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Introduction The Package of Essential Noncommunicable Disease Interventions—Plus (PEN-Plus) is a strategy decentralising care for severe non-communicable diseases (NCDs) including type 1 diabetes, rheumatic heart disease and sickle cell disease, to increase access to care. In the PEN-Plus model, mid-level clinicians in intermediary facilities in low and lower middle income countries are trained to provide integrated care for conditions where services traditionally were only available at tertiary referral facilities. For the upcoming phase of activities, 18 first-level hospitals in 9 countries and 1 state in India were selected for PEN-Plus expansion and will treat a variety of severe NCDs. Over 3 years, the countries and state are expected to: (1) establish PEN-Plus clinics in one or two district hospitals, (2) support these clinics to mature into training sites in preparation for national or state-level scale-up, and (3) work with the national or state-level stakeholders to describe, measure and advocate for PEN-Plus to support development of a national operational plan for scale-up.Methods and analysis Guided by Proctor outcomes for implementation research, we are conducting a mixed-method evaluation consisting of 10 components to understand outcomes in clinical implementation, training and policy development. Data will be collected through a mix of quantitative surveys, routine reporting, routine clinical data and qualitative interviews.Ethics and dissemination This protocol has been considered exempt or covered by central and local institutional review boards. Findings will be disseminated throughout the project’s course, including through quarterly M&E discussions, semiannual formative assessments, dashboard mapping of progress, quarterly newsletters, regular feedback loops with national stakeholders and publication in peer-reviewed journals.

Abstract Background The electrocardiogram (ECG) is the most relied upon tool for cardiovascular diagnosis, especially in low-resource settings because of its low cost and straightforward usability. It is imperative that internal medicine (IM) and emergency medicine (EM) specialists are competent in ECG interpretation. Our study was designed to improve proficiency in ECG interpretation through a competition among IM and EM residents at a teaching hospital in rural central Haiti in which over 40% of all admissions are due to CVD. Methodology The 33 participants included 17 EM residents and 16 IM residents from each residency year at the Hôpital Universitaire de Mirebalais (HUM). Residents were divided into 11 groups of 3 participants with a representative from each residency year and were given team-based online ECG quizzes to complete weekly. The format included 56 ECG cases distributed over 11 weeks, and each case had a pre-specified number of points based on abnormal findings and complexity. All ECG cases represented cardiovascular pathology in Haiti adapted from the Association of Program Directors in Internal Medicine evaluation list. The main intervention was sharing group performance and ECG solutions to all participants each week to promote competition and self-study without specific feedback or discussion by experts. To assess impact, pre- and post-intervention assessments measuring content knowledge and comfort for each participant were performed. Results Overall group participation was heterogeneous with groups participating a median of 54.5% of the weeks (range 0–100%). 22 residents completed the pre- and post-test assessments. The mean pre- and post-intervention assessment knowledge scores improved from 27.3% to 41.7% (p = 0.004). 70% of participants improved their test scores. The proportion of participants who reported comfort with ECG interpretation increased from 57.6% to 66.7% (p = 0.015). Conclusion This study demonstrates improvement in ECG interpretation through a team-based, asynchronous ECG competition approach. This method is easily scalable and could help to fill gaps in ECG learning. This approach can be delivered to other hospitals both in and outside Haiti. Further adaptations are needed to improve weekly group participation.

Tibial plateau fractures typically are associated with high-energy mechanisms in young patients, as in the case of this 35-year-old softball player, and with low-energy trauma in the older population. A detailed history, careful physical examination, and plain radiographs constitute the essential initial study; MRI and CT are indicated to refine the diagnosis. Concomitant injuries are common, including ligament tears, meniscal damage, and other soft-tissue compromise. Less common but more devastating injuries include vascular disruption, nerve injury, and compartment syndrome. Injury severity determines whether treatment will be nonoperative or operative, but the goal is to restore the patient's normal function. [ABSTRACT FROM AUTHOR]

Abstract Background Guidelines recommend inhaled corticosteroids (ICS) for patients with severe chronic obstructive pulmonary disease (COPD). Most COPD patients are managed in primary care and receive ICS long-term and irrespective of severity. The effect of withdrawing ICS from COPD patients in primary care is unknown. Methods In a pragmatic randomised, double-blind, placebo-controlled trial in 31 practices, 260 COPD patients stopped their usual ICS (median duration of use 8 years) and were allocated to 500 mcg fluticasone propionate twice daily (n = 128), or placebo (n = 132). Follow-up assessments took place at three monthly intervals for a year at the patients' practice. Our primary outcome was COPD exacerbation frequency. Secondary outcomes were time to first COPD exacerbation, reported symptoms, peak expiratory flow rate and reliever inhaler use, and lung function and health related quality of life. Results In patients randomised to placebo, COPD exacerbation risk over one year was RR: 1.11 (CI: 0.91–1.36). Patients taking placebo were more likely to return to their usual ICS following exacerbation, placebo: 61/128 (48%); fluticasone: 34/132 (26%), OR: 2.35 (CI: 1.38–4.05). Exacerbation risk whilst taking randomised treatment was significantly raised in the placebo group 1.48 (CI: 1.17–1.86). Patients taking placebo exacerbated earlier (median time to first exacerbation: placebo (days): 44 (CI: 29–59); fluticasone: 63 (CI: 53–74), log rank 3.81, P = 0.05) and reported increased wheeze. In a post-hoc analysis, patients with mild COPD taking placebo had increased exacerbation risk RR: 1.94 (CI: 1.20–3.14). Conclusion Withdrawal of long-term ICS in COPD patients in primary care increases risk of exacerbation shortens time to exacerbation and causes symptom deterioration. Patients with mild COPD may be at increased risk of exacerbation after withdrawal. Trial Registration ClinicalTrials.gov NCT00440687

Snakebites affect about 1.8 million people annually. The current standard of care involves antibody-based antivenoms, which can be difficult to access and are generally not effective against local tissue injury, the primary cause of morbidity. Here, we used a pooled whole-genome CRISPR knockout screen to define human genes that, when targeted, modify cell responses to spitting cobra venoms. A large portion of modifying genes that conferred resistance to venom cytotoxicity was found to control proteoglycan biosynthesis, including EXT1 , B4GALT7 , EXT2 , EXTL3 , XYLT2 , NDST1 , and SLC35B2 , which we validated independently. This finding suggested heparinoids as possible inhibitors. Heparinoids prevented venom cytotoxicity through binding to three-finger cytotoxins, and the US Food and Drug Administration-approved heparinoid tinzaparin was found to reduce tissue damage in mice when given via a medically relevant route and dose. Overall, our systematic molecular dissection of cobra venom cytotoxicity provides insight into how we can better treat cobra snakebite envenoming.

In rich false memory studies, familial informants often provide information to support researchers in planting vivid memories of events that never occurred. The goal of the current study was to assess how effectively we can retract these false memories via debriefing - i.e., to what extent can we put participants back the way we found them? We aimed to establish (1) what proportion of participants would retain a false memory or false belief following debriefing, and (2) whether richer, more detailed memories would be more difficult to retract. Participants (N = 123) completed a false memory implantation protocol as part of a replication of the "Lost in the Mall" study (Loftus & Pickrell, Psychiatric Annals, 25, 720-725, 1995). By the end of the protocol, 14% of participants self-reported a memory for the fabricated event, and a further 52% believed it had happened. Participants were then fully debriefed, and memory and belief for the false event were assessed again. In a follow-up assessment 3 days post-debriefing, the false memory rate had dropped to 6% and false belief rates also fell precipitously to 7%. Moreover, virtually all persistent false memories were found to be nonbelieved memories, where participants no longer accepted that the fabricated event had occurred. Richer, more detailed memories were more resistant to correction, but were still mostly retracted. This study provides evidence that participants can be "dehoaxed", and even very convincing false memories can be retracted., (© 2024. The Author(s).)

Snakebite envenoming is a neglected tropical disease that causes substantial mortality and morbidity globally. The venom of African spitting cobras often causes permanent injury via tissue-destructive dermonecrosis at the bite site, which is ineffectively treated by current antivenoms. To address this therapeutic gap, we identified the etiological venom toxins in Naja nigricollis venom responsible for causing local dermonecrosis. While cytotoxic three-finger toxins were primarily responsible for causing spitting cobra cytotoxicity in cultured keratinocytes, their potentiation by phospholipases A 2 toxins was essential to cause dermonecrosis in vivo. This evidence of probable toxin synergism suggests that a single toxin-family inhibiting drug could prevent local envenoming. We show that local injection with the repurposed phospholipase A 2 -inhibiting drug varespladib significantly prevents local tissue damage caused by several spitting cobra venoms in murine models of envenoming. Our findings therefore provide a therapeutic strategy that may effectively prevent life-changing morbidity caused by snakebite in rural Africa., Competing Interests: Competing interests statement:The authors declare no competing interest.

Snakebite envenoming results in ∼100,000 deaths per year, with close to four times as many victims left with life-long sequelae. Current antivenom therapies have several limitations including high cost, variable cross-snake species efficacy and a requirement for intravenous administration in a clinical setting. Next-generation snakebite therapies are being widely investigated with the aim to improve cost, efficacy, and safety. In recent years several small molecule drugs have shown considerable promise for snakebite indication, with oral bioavailability particularly promising for community delivery rapidly after a snakebite. However, only two such drugs have entered clinical development for snakebite. To offset the risk of attrition during clinical trials and to better explore the chemical space for small molecule venom toxin inhibitors, here we describe the first high throughput drug screen against snake venom metalloproteinases (SVMPs)-a pathogenic toxin family responsible for causing haemorrhage and coagulopathy. Following validation of a 384-well fluorescent enzymatic assay, we screened a repurposed drug library of 3,547 compounds against five geographically distinct and toxin variable snake venoms. Our drug screen resulted in the identification of 14 compounds with pan-species inhibitory activity. Following secondary potency testing, four SVMP inhibitors were identified with nanomolar EC 50 s comparable to the previously identified matrix metalloproteinase inhibitor marimastat and superior to the metal chelator dimercaprol, doubling the current global portfolio of SVMP inhibitors. Following analysis of their chemical structure and ADME properties, two hit-to-lead compounds were identified. These clear starting points for the initiation of medicinal chemistry campaigns provide the basis for the first ever designer snakebite specific small molecules., 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 © 2024 Clare, Dawson, Westhorpe, Albulescu, Woodley, Mosallam, Chong, Kool, Berry, O’Neill and Casewell.)

Understanding the suitable habitat of endangered species is crucial for agencies such as the Bureau of Land Management to plan management and conservation. However, few species distribution models are directly validated, potentially limiting their application in management. In preparation for a Species Status Assessment of clay-loving wild buckwheat ( Eriogonum pelinophilum ), an endangered subshrub found in southwest Colorado, we ran a series of species distribution models to estimate the species' potential occupied habitat and validated these models in the field. A 1-meter resolution digital elevation model derived from LiDAR and a high-resolution geology mapping helped identify biologically relevant characteristics of the species' habitat. We employed a weighted ensemble model based on two Random Forest and one Boosted Regression Tree model, and discrimination performance of the ensemble model was high (AUC-PR = 0.793). We then conducted a systematic field survey of model habitat suitability predictions, during which we discovered 55 new subpopulations of the species and demonstrated that new species observations were strongly associated with model predictions ( p  < .0001, Cliff's delta = 0.575). We further refined our original models by incorporating the additional species occurrences collected in the field survey, a new explanatory variable, and a more diverse set of models. These iterative changes marginally improved performance of the ensemble model (AUC-PR = 0.825). Direct validation of species distribution models is extremely rare, and our field survey provides strong validation of our model results. This helps increase confidence to utilize predictions in planning. The final model predictions greatly improve the Bureau of Land Management's understanding of the species' habitat and increase our ability to consider potential habitat in planning land use activities such as road development and travel management., Competing Interests: The authors declare no competing interests., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Ecology and Evolution published by John Wiley & Sons Ltd.)

Morbidity from snakebite envenoming affects approximately 400,000 people annually. Tissue damage at the bite-site often leaves victims with catastrophic life-long injuries and is largely untreatable by current antivenoms. Repurposed small molecule drugs that inhibit specific snake venom toxins show considerable promise for tackling this neglected tropical disease. Using human skin cell assays as an initial model for snakebite-induced dermonecrosis, we show that the drugs 2,3-dimercapto-1-propanesulfonic acid (DMPS), marimastat, and varespladib, alone or in combination, inhibit the cytotoxicity of a broad range of medically important snake venoms. Thereafter, using preclinical mouse models of dermonecrosis, we demonstrate that the dual therapeutic combinations of DMPS or marimastat with varespladib significantly inhibit the dermonecrotic activity of geographically distinct and medically important snake venoms, even when the drug combinations are delivered one hour after envenoming. These findings strongly support the future translation of repurposed drug combinations as broad-spectrum therapeutics for preventing morbidity caused by snakebite., (© 2023. The Author(s).)

Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snake's venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tooL (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis in mice. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease., (© 2023. The Author(s).)

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Background: Ecological momentary assessment (EMA) is used to capture daily lived experiences, states, and environments. Although EMA is commonly used in behavioral health research, there remains a dearth of literature on how researchers account for design considerations of EMA techniques when designing studies. The goal of this formative mixed methods study was to elicit feedback on EMA study procedures and materials from the target populations for a larger study about binge eating among sexual minority and heterosexual young women, in which data are collected entirely remotely., Methods: Sexual minority (n=12) and heterosexual (n=9) women ages 18-30 who binge ate took part in a pilot EMA study and exit interview and survey. As part of the consent and orientation process, participants reviewed video and written materials describing the study purpose and procedures. Using a smartphone app, for seven consecutive days they completed a survey each morning, 5 random surveys per day, and self-initiated a survey each time they binge ate. Participants then provided feedback on the study via a 1-hour virtual interview and online survey. Interviews were transcribed and reviewed by two coders to identify themes on the acceptability and feasibility of the EMA procedures with a focus on: (I) the training and study description materials; (II) general smartphone app and survey preferences; and (III) specific EMA survey question content and wording., Results: The qualitative and quantitative data converged to suggest participants were able to easily download and use the app to complete surveys and report on binge eating events. Participants provided feedback that was incorporated into revisions on general study procedures, the training video content, and EMA question content for binge eating, identity-related stressors, and appearance-related pressures. No systematic themes in the quantitative or qualitative data emerged to suggest questions were perceived differently by sexual minority and heterosexual young women., Conclusions: These findings provide evidence for the feasibility of conducting a remote EMA study to assess young women's experiences around binge eating. This formative study provides an example of how a mixed methods approach can be used to refine EMA study methods and questions to improve study design., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://mhealth.amegroups.com/article/view/10.21037/mhealth-23-16/coif). KEH serves as an unpaid editorial board member of mHealth from March 2023 to February 2025. She also received funding from National Institute on Minority Health and Health Disparities (NIMHD) of the National Institutes of Health (NIH) to conduct this study and for related activities (No. R01MD012598). KEH is also a member of the Executive Committee for the Society of Ambulatory Assessment. ALB and RJL received funding as co-investigators from NIMHD of NIH (No. R01MD012598). The other authors have no conflicts of interest to declare., (2023 mHealth. All rights reserved.)

"γc" cytokines are a family whose receptors share a "common-gamma-chain" signaling moiety, and play central roles in differentiation, homeostasis, and communications of all immunocyte lineages. As a resource to better understand their range and specificity of action, we profiled by RNAseq the immediate-early responses to the main γc cytokines across all immunocyte lineages. The results reveal an unprecedented landscape: broader, with extensive overlap between cytokines (one cytokine doing in one cell what another does elsewhere) and essentially no effects unique to any one cytokine. Responses include a major downregulation component and a broad Myc-controlled resetting of biosynthetic and metabolic pathways. Various mechanisms appear involved: fast transcriptional activation, chromatin remodeling, and mRNA destabilization. Other surprises were uncovered: IL2 effects in mast cells, shifts between follicular and marginal zone B cells, paradoxical and cell-specific cross-talk between interferon and γc signatures, or an NKT-like program induced by IL21 in CD8+ T cells., (© 2023 Benoist et al.)

The seminal Lost in the Mall study has been enormously influential in psychology and is still cited in legal cases. The current study directly replicated this paper, addressing methodological weaknesses including increasing the sample size fivefold and preregistering detailed analysis plans. Participants ( N  = 123) completed a survey and two interviews where they discussed real and fabricated childhood events, based on information provided by an older relative. We replicated the findings of the original study, coding 35% of participants as reporting a false memory for getting lost in a mall in childhood (compared to 25% in the original study). In an extension, we found that participants self-reported high rates of memories and beliefs for the fabricated event. Mock jurors were also highly likely to believe the fabricated event had occurred and that the participant was truly remembering the event, supporting the conclusions of the original study.

ABSTRACT Deception is often a necessity in rich false memory studies, but is this deception acceptable to participants? In the current study, we followed up with 175 participants who had taken part in a replication of the Lost in the Mall childhood false memory study (Loftus & Pickrell, 1995), as either a research subject or a familial informant. We found that both participants and informants were generally very positive about their experience, did not regret taking part and found the deceptive methods acceptable. Importantly, the vast majority reported that they would still have taken part had they known the true objectives from the beginning. Participants also reported learning something interesting about memory and enjoying the nostalgia and family discussions that were prompted by the study. We would encourage other researchers to assess the ethical implications of false memory research paradigms and to incorporate the valuable feedback from participants and informants.

Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Previous research has identified health disparities between sexual minority and heterosexual women, including increased rates of obesity and binge eating in sexual minority women. Established predictors of binge eating behavior include negative emotions and sociocultural processes; however, these studies are generally conducted in samples of young women where sexual identity is not known or reported. There is a dearth of research evaluating how sexual minority-specific factors (eg, minority stress and connectedness to the lesbian, gay, bisexual, transgender, and queer community) may affect binge eating in sexual minority women. In addition, no studies have examined these processes in racially diverse samples or considered how intersecting minority identities (eg, Black and sexual minority) may affect eating behaviors., Objective: The Health and Experiences in Real Life (HER Life) Project aims to clarify real-world predictors of binge eating in young heterosexual and sexual minority women using ecological momentary assessment. The role of affective, social, and health behavior factors in binge eating will be examined for all women (aim 1), and sexual minority-specific predictors will also be considered for sexual minority women participants (aim 2). Person-level moderators of race, body- and eating-related factors, and sexual minority-specific factors will also be examined to better understand how real-world binge eating predictors may differ for various demographic groups (aim 3)., Methods: Researchers aim to recruit 150 sexual minority and 150 heterosexual women from across the United States, including at least 50 Black women for each group, using web-based recruitment methods. The eligibility criteria include identifying as a woman, being aged between 18 and 30 years, and having had at least two binge eating episodes in the last 2 weeks. Participants must endorse being only or mostly attracted to men (considered heterosexual) or only or mostly attracted to women or having a current or most recent female partner (considered sexual minority). Eligible participants complete an initial web-based baseline survey and then 14 days of ecological momentary assessment involving the completion of a morning and before-bed survey and 5 prompted surveys per day as well as a user-initiated survey after binge eating episodes. The data will be analyzed using a series of multilevel models., Results: Data collection started in February 2021. We have currently enrolled 129 sexual minority women and 146 heterosexual women. Data collection is expected to conclude in fall 2022., Conclusions: The Health and Experiences in Real Life Project aims to elucidate potential differences between sexual minority and heterosexual women in within-person factors predicting binge eating and inform eating disorder interventions for sexual minority women. The challenges in recruiting sexual minority women, including the determination of eligibility criteria and considerations for remote data collection, are discussed., International Registered Report Identifier (irrid): DERR1-10.2196/41199., (©Kristin E Heron, Abby L Braitman, Charlotte A Dawson, Cassidy M Sandoval, Lauren V Butler, Alicia Moulder, Robin J Lewis. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 21.10.2022.)

Nearly all past research about body dissatisfaction and romantic relationship factors is among heterosexual couples; little is known about these associations in sexual minority couples. The present study aimed to fill gaps in the current literature by using actor-partner interdependence models (APIMs) to examine dyadic patterns of association between body dissatisfaction and different aspects of relationship functioning among same-sex female couples. Participants were 163 same-sex female romantic dyads (326 women) between the ages of 18-35 years who completed measures of body dissatisfaction and relationship factors. Results from significance testing of actor and partner effects indicated higher levels of women's own body dissatisfaction were associated with lower levels of their own, but not their partner's, relationship satisfaction, closeness, sexual satisfaction, and intimacy/connectedness. Significance testing alone indicated that the association between one's own body dissatisfaction and their partner's relationship satisfaction was not significant. However, dyadic pattern testing identified a partner pattern for this effect, which suggests that the association between one's own body dissatisfaction and one's own relationship satisfaction is similar in magnitude and direction as that between an individuals' own body dissatisfaction and their partner's relationship satisfaction. In this study, women's own body dissatisfaction was found to be negatively associated with their own relationship functioning, which is consistent with findings of women in male-female couples. Thus, these findings highlight the important role that body dissatisfaction plays in women's relationship experiences. More research is needed to better understand potential cross-partner effects of body dissatisfaction and relationship factors in same-sex female couples. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Antivenom is currently the first-choice treatment for snakebite envenoming. However, only a low proportion of antivenom immunoglobulins are specific to venom toxins, resulting in poor dose efficacy and potency. We sought to investigate whether linear venom epitopes displayed on virus like particles can stimulate an antibody response capable of recognising venom toxins from diverse medically important species. Bioinformatically-designed epitopes, corresponding to predicted conserved regions of group I phospholipase A 2 and three finger toxins, were engineered for display on the surface of hepatitis B core antigen virus like particles and used to immunise female CD1 mice over a 14 weeks. Antibody responses to all venom epitope virus like particles were detectable by ELISA by the end of the immunisation period, although total antibody and epitope specific antibody titres were variable against the different epitope immunogens. Immunoblots using pooled sera demonstrated recognition of various venom components in a diverse panel of six elapid venoms, representing three continents and four genera. Insufficient antibody yields precluded a thorough assessment of the neutralising ability of the generated antibodies, however we were able to test polyclonal anti-PLA 2 IgG from three animals against the PLA 2 activity of Naja nigricollis venom, all of which showed no neutralising ability. This study demonstrates proof-of-principle that virus like particles engineered to display conserved toxin linear epitopes can elicit specific antibody responses in mice which are able to recognise a geographically broad range of elapid venoms., (© 2022. The Author(s).)

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

SHARPIN regulates signaling from the tumor necrosis factor (TNF) superfamily and pattern-recognition receptors. An inactivating Sharpin mutation in mice causes TNF-mediated dermatitis. Blocking cell death prevents the phenotype, implicating TNFR1-induced cell death in causing the skin disease. However, the source of TNF that drives dermatitis is unknown. Immune cells are a potent source of TNF in vivo and feature prominently in the skin pathology; however, T cells, B cells, and eosinophils are dispensable for the skin phenotype. We use targeted in vivo cell ablation, immune profiling, and extensive imaging to identify immune populations driving dermatitis. We find that systemic depletion of Langerin + cells significantly reduces disease severity. This is enhanced in mice that lack Langerhans cells (LCs) from soon after birth. Reconstitution of LC-depleted Sharpin mutant mice with TNF-deficient LCs prevents dermatitis, implicating LCs as a potential cellular source of pathogenic TNF and highlighting a T cell-independent role in driving skin inflammation., Competing Interests: Declaration of interests The authors claim no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Breast cancer is a heterogeneous disease that comprises multiple histological and molecular subtypes. To gain insight into mutations that drive breast tumorigenesis, we describe a pipeline for the identification and validation of tumor suppressor genes. Based on an in vivo genome-wide CRISPR/Cas9 screen in Trp53 +/- heterozygous mice, we identified tumor suppressor genes that included the scaffold protein Axin1, the protein kinase A regulatory subunit gene Prkar1a, as well as the proof-of-concept genes Pten, Nf1, and Trp53 itself. Ex vivo editing of primary mammary epithelial organoids was performed to further interrogate the roles of Axin1 and Prkar1a. Increased proliferation and profound changes in mammary organoid morphology were observed for Axin1/Trp53 and Prkar1a/Trp53 double mutants compared to Pten/Trp53 double mutants. Furthermore, direct in vivo genome editing via intraductal injection of lentiviruses engineered to express dual short-guide RNAs revealed that mutagenesis of Trp53 and either Prkar1a, Axin1, or Pten markedly accelerated tumor development compared to Trp53-only mutants. This proof-of-principle study highlights the application of in vivo CRISPR/Cas9 editing for uncovering cooperativity between defects in tumor suppressor genes that elicit mammary tumorigenesis., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The underlying mechanisms of sexual minority women's (SMW's) numerous physical and mental health disparities compared to heterosexual women are not well understood. The contribution of relationship factors is particularly understudied; few studies collect data from both same-sex female partners. Further, most research among SMW is cross sectional which limits our understanding of day-to-day experiences of same-sex women's couples. This paper aimed to describe the feasibility of recruiting a large sample of SMW and their female partners for a disparity-focused daily diary study investigating alcohol use and mental health. A firm specializing in sexual minority market research was enlisted to help with recruitment from multiple sources and conducted an initial pre-screening of SMW and their female partners, at least one of whom drank alcohol regularly. A total of 4182 individuals completed the pre-screener, with information for 930 individuals (465 couples) being sent to the research team. From this, 376 individuals (188 couples) completed the study screener, met the inclusion criteria, and were invited to participate. Ultimately, 326 individuals (163 couples) consented and completed baseline. A total of 321 individuals, from 162 couples, began the daily diary portion of the study. Compliance with study procedures was excellent. The use of multiple recruitment sources increased the diversity of the sample. Challenges to recruitment, changes in protocol, and characteristics of the final sample are discussed.

Background: To support the development of social medicine curricula that empower medical school graduates to redress health inequities, we conducted a mixed methods student and faculty evaluation of an expanded and innovative preclinical social medicine curriculum., Methods: We implemented a longitudinal, interactive preclinical social medicine curriculum that was closely integrated with foundational science teaching then conducted a survey-based mixed methods student and faculty curriculum evaluation. Based on these results, we propose a novel conceptual roadmap for social medicine curriculum design., Results: Student and faculty evaluations of an expanded and innovative longitudinal preclinical social medicine curriculum were strongly favorable. Both student and faculty respondents indicated a particular desire for deeper coverage of race and poverty among other social medicine domains. Qualitative student evaluations highlighted the importance of faculty champions to social medicine teaching as well as the educational impact of stories that exemplify the practical impact of the social determinants of health on specific patient experiences. Qualitative faculty evaluations pointed to the challenges of curriculum integration and the need for faculty career development in social medicine teaching., Conclusions: Based on mixed methods student and faculty curriculum evaluation data, we propose a novel conceptual roadmap for the design of social medicine curricula at other institutions., (© 2021. The Author(s).)

Background: Ecological momentary assessment (EMA) methods can be used to remotely assess physical and mental health in daily life for hard-to-reach, marginalized, and geographically dispersed populations in the U.S., such as sexual minority women (e.g., lesbian, bisexual). However, EMA studies are often complex, and engaging participants from afar can be a challenge. This study experimentally examined whether adding videos to written recruitment materials would improve consent rates, reduce dropout rates, and improve survey completion rates for an online daily diary study., Methods: As part of a 2-week study of same-sex female couples' health, 376 women ages 18-35 were recruited from across the U.S. using a market research firm. Couples were randomized to an introductory information condition (written + video materials or written-only materials) prior to informed consent., Results: Overall, 97.1% of eligible women reviewed introductory materials and of these 96.7% consented; consent rates did not differ by condition (written + video: 97.1%, written-only: 97.1%). Dropout rates were low (5.4%) and survey completion rates were high (90.4% of surveys completed); there were no group differences for study dropout (written + video: 3.6%, written-only: 7.0%) or survey completion (written + video: 92.5%, written-only: 88.4%). Data from women randomized to receive videos indicated more than half (53.3%) did not watch any of the five videos in full. However, among those who viewed the videos, time spent watching videos, watching more videos in full, and watching at least one video in full were each positive associated with survey completion rates., Conclusions: In summary, we had high consent rates, low dropout rates, and high survey completion rates regardless of video instructions. Although sexual minority women can be hard to reach, our potential participants appeared highly motivated to take part in research, and thus video recruitment materials were not necessary to improve participation. Future experimental research to maximize EMA study design and implementation could be important for populations less inclined to participate in EMA studies, or who are less familiar with research., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/mhealth-20-116). Kristin E. Heron serves as an unpaid editorial board member of mHealth from February 2019 to January 2021. The other authors have no conflicts of interest to declare., (2021 mHealth. All rights reserved.)

Background: Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty., Methods: The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points., Results: The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states., Conclusions: This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.

Despite rapid advances in our knowledge of the cellular heterogeneity and molecular regulation of the mammary gland, how these relate to 3D cellular organization remains unclear. In addition to hormonal regulation, mammary gland development and function is directed by para- and juxtacrine signaling among diverse cell-types, particularly the immune and mesenchymal populations. Precise mapping of the cellular landscape of the breast will help to decipher this complex coordination. Imaging of thin tissue sections has provided foundational information about cell positioning in the mammary gland and now technological advances in tissue clearing and subcellular-resolution 3D imaging are painting a more complete picture. In particular, confocal, light-sheet and multiphoton microscopy applied to intact tissue can fully capture cell morphology, position and interactions, and have the power to identify spatially rare events. This review will summarize our current understanding of mammary gland cellular organization as revealed by microscopy. We focus on the mouse mammary gland and cover a broad range of immune and stromal cell types at major developmental stages and give insights into important tissue niches and cellular interactions.

Background: Despite an abundant literature advocating that social determinants of health (SDH) be taught during undergraduate medical education, there are few detailed descriptions of how to design and implement longitudinal core curricula that is delivered to all students and accomplishes this goal., Methods: In this paper, we describe the design and implementation of a social medicine curriculum at the University of Vermont's Larner College of Medicine (UVM Larner). Using Kern's principles, we designed a longitudinal curriculum that extends through both preclinical and clinical training for all students and focused on integrating SDH material directly into basic science and clinical training., Results: We successfully developed and implemented two primary tools, a "Social Medicine Theme of the Week" (SMTW) in preclinical training, and SDH rounds in the clinical setting to deliver SDH content to all learners at UVM Larner., Conclusions: Extensive student-faculty partnerships, robust needs assessment, and focusing on longitudinal and integrated SDH content delivery to all students were key features that contributed to successful design and implementation.

Snakebite is a medical emergency causing high mortality and morbidity in rural tropical communities that typically experience delayed access to unaffordable therapeutics. Viperid snakes are responsible for the majority of envenomings, but extensive interspecific variation in venom composition dictates that different antivenom treatments are used in different parts of the world, resulting in clinical and financial snakebite management challenges. Here, we show that a number of repurposed Phase 2-approved small molecules are capable of broadly neutralizing distinct viper venom bioactivities in vitro by inhibiting different enzymatic toxin families. Furthermore, using murine in vivo models of envenoming, we demonstrate that a single dose of a rationally-selected dual inhibitor combination consisting of marimastat and varespladib prevents murine lethality caused by venom from the most medically-important vipers of Africa, South Asia and Central America. Our findings support the translation of combinations of repurposed small molecule-based toxin inhibitors as broad-spectrum therapeutics for snakebite.

How platelets are produced by megakaryocytes in vivo remains controversial despite more than a century of investigation. Megakaryocytes readily produce proplatelet structures in vitro; however, visualization of platelet release from proplatelets in vivo has remained elusive. We show that within the native prenatal and adult environments, the frequency and rate of proplatelet formation is incompatible with the physiological demands of platelet replacement. We resolve this inconsistency by performing in-depth analysis of plasma membrane budding, a cellular process that has previously been dismissed as a source of platelet production. Our studies demonstrate that membrane budding results in the sustained release of platelets directly into the peripheral circulation during both fetal and adult life without induction of cell death or proplatelet formation. In support of this model, we demonstrate that in mice deficient for NF-E2 (the thrombopoietic master regulator), the absence of membrane budding correlates with failure of in vivo platelet production. Accordingly, we propose that membrane budding, rather than proplatelet formation, supplies the majority of the platelet biomass., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Potts et al.)

Bisexual women report more physical and psychological health problems than lesbian women do, which may be attributed to greater sexual minority stress and less social support. However, many studies combine lesbian and bisexual women into a single group. The current study examined if sexual minority stress and social support mediated the association between women's sexual identity (lesbian or bisexual) and health-related outcomes. A total of 650 U.S. young adult lesbian ( n = 227) and bisexual ( n = 423) women completed an online survey about sexual minority stress, social support, and physical and mental health problems. Bisexual women reported more physical and mental health problems. A sequential mediation model showed that bisexual women reported greater sexual minority stress than lesbian women, which in turn was associated with less social support, which was associated with more physical and mental health problems. Greater sexual minority stress and lower social support may help explain why bisexual women report more health-related problems than lesbian women. The results of the present study support the importance of examining risk and protective factors for health problems separately for lesbian and bisexual women. Health-related intervention programs that target sexual minority women may need to be tailored differently for lesbian and bisexual women.

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary-gland function, their diversity has not been fully addressed. By exploiting high-resolution three-dimensional imaging and flow cytometry, we identified a unique population of tissue-resident ductal macrophages that form a contiguous network between the luminal and basal layers of the epithelial tree throughout postnatal development. Ductal macrophages are long lived and constantly survey the epithelium through dendrite movement, revealed via advanced intravital imaging. Although initially originating from embryonic precursors, ductal macrophages derive from circulating monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, when they are poised to phagocytose milk-producing cells post-lactation and facilitate remodelling. Interestingly, ductal macrophages strongly resemble mammary tumour macrophages and form a network that pervades the tumour. Thus, the mammary epithelium programs specialized resident macrophages in both physiological and tumorigenic contexts.

Background: The Healthy People 2020 initiative aims to reduce health disparities, including alcohol use, among sexual minority women (SMW; eg, lesbian, bisexual, queer, and pansexual). Compared with heterosexual women, SMW engage in more hazardous drinking and report more alcohol-related problems. Sexual minority stress (ie, the unique experiences associated with stigmatization and marginalization) has been associated with alcohol use among SMW. Among heterosexuals, relationship factors (eg, partner violence and drinking apart vs together) have also been associated with alcohol use. Negative affect has also been identified as a contributor to alcohol use. To date, most studies examining alcohol use among SMW have used cross-sectional or longitudinal designs., Objective: Project Relate was designed to increase our understanding of alcohol use among young SMW who are at risk for alcohol problems. The primary objectives of this study are to identify daily factors, as well as potential person-level risk and protective factors, which may contribute to alcohol use in SMW. Secondary objectives include examining other physical and mental concerns in this sample (eg, other substance use, eating, physical activity, and stress)., Methods: Both partners of a female same-sex couple (aged 18-35 years; n=150 couples) are being enrolled in the study following preliminary screening by a market research firm that specializes in recruiting sexual minority individuals. Web-based surveys are being used to collect information about the primary constructs of interest (daily experiences of alcohol use, sexual minority stress, relationship interactions, and mood) as well as secondary measures of other physical and mental health constructs. Data are collected entirely remotely from women across the United States. Each member of eligible couples completes a baseline survey and then 14 days of daily surveys each morning. Data will be analyzed using multilevel structural equation modeling., Results: To date, 208 women (ie, 104 couples) were successfully screened and enrolled into the study. In total, 164 women have completed the 14-day daily protocol. Compliance with completing the daily diaries has been excellent, with participants on average completing 92% of the daily diaries. Data collection will be completed in fall 2018, with results published as early as 2019 or 2020., Conclusions: Project Relate is designed to increase our understanding of between- and within-person processes underlying hazardous drinking in understudied, at-risk SMW. The study includes a remote daily diary methodology to provide insight into variables that may be associated with daily hazardous alcohol use. Before the development of programs that address hazardous alcohol use among young SMW, there is a need for better understanding of individual and dyadic variables that contribute to risk in this population. The unique challenges of recruiting and enrolling SMW from across the United States in a daily diary study are discussed., International Registered Report Identifier (irrid): DERR1-10.2196/11718., (©Kristin E Heron, Robin J Lewis, Alexander T Shappie, Charlotte A Dawson, Rachel Amerson, Abby L Braitman, Barbara A Winstead, Michelle L Kelley. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 04.02.2019.)

Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type-specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states., Competing Interests: The authors have declared that no competing interests exist.

The thrombopoietic environment of the neonate is established during prenatal life; therefore, a comprehensive understanding of platelet-forming cell development during embryogenesis is critical to understanding the etiology of early-onset thrombocytopenia. The recent discovery that the first platelet-forming cells of the conceptus are not megakaryocytes (MKs) but diploid platelet-forming cells (DPFCs) revealed a previously unappreciated complexity in thrombopoiesis. This raises important questions, including the following. When do conventional MKs appear? Do pathogenic genetic lesions of adult MKs affect DPFCs? What role does myeloproliferative leukemia virus (MPL), a key regulator of adult megakaryopoiesis, play in prenatal platelet-forming lineages? We performed a comprehensive study to determine the spatial and temporal appearance of prenatal platelet-forming lineages. We demonstrate that DPFCs originate in the yolk sac and then rapidly migrate to other extra- and intraembryonic tissues. Using gene disruption models of Gata1 and Nfe2, we demonstrate that perturbing essential adult MK genes causes an analogous phenotype in the early embryo before the onset of hematopoietic stem/progenitor cell-driven (definitive) hematopoiesis. Finally, we present the surprising finding that DPFC and MK commitment from their respective precursors is MPL independent in vivo but that completion of MK differentiation and establishment of the prenatal platelet mass is dependent on MPL expression., (© 2015 by The American Society of Hematology.)

Methods for the spatial estimation of risk of harm to soil by erosion by water and wind and by soil organic matter decline are explored. Rates of harm are estimated for combinations of soil type and land cover (as a proxy for hazard frequency) and used to estimate risk of soil erosion and loss of soil organic carbon (SOC) for 1 km(2)pixels. Scenarios are proposed for defining the acceptability of risk of harm to soil: the most precautionary one corresponds to no net harm after natural regeneration of soil (i.e. a 1 in 20 chance of exceeding an erosion rate of <1 tha(-1)y(-1) and SOC content decline of 0 kg t(-1)y(-1) for mineral soils and a carbon stock decline of 0 tha(-1)y(-1) for organic soils). Areas at higher and lower than possible acceptable risk are mapped. The veracity of boundaries is compromised if areas of unacceptable risk are mapped to administrative boundaries. Errors in monitoring change in risk of harm to soil and inadequate information on risk reduction measures' efficacy, at landscape scales, make it impossible to use or monitor quantitative targets for risk reduction adequately. The consequences for priority area definition of expressing varying acceptable risk of harm to soil as a varying probability of exceeding a fixed level of harm, or, a varying level of harm being exceeded with a fixed probability, are discussed. Soil data and predictive models for rates of harm to soil would need considerable development and validation to implement a priority area approach robustly., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rationale: Mechanical ventilation with large tidal volumes causes ventilator-induced lung injury in animal models. Little direct evidence exists regarding the deformation of airways in vivo during mechanical ventilation, or in the presence of positive end-expiratory pressure (PEEP)., Objectives: To measure airway strain and to estimate airway wall tension during mechanical ventilation in an intact animal model., Methods: Sprague-Dawley rats were anesthetized and mechanically ventilated with tidal volumes of 6, 12, and 25 cm(3)/kg with and without 10-cm H(2)O PEEP. Real-time tantalum bronchograms were obtained for each condition, using microfocal X-ray imaging. Images were used to calculate circumferential and longitudinal airway strains, and on the basis of a simplified mathematical model we estimated airway wall tensions., Measurements and Main Results: Circumferential and longitudinal airway strains increased with increasing tidal volume. Levels of mechanical strain were heterogeneous throughout the bronchial tree. Circumferential strains were higher in smaller airways (less than 800 mum). Airway size did not influence longitudinal strain. When PEEP was applied, wall tensions increased more rapidly than did strain levels, suggesting that a "strain limit" had been reached. Airway collapse was not observed under any experimental condition., Conclusions: Mechanical ventilation results in significant airway mechanical strain that is heterogeneously distributed in the uninjured lung. The magnitude of circumferential but not axial strain varies with airway diameter. Airways exhibit a "strain limit" above which an abrupt dramatic rise in wall tension is observed.

Little is known about the constituent hemodynamic consequences of structural changes that occur in the pulmonary arteries during the onset and progression of pulmonary arterial remodeling. Many disease processes are known to be responsible for vascular remodeling that leads to pulmonary arterial hypertension, cor pulmonale, and death. Histology has been the primary tool for evaluating pulmonary remodeling, but it does not provide information on intact vascular structure or the vessel mechanical properties. This study is an extension of our previous work in which we developed an alternative imaging technique to evaluate pulmonary arterial structure. The lungs from Sprague-Dawley rats were removed, perfusion analysis was performed on the isolated lungs, and then an X-ray contrast agent was used to fill the arterial network for imaging. The lungs were scanned over a range of intravascular pressures by volumetric micro-computed tomography, and the arterial morphometry was mapped and measured in the reconstructed isotropic volumes. A quantitative assessment of hemodynamic, structural, and biomechanical differences between rats exposed for 21 days to hypoxia (10% O(2)) or normoxia (21.0% O(2)) was performed. One metric, the normalized distensibility of the arteries, is significantly (P < 0.001) larger [0.025 +/- 0.0011 (SE) mmHg(-1)] (n = 9) in normoxic rats compared with hypoxic [0.015 +/- 0.00077 (SE) mmHg(-1)] (n = 9). The results of the study show that these models can be applied to the Sprague-Dawley rat data and, specifically, can be used to differentiate between the hypoxic and the control groups.

Rationale and Objectives: Micro computed tomography is an important tool for small animal imaging. On many occasions, it is desirable to image lungs in a live instead of postmortem small animal to perform a pulmonary physiology study. Because the lungs are moving, gating with respect to the ventilatory phase has to be performed to reduce motion artifacts. Precapture ventilation gating may be difficult to achieve in some situations, which motivates us to propose and implement a simple postacquisition gating method., Materials and Methods: Rats were used as the subjects in this study. A sequence of low-dose projection images were acquired at 30 frames per second for each view angle. During each capture sequence the rat undergoes multiple ventilation cycles. Using the sequence of projection images, an automated region of interest algorithm, based on integrated grayscale intensity, tracts the ventilatory phase of the lungs. In the processing of an image sequence, multiple projection images are identified at a particular phase and averaged to improve the signal-to-noise ratio. The resulting averaged projection images from different view angles are input to a Feldkamp cone-beam algorithm reconstruction algorithm to obtain isotropic image volumes., Results: Reconstructions with reduced movement artifacts are obtained. In the gated reconstruction, registration of the bone is much better, the edge of the lung is clearly defined, and structures within the lung parenchyma are better resolved. Also, different phases of a breathing cycle can be reconstructed from one single tomographic scan by the proposed gating method., Conclusion: A postacquisition gating method using the phase information encoded in the 2-dimensional cone beam projections is proposed. This method is simple to implement and does not require additional experimental set-up to monitor the respiration. It may find applications in lung tumor detection, dynamic pulmonary physiology studies, and the respiratory systems modeling. Minimal motion artifact data sets improve qualitative and quantitative analysis techniques that are useful in physiologic studies of pulmonary structure and function.

Liquid can be instilled into the pulmonary airways during medical procedures such as surfactant replacement therapy, partial liquid ventilation, and pulmonary drug delivery. For all cases, understanding the dynamics of liquid distribution in the lung will increase the efficacy of treatment. A recently developed imaging technique for the study of real-time liquid transport dynamics in the pulmonary airways was used to investigate the effect of respiratory rate on the distribution of an instilled liquid, surfactant, in a rat lung. Twelve excised rat lungs were suspended vertically, and a single bolus (0.05 ml) of exogenous surfactant (Survanta, Ross Laboratories, Columbus, OH) mixed with radiopaque tracer was instilled as a plug into the trachea. The lungs were ventilated with a 4-ml tidal volume for 20 breaths at one of two respiratory rates: 20 or 60 breaths/min. The motion of radiodense surfactant was imaged at 30 frames/s with a microfocal X-ray source and an image intensifier. Dynamics of surfactant distribution were quantified for each image by use of distribution statistics and a homogeneity index. We found that the liquid distribution depended on the time to liquid plug rupture, which depends on ventilation rate. At 20 breaths/min, liquid was localized in the gravity-dependent region of the lung. At 60 breaths/min, the liquid coated the airways, providing a more vertically uniform liquid distribution.

Introduction: The murine model is a well-established surrogate for studying human cranial suture biology. In mice, all sutures with the exception of the posterior frontal (PF) suture remain patent throughout life. Histology is regarded as the gold standard for analyzing sutures. On this basis, PF suture fusion begins on day of life 25 and is complete by day 45. Cranial suture histology, however, requires sacrifice of the animal to obtain tissue for analysis. As a result, knowledge of the kinetics of cranial suture fusion is based on a patchwork analysis of many sutures from many different animals. The behavior of a single suture through time is unknown. Our goal is to develop a noninvasive means to repeatedly image mouse cranial sutures in vivo. As a first step, the present study was performed to evaluate microfocal computer tomography (micro-CT) technology for the use of capturing images of a mouse cranium in situ., Methods: The micro-CT system consists of a microfocal X-ray source and a large format CCD camera optically coupled to a high-resolution X-ray image intensifier, digitally linked to a computer. The PF and sagittal sutures lie in continuity along the midline of the skull. Holes were drilled in the calvaria on both sides of the PF and sagittal sutures of a 45-day-old euthanized mouse. A micro-CT scan of this animal was performed and hundreds of cross-sectional images were generated for the cranium. These images were used to reconstruct three-dimensional volumetric images of the entire cranium. Comparisons were made between (1). the gross specimen and the three dimensional reconstructions; (2). two-dimensional coronal images obtained by micro-CT and those obtained by histology., Results: Analysis of PF and sagittal sutures demonstrated the following: (1). The drilled holes were accurately rendered by micro-CT, when compared to both the gross specimen and the histology. (2). The sagittal suture was found to be patent by both micro-CT and histology. (3). The PF suture is fused by histology, but unexpectedly, the PF suture appears incompletely fused by micro-CT. By micro-CT, however, the anterior and endocranial regions appear more extensively fused than the remainder of the PF suture, a finding consistent with published histologic analysis., Conclusions: We successfully imaged 45-day-old mouse cranial sutures in situ using micro-CT technology. Precise correlation between histologic sections and radiologic images is difficult, but convincing similarities exist between the gross specimen and images from micro-CT and histology. PF suture fusion in a 45-day-old animal appears different by micro-CT than by histology. One possible explanation for this apparent discrepancy is that suture fusion in histology is determined based on the appearance of bone morphology and not tissue density, as the specimens are necessarily decalcified to section the bone. Micro-CT, on the other hand, distinguishes tissues on the basis of density. Newly forming bone may require bone matrix formation prior to complete calcification; PF suture in 45-day-old mice may be morphologically complete but incompletely ossified. Studies correlating histologic and micro-CT assessment of suture development are underway. Micro-CT appears to be a promising method for noninvasive imaging of mouse cranial suture.

The lungs can substantially influence the redox status of redox-active plasma constituents. Our objective was to examine aspects of the kinetics and mechanisms that determine pulmonary disposition of redox-active compounds during passage through the pulmonary circulation. Experiments were carried out on rat and mouse lungs with 2,3,5,6-tetramethyl-1,4-benzoquinone [duroquinone (DQ)] as a model amphipathic quinone reductase substrate. We measured DQ and durohydroquinone (DQH2) concentrations in the lung venous effluent after injecting, or while infusing, DQ or DQH2 into the pulmonary arterial inflow. The maximum net rates of DQ reduction to DQH2 in the rat and mouse lungs were approximately 4.9 and 2.5 micromol. min(-1).g dry lung wt(-1), respectively. The net rate was apparently the result of freely permeating access of DQ and DQH2 to tissue sites of redox reactions, dominated by dicumarol-sensitive DQ reduction to DQH2 and cyanide-sensitive DQH2 reoxidation back to DQ. The dicumarol sensitivity along with immunodetectable expression of NAD(P)H-quinone oxidoreductase 1 (NQO1) in the rat lung tissue suggest cytoplasmic NQO1 as the dominant site of DQ reduction. The effect of cyanide on DQH2 oxidation suggests that the dominant site of oxidation is complex III of the mitochondrial electron transport chain. If one envisions DQ as a model compound for examining the disposition of amphipathic NQO1 substrates in the lungs, the results are consistent with a role for lung NQO1 in determining the redox status of such compounds in the circulation. For DQ, the effect is conversion of a redox-cycling, oxygen-activating quinone into a stable hydroquinone.

Inhaled nitric oxide (iNO) is used as a selective pulmonary vasodilator, and often under conditions when a high fraction of inspired oxygen is indicated. However, little is known about the potential toxicity of iNO therapy with or without concomitant oxygen therapy. NO can combine with superoxide (O2-) to form peroxynitrite (ONOO-), which can in turn decompose to form hydroxyl radical (OH.). Both OH. and ONOO- are involved in various forms of lung injury. To begin evaluation of the effect of iNO under either normoxic or hyperoxic conditions on OH. and/or ONOO- formation, rats were exposed for 58 h to either 21% O2, 21% O2 + 10 parts per million (ppm) NO, 21% O2 + 100 ppm NO, 50% O2, 90% O2, 90% O2 + 10 ppm NO, or 90% O2 + 100 ppm NO. We used a salicylate hydroxylation assay to detect the effects of these exposures on lung OH. and/or ONOO- formation measured as the appearance of 2,3-dihydroxybenzoic acid (2,3-DHBA). Exposure to 90% O2 and 90% O2 + 100 ppm NO resulted in significantly (p < 0.05) greater lung wet weight (1.99 +/- 0.14 g and 3.14 +/- 0.30 g, respectively) compared with 21% O2 (1.23 +/- 0.01 g). Exposure to 21% O2 + 100 ppm NO led to 2.5 times the control (21% O2 alone) 2,3 DHBA formation (p < 0.05) and exposure to 90% O2 led to 2.4 times the control 2,3-DHBA formation (p < 0.05). However, with exposure to both 90% O2 and 100 ppm NO, the 2,3-DHBA formation was no greater than the control condition (21% O2). Thus, these results indicate that, individually, both the hyperoxia and the 100 ppm NO led to greater salicylate hydroxylation, but that the combination of hyperoxia and 100 ppm NO led to less salicylate hydroxylation than either did individually. The production of OH. and/or ONOO- in the lung during iNO therapy may depend on the ratio of NO to O2.