Dermonecrosis caused by a spitting cobra snakebite results from toxin potentiation and is prevented by the repurposed drug varespladib.

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 A2 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 A2-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.

Refining an ecological momentary assessment study of binge eating among sexual minority and heterosexual young women: a mixed methods pilot study.

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.

Snakebite drug discovery: high-throughput screening to identify novel snake venom metalloproteinase toxin inhibitors.

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 EC50s 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.

Repurposed drugs and their combinations prevent morbidity-inducing dermonecrosis caused by diverse cytotoxic snake venoms.

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.

Machine-learning guided Venom Induced Dermonecrosis Analysis tooL: VIDAL.

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.

Recruiting Cisgender Female Couples for Health Disparity-Focused Daily Diary Research: Challenges, Successes, and Lessons Learned.

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.

Rationale and Design of an Ecological Momentary Assessment Study Examining Predictors of Binge Eating Among Sexual Minority and Heterosexual Young Women: Protocol for the Health and Experiences in Real Life (HER Life) Study.

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.

Associations between body dissatisfaction and relationship functioning among same-sex female couples: An actor-partner interdependence model.

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).

Virus-like particles displaying conserved toxin epitopes stimulate polyspecific, murine antibody responses capable of snake venom recognition.

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 A2 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-PLA2 IgG from three animals against the PLA2 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.

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms.

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.

Proteomics and histological assessment of an organotypic model of human skin following exposure to Naja nigricollis venom.

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.

Evaluating study procedure training methods for a remote daily diary study of sexual minority women.

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.

Sexual Minority Stress and Social Support Explain the Association between Sexual Identity with Physical and Mental Health Problems among Young Lesbian and Bisexual Women.

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.

A therapeutic combination of two small molecule toxin inhibitors provides broad preclinical efficacy against viper snakebite.

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.

Rationale and Design of a Remote Web-Based Daily Diary Study Examining Sexual Minority Stress, Relationship Factors, and Alcohol Use in Same-Sex Female Couples Across the United States: Study Protocol of Project Relate.

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.