β pathway to emphysema in humans., Competing Interests: MP, YH, FG, BP, RC, JP, VT, JC, XZ No competing interests declared, MC reports grants from GSK and personal fees from Genentech, CH reports personal fees from Mylan, personal fees from AstraZeneca, Concert Pharmaceuticals, 23andMe, grants from Novartis, and Boehringer-Ingelheim, GW reports grants and other support from Boehringer Ingelheim, PulmonX, BTG Interventional Medicine, Janssen Pharmaceuticals and GSK, SR reports receiving personal fees from Amgen, ES received honoraria from Novartis for Continuing Medical Education Seminars and grant and travel support from GlaxoSmithKline, RS reports personal fees from Boehringer Ingelheim, Eolo Medical and Toshiba, PC has received research support and consulting fees from GSK and Novartis, (© 2019, Parker et al.)