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Chatbot use is increasing for mobile health interventions on sensitive and stigmatized topics like mental health because of their anonymity and privacy. This anonymity provides acceptability to sexual and gendered minority youth (ages 16-24) at increased risk of HIV and other STIs with poor mental health due to higher levels of stigma, discrimination, and social isolation. This study evaluates the usability of Tabatha-YYC, a pilot chatbot navigator created to link these youth to mental health resources. Tabatha-YYC was developed using a Youth Advisory Board (n=7), and the final design underwent user testing (n=20) through a think-aloud protocol, semi-structured interview, and a brief survey post-exposure which included the Health Information Technology Usability Evaluation Scale. The chatbot was found to be an acceptable mental health navigator by participants. This study provides important design methodology considerations and key insights into chatbot design preferences of youth at risk of STIs seeking mental health resources.

BACKGROUND Suicide attempts and suicide death disproportionately affect sexual and gender minority emerging adults (ages 18-24). However, suicide prevention strategies tailored for emerging adult sexual and gender minorities (EA-SGM) are not widely available. OBJECTIVE We will pilot test the combination of peer mentoring alongside an app-based intervention (Supporting Transitions to Adulthood and Reducing Suicide; STARS) designed to reduce suicidal ideation and behaviors. STARS will include suicide prevention content and will target positive affect, discrimination, and social support. After an in-person Safety Planning Intervention (SPI) with a clinician, STARS users can access content and activities to increase their intentions to use the SPI and overcome obstacles to its use. METHODS Guided by the RE-AIM framework, we will recruit and enroll a racially/ethnically diverse sample of 60 EA-SGM reporting past-month suicidal ideation. Using a Type 1 Effectiveness-Implementation Hybrid Design, participants will be randomized to receive the SP intervention (Control arm) or to receive the SP intervention alongside STARS (Intervention arm). We will follow participants for six months, with evaluations at 2, 4, and 6 months. Preliminary effectiveness outcomes (suicidal ideation and behavior) and hypothesized mechanisms of change (positive affect, coping with discrimination, and social support) will serve as our primary outcomes. Secondary outcomes include key implementation indicators, including participants’ willingness and adoption of SPI and STARS, and staff’s experiences delivering the program. RESULTS Research activities began in September 2021 and are ongoing. The University of Pennsylvania is the central institutional review board for this study (protocol #849500). Study recruitment began on October 14, 2022. CONCLUSIONS This project will be among the first tailored, mobile-based interventions for EA-SGM at risk for suicide. This project is responsive to documented gaps for this population: 1) approaches that address chosen family; 2) focus on a life-course perspective; 3) online approaches; and 4) focus on health equity and provision of additional services relevant to sexual minority youth. CLINICALTRIAL ClinicalTrials.gov NCT05018143

Cases of new-onset pernio and recurrences in our cohort align tightly with trends in mean 7-day COVID-19 positivity in Wisconsin and mean temperature in Madison, Wisconsin by month.

BACKGROUND Behavioral health provider supply has not kept pace with demand. Simply training more professionals will not be enough to address the strain on the U.S. behavioral healthcare workforce and exacerbated behavioral health challenges among U.S. adults due to COVID-19. OBJECTIVE Our objective was to pilot test a mental health chatbot designed to screen users for psychological distress and refer to resources. METHODS Data were collected via a national, cross-sectional, internet-based survey of U.S. adults. Measures included demographics, symptoms, stigma, technology acceptance, willingness to use the chatbot, and chatbot acceptability. Relationships between these variables were explored using chi-square tests, correlations, and logistic regression. RESULTS Of 222 participants, 75.7% completed mental health screening within the chatbot. Participants found the chatbot to be acceptable. Demographic predictors of chatbot use included being White or Black/African American, identifying as Hispanic/Latino, having dependents, having insurance coverage, having used mental health services in the past, having a diagnosed mental health condition, and reporting current distress. Logistic regression produced a significant model with perceived usefulness and symptoms as significant positive predictors of chatbot use for the overall sample, and label avoidance as the only significant predictor of chatbot use for those currently experiencing distress. CONCLUSIONS Chatbot technology may be a feasible and acceptable way to screen large numbers of people for psychological distress and disseminate mental health resources. Since label avoidance was identified as the single significant predictor of chatbot use among currently distressed individuals, chatbot technology may be one way to circumnavigate stigma as a barrier to engagement in behavioral health care.

The health care transition (HCT) from pediatric to adult care is pivotal for childhood cancer survivors (CCS) and their parents. However, there is little research examining parental needs during HCT, despite this being a key predictor of successful HCT. The goal of this study was to investigate the needs of parents of CCS during HCT. Using an integrative review of the literature structured around the social-ecological model (SEM) of CCS transition readiness yielded 454 articles, including three hand-searched articles. Six articles were included in the final analysis. Data were extracted into nine factors derived from SEM. Articles were published within the last decade, largely qualitative, and mainly examined parents and CCS together. Parents most frequently mentioned relationships with their practitioner and CCS as contributing to HCT readiness, while abstract factors, such as goal-setting and expectations around HCT, were not mentioned. Our results are limited by the dearth of research on this topic, the homogeneity of samples, and joint presentation of CCS and parent data. Nonetheless, our results indicate that parents do not weigh all aspects of SEM equally, with macrolevel barriers, such as sociodemographic factors being viewed as less salient for HCT readiness. Parents mostly focused on interpersonal factors, such as their relationships with practitioners and CCS, indicating that practitioners should emphasize these in preparing parents for HCT.

Objectives To pilot test a mental health chatbot designed to screen users for psychological distress and refer to resources, addressing the strain on the U.S. behavioral healthcare workforce and exacerbated behavioral health challenges among U.S. adults due to COVID-19. Methods Data were collected via a national, cross-sectional, internet-based survey of U.S. adults. Measures included demographics, symptoms, stigma, technology acceptance, willingness to use the chatbot, and chatbot acceptability. Relationships between these variables were explored using chi-square tests, correlations, and logistic regression. Results Of 222 participants, 75.7% completed mental health screening within the chatbot. Participants found the chatbot to be acceptable. Demographic predictors of chatbot use included being White or Black/African American, identifying as Hispanic/Latino, having dependents, having insurance coverage, having used mental health services in the past, having a diagnosed mental health condition, and reporting current distress. Logistic regression produced a significant model with perceived usefulness and symptoms as significant positive predictors of chatbot use for the overall sample, and label avoidance as the only significant predictor of chatbot use for those currently experiencing distress. Discussion Chatbot technology may be a feasible and acceptable way to screen large numbers of people for psychological distress and disseminate mental health resources. Since label avoidance was identified as the single significant predictor of chatbot use among currently distressed individuals, chatbot technology may be one way to circumnavigate stigma as a barrier to engagement in behavioral health care. Limitations Recruitment through Amazon’s mTurk limits generalizability of our findings, and chi-square test effect sizes were small.

The Pinellas County Empowerment Team (PCET) was an adapted assertive community treatment (ACT) program created to meet the needs of Pinellas County residents with serious behavioral health concerns and high frequency of hospitalization (medical and psychiatric) and incarceration. Recent research demonstrates that individuals participating in ACT programs can transition to lower-intensity services. To understand the needs and barriers in transitioning PCET clients to lower-intensity services and the unique experiences during the coronavirus (COVID-19) pandemic, the researchers conducted a qualitative evaluation which includes a case record review and in-depth interviews with clients of PCET and staff members. Our findings indicated several barriers to transitioning PCET clients, including a lack of sufficient behavioral health support outside the ACT program and some clients' concerns regarding their abilities once out of the program.

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities in relevant pathogens. The Gram-negative pathogen,Acinetobacter baumannii, poses an urgent threat by evading antibiotic treatment through both intrinsic and acquired mechanisms. Antibiotics kill bacteria by targeting essential gene products, but antibiotic-essential gene interactions have not been studied systematically inA. baumannii. Here, we use CRISPR interference (CRISPRi) to comprehensively phenotypeA. baumanniiessential genes. We show that certain essential genes are acutely sensitive to knockdown, providing a set of promising therapeutic targets. Screening our CRISPRi library against last-resort antibiotics revealed essential pathways that modulate beta-lactam resistance, an unexpected link between NADH dehydrogenase function and polymyxin killing, and the genetic basis for synergy between polymyxins and rifamycins. Our results demonstrate the power of systematic genetic approaches to identify weaknesses in Gram-negative pathogens and uncover antibiotic mechanisms that better inform combination therapies. Significance Acinetobacter baumanniiis a hospital-acquired pathogen that is resistant to common antibiotic treatments.A. baumanniiinfections, we need to identify promising therapeutic targets and effective antibiotic combinations. Here, we characterize genes critical forA. baumanniiviability and their interactions with antibiotics. We find that genes involved in proton gradient formation required for oxygen-dependent energy generation are central toA. baumanniiphysiology and represent appealing drug targets. We show that polymyxins interact with proton gradient genes, explaining how these antibiotics inhibit growth at sub-lethal concentrations and their efficacy in combination therapies. Our studies reveal antibiotic-gene interactions inA. baumanniithat can inform future therapies.

[Image: see text] Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD(60)). In rabbits challenged with 200 LD(50) of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2 000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.

Mosaic KRAS variants and other RASopathy genes cause oculoectodermal, encephalo-cranio-cutaneous lipomatosis, and Schimmelpenning-Feuerstein-Mims syndromes, and a spectrum of vascular malformations, overgrowth and other associated anomalies, the latter of which are only recently being characterized. We describe eight individuals in total (six unreported cases and two previously reported cases) with somatic KRAS variants and variably associated features. Given the findings of somatic overgrowth (in seven individuals) and vascular or lymphatic malformations (in eight individuals), we suggest mosaic RASopathies (mosaic KRAS variants) be considered in the differential diagnosis for individuals presenting with asymmetric overgrowth and lymphatic or vascular anomalies. We expand the association with embryonal tumors, including the third report of embryonal rhabdomyosarcoma, as well as novel findings of Wilms tumor and nephroblastomatosis in two individuals. Rare or novel findings in our series include the presence of epilepsy, polycystic kidneys, and T-cell deficiency in one individual, and multifocal lytic bone lesions in two individuals. Finally, we describe the first use of targeted therapy with a MEK inhibitor for an individual with a mosaic KRAS variant. The purposes of this report are to expand the phenotypic spectrum of mosaic KRAS-related disorders, and to propose possible mechanisms of pathogenesis, and surveillance of its associated findings.

Port-wine birthmarks (PWBs) are progressive vascular malformations with significant disfigurement and psychosocial morbidity; early light-based treatment has shown improved outcomes in the pediatric population. Somatic mosaic mutations underly the progressive nature of PWBs and explain the significant differences in response and heterogeneity of vessel architecture in the pediatric population when compared to the adult cohort. Here, we summarize a review of pediatric specific literature on the various light-based treatment modalities, including pulsed dye laser, near-infrared lasers, and intense pulsed light, providing the various indications, tips, advantages, and disadvantages for the pediatric dermatologist.

Molecular photochemistry is an integral field in chemistry. Despite this, it is often difficult to demonstrate an effective hands-on experiment that offers a unique opportunity to students to obser...

This qualitative study investigated the social class worldviews of Chinese International Students (CIS) in the United States. Social class worldviews are a constellation of beliefs, attitudes, and values that individuals use to maintain a sense of psychological equilibrium within their perceived social class groups and culture. Having interviewed eleven CIS in aMidwestern city in the United States, the researchers found that their experiences reflected five themes: (a) Social Class Consciousness; (b) Social Class Socialization; (c) Social Class Resources; (d) Social Class Values; and (e) Social Class Challenges. Suggestions for research and implications for higher education are discussed.

Metastatic prostate cancer (PCa) is the leading cause of PCa-associated deaths and one of the leading causes of cancer mortality in men. Novel therapeutics, such as Prostate Specific Membrane Antigen (PSMA) ligand therapy, are emerging as effective treatment strategies for metastatic prostate cancer. However, neuroendocrine differentiated (NED) metastatic PCa, which lacks the PSMA antigen, is deprived of effective biomarkers for disease treatment. Magmas, a 13.8 kDa mitochondrial protein, imports nuclear-encoded mitochondrial proteins into the mitochondrial matrix. Magmas is overexpressed in several aggressive tumors, such as breast cancer, glioblastoma, and prostate cancer. When overexpressed, Magmas plays a role as a cytoprotective protein by acting as a reactive oxygen species (ROS) scavenger that maintains sufficient ROS levels to promote cell proliferation, but below the threshold levels that could lead to apoptosis. This study was designed to investigate the pro-survival and therapy-resistance functions of Magmas in PCa cells expressing NED markers. Predictively, we observed that the docetaxel (DTX)-resistant PC3-DR and DU145-DR PCa cell lines expressed significantly higher levels of Magmas compared to their drug-sensitive parental cell lines. We hypothesized that inhibiting Magmas reduces the aggressive properties of highly proliferative DTX- resistant metastatic PCa cell lines and re-sensitize them to DTX. Magmas knockdown in DTX-sensitive and DTX-resistant PC3 and DU145 cells in the presence of increasing DTX treatments enhanced sensitivity to DTX. Consistent with these results, pharmacological inhibition of Magmas with the novel BT#9 inhibitor in combination with increasing DTX concentrations also enhanced sensitivity to DTX in these cell lines. Additional studies using clonogenic assays, cell migration/invasion assays, and tumorsphere formation assays are currently underway to further establish Magmas as a potential therapeutic target to attenuate the aggressive properties of chemoresistant PCa cells. Lastly, we will investigate the diagnostic potential of Magmas as a biomarker for NED and BT#9 as an imaging/therapeutic-specific agent for NED. Citation Format: Alfonso M. Durán, Christian H. Yoo, Jennifer D. Tran, Krystal Santiago, Adil S. Mohammed, Carlos A. Casiano, Frankis A. Almaguel. Targeting the mitochondrial protein MAGMAS increases sensitivity to docetaxel in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1754.

Despite thousands of reported patients with pandemic-associated pernio, low rates of seroconversion and PCR positivity have defied causative linkage to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pernio in uninfected children is associated with monogenic disorders of excessive IFN-1 immunity, whereas severe COVID-19 pneumonia can result from insufficient IFN-1. Moreover, SARS-CoV-2 spike protein and robust IFN-1 response are seen in the skin of patients with pandemic-associated pernio, suggesting an excessive innate immune skin response to SARS-CoV-2. Understanding the pathophysiology of this phenomenon may elucidate the host mechanisms that drive a resilient immune response to SARS-CoV-2 and could produce relevant therapeutic targets.

Breast cancer is the most common malignancy in women. Young African American (AA) women are affected by a higher incidence and mortality rate of triple-negative breast cancer (TNBC) compared to Non-Hispanic White (NHW) women. Currently, available treatment options are suboptimal, with a 5-year survival of less than 12% for metastatic TNBC. Our laboratory demonstrated that IGF-2 plays a pivotal role in the tumor aggressiveness of AA women. Moreover, tissues from AA women express high levels of IGF-2 and exhibit high activation of IGF-2 signaling pathways associated with tumorgenesis and chemoresistance. An emerging pathway linked to IGF-2 mediated chemoresistance and cell survival in TNBC is the regulation of mitochondrial and survival proteins. Mitochondrial Magmas is a mitochondrial protein found in all eukaryotic cells that is required for cell viability. It functions to transport nuclear proteins from the inner mitochondrial membrane to the mitochondrial matrix. Magmas can buffer increased reactive oxidative species (ROS) and promote survival of cancer cells. Overexpression of Magmas reduced ROS and enhanced cellular tolerance to oxidative stress; however, its downregulation raised cellular ROS and made cells more vulnerable to ROS-mediated death. Given the role of IGF-2 in regulating mitochondrial proteins, we investigated if IGF-2 regulated Magmas. We hypothesize that IGF-2 increases Magmas, and since AA women express higher levels of IGF-2, they will express higher Magmas levels. We used the CRL-2335 AA TNBC cell line and an IGF-2 knockdown of those same cells. Western blotting was used to detect the specific isoforms of IGF-2 and Magmas from cell lysates. Immunohistochemistry (IHC) characterized Magmas expression in paired, normal-tumor breast cancer tissues. Real time PCR was used to evaluate the expression of IGF2 and MAGMAS. The preliminary results showed that IGF-2 knockdown in CRL-2335 AA TNBC cells significantly decreased Magmas protein expression and its corresponding mRNA. In addition, tissue analysis demonstrated high levels of Magmas staining in TNBC tissue while little or no staining was detected in the normal tissues. Furthermore, tumor tissues of AA women showed significantly higher expression of Magmas as compared to tissues from NHW women. Targeting Magmas represents a novel and much needed tool to treat TNBC. Our team is currently validating Magmas as a target for Theranostics treatment, a promising emerging radiotherapy for TNBC patients. Citation Format: Jennifer D. Tran, Alfonso D. Duran, Qian-Wei Tan, Frankis G. Almaguel, Daisy De León. IGF-II regulates a critical mitochondrial protein, Magmas, with differential expression between African American and Non-Hispanic White women with TNBC [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C064.

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

LGBTQ+ individuals experience stigma due to their sexual orientation and/or gender identity which has clear, profound, far-ranging effects on LGBTQ+ health including greater risk of suicidality, depression, anxiety, substance use, and poor physical health. Several stigma change strategies have been used to attempt to mitigate the profound impact of LGBTQ+ related stigma in marginalized sexual and gender minority communities. Contact-based interventions to address LGBTQ+ related stigma rest on contact theory, which posits that intergroup contact between individuals of an in-group (majority group) and individuals of an out-group (minority or marginalized group) aids in the reduction of negative stereotypes, prejudice, and resulting discrimination. The definition of “contact” has progressed to encompass various forms of exposure including in-vivo interpersonal contact, vicarious contact, electronic or e-contact and imagined contact. Guided by the Cochrane Handbook for Systematic Reviews of Interventions, we summarize the literature between 2001 and 2020, examining and synthesizing 20 studies that explore contact-based interventions to reduce LGBTQ+ related stigma, stereotyping, prejudice, and discrimination. Overall, several forms of contact-based interventions have demonstrated positive effects on reducing LGBTQ+ related stigma. Future research should include examining the use of contact-based interventions in reducing the stigma faced by individuals within LGBTQ+ communities with intersecting marginalized identities.

To describe adolescents' experience with treatment adherence when prescribed antibiotics for sexually transmitted infections (STIs) in emergency departments (ED).Adolescents diagnosed with STIs in two EDs and prescribed outpatient treatment were interviewed to identify barriers and facilitators to treatment adherence.Of 26 participants, 46.2% filled their prescriptions, 38.5% returned to the ED for treatment, 7.7% received treatment elsewhere, and 7.7% received no treatment. All adolescents who filled their prescriptions notified caregivers of their diagnosis, compared with 50.0% who returned to the ED (P = 0.01). Adolescents identified cost, transportation, lack of insurance card, and lack of knowledge as perceived barriers to treatment adherence. Less than half of the participants were interested in school-based health clinics due to anonymity concerns. Most expressed interest in using mobile health (mHealth) to overcome adherence challenges.Adolescents identified multiple barriers to treatment adherence. Future work should explore the role of caregiver communication and incorporation of mHealth.

BackgroundChronic pain affects approximately 50 million adults in the United States and impacts mood, everyday functioning, and quality of life. The challenges of analgesic clinical trials and, therefore, the approval of new non-opioid analgesics, are based in part on a fundamental lack of understanding of those outcomes that are relevant to an individual’s overall functioning.ObjectivesTo determine the behaviors and health outcomes associated with chronic pain. This manuscript presents an overview of the study design, baseline health and behavioral characteristics of our sample, and preliminary findings of how behavioral characteristics differ between individuals with and without chronic pain.MethodsThe study is a decentralized digital longitudinal cohort study of 10,036 individuals (5,832 with chronic pain [CP] and 4,204 with no chronic pain [NCP]), age 18 years or older, living in the United States. The study period was one year. Data were collected from wearable activity trackers and health or fitness mobile applications to capture passively collected behavioral data including steps, sleep, and heart rate. Patient-reported outcomes on mood and pain, including the BPI-SF, PHQ-9, and GAD-7, were collected at various timepoints during the study.ResultsThe data suggest greater levels of depression and anxiety, lower quality of life, less physical activity, more variable sleep, and higher resting heart rate are associated with CP.ConclusionsThe longitudinal data from the larger study will yield substantial contributions to the body of literature in chronic pain, particularly in delineating relational and causal factors relevant to the impact of chronic pain, and potential development of a digital biomarker to assess and monitor patients’ everyday experience with chronic pain.

Summary We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities., Graphical abstract, Highlights • Cities possess a consistent “core” set of non-human microbes • Urban microbiomes echo important features of cities and city-life • Antimicrobial resistance genes are widespread in cities • Cities contain many novel bacterial and viral species, This systematic, worldwide catalog of urban microbiomes represents a metagenomic atlas important for understanding the ecology, virulence, and antibiotic resistance of city-specific microbial communities.

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICEBs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3: Tn7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4: ICEBs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1: Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2: Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3: Transformation of E. coli by electroporation Support Protocol 4: Transformation of CaCl2‐competent E. coli

Leviviruses are bacteriophages with small single-stranded RNA genomes consisting of 3-4 genes, one of which (sgl) encodes a protein that induces the host to undergo autolysis and liberate progeny virions. Recent meta-transcriptomic studies have uncovered thousands of leviviral genomes, but most of these lack an annotated sgl, mainly due to the small size, lack of sequence similarity, and embedded nature of these genes. Here, we identify sgl genes in 244 leviviral genomes and functionally characterize them in Escherichia coli. We show that leviviruses readily evolve sgl genes and sometimes have more than one per genome. Moreover, these genes share little to no similarity with each other or to previously known sgl genes, thus representing a rich source for potential protein antibiotics., Leviviruses are phages with ssRNA genomes that encode a protein (Sgl) that induces host autolysis by interfering with bacterial cell wall synthesis. Identification of sgl genes is complicated by their small size and lack of sequence similarity. Here, Chamakura et al. use bioinformatic and experimental approaches to identify sgl genes in 244 leviviral genomes.

Background Depression and anxiety greatly impact daily behaviors, such as sleep and activity levels. With the increasing use of activity tracking wearables among the general population, there has been a growing interest in how data collected from these devices can be used to further understand the severity and progression of mental health conditions. Objective This virtual 1-year observational study was designed with the objective of creating a longitudinal data set combining self-reported health outcomes, health care utilization, and quality of life data with activity tracker and app-based behavioral data for individuals with depression and anxiety. We provide an overview of the study design, report on baseline health and behavioral characteristics of the study population, and provide initial insights into how behavioral characteristics differ between groups of individuals with varying levels of disease severity. Methods Individuals who were existing members of an online health community (Achievement, Evidation Health Inc) and were 18 years or older who had self-reported a diagnosis of depression or anxiety were eligible to enroll in this virtual 1-year study. Participants agreed to connect wearable activity trackers that captured data related to physical activity and sleep behavior. Mental health outcomes such as the Patient Health Questionnaire (PHQ-9), the Generalized Anxiety Disorder Questionnaire (GAD-7), mental health hospitalizations, and medication use were captured with surveys completed at baseline and months 3, 6, 9, and 12. In this analysis, we report on baseline characteristics of the sample, including mental health disease severity and health care utilization. Additionally, we explore the relationship between passively collected behavioral data and baseline mental health status and health care utilization. Results Of the 1304 participants enrolled in the study, 1277 individuals completed the baseline survey and 1068 individuals had sufficient activity tracker data. Mean age was 33 (SD 9) years, and the majority of the study population was female (77.2%, 994/1288) and identified as Caucasian (88.3%, 1137/1288). At baseline, 94.8% (1211/1277) of study participants reported experiencing depression or anxiety symptoms in the last year. This baseline analysis found that some passively tracked behavioral traits are associated with more severe forms of anxiety or depression. Individuals with depressive symptoms were less active than those with minimal depressive symptoms. Severe forms of depression were also significantly associated with inconsistent sleep patterns and more disordered sleep. Conclusions These initial findings suggest that longitudinal behavioral and health outcomes data may be useful for developing digital measures of health for mental health symptom severity and progression.

Purpose Transforming growth factor-beta (TGF-β) isoform 1 (T1) is involved in corneal fibrotic wound healing by stimulating myofibroblast transformation and altering fibrotic gene expression. In this study, two specific inhibitors were used to dissect the relationship between myofibroblast generation and the TGF-β/Smad- or TGF-β/p38-signaling pathway in human corneal fibroblasts (HCF). Methods In HCF, Trx-SARA (Smad-pathway inhibitor) was used to block the TGF-β/Smad-signaling pathway, and the p38 inhibitor (p38inh, SB202190) was used to inhibit p38MAPK, thus blocking the TGF-β/p38-signaling pathway. HCF ± Trx-SARA or Trx-GA (SARA control) were serum starved overnight in Eagle's minimum essential medium (EMEM) ± p38inh, grown in EMEM ± T1 ± p38inh for 24 hours, and then processed for indirect-immunofluorescence, Western blot, or quantitative real-time polymerase chain reaction to examine α-smooth muscle actin (αSMA) and other fibrotic genes, such as fibronectin, thrombospondin1, and type III collagen. In addition, the morphology and the effect of p38inh on myofibroblast phenotype after myofibroblast formation were examined. Results We observed that Trx-SARA had little effect on αSMA expression, indicating that blocking the Smad pathway did not significantly inhibit myofibroblast formation. However, p38inh did significantly inhibit αSMA and other fibrotic genes, thus efficiently preventing the transition of HCFs to myofibroblasts. In addition, morphology changed and αSMA decreased in myofibroblasts exposed to p38inh medium, as compared with controls. Conclusions HCF transition to myofibroblasts was mainly through the p38 pathway. Therefore, blocking the p38 pathway may be a potential therapeutic tool for human corneal fibrosis prevention/treatment, because it controls myofibroblast formation in human corneal cells, while leaving other functions of T1 unaffected.

Coincident with the start of the COVID-19 pandemic, dermatologists worldwide have reported an uncharacteristic increase in pernio or chilblains (aka ‘COVID toes’). However, the lack of systemic illness, low PCR positivity and lack of consistent seroconversion have led some authors to postulate an epiphenomenon. SARS-CoV-2 spike protein has been identified in a limited number of skin biopsies in few publications, yet there remain conflicting reports regarding other SARS-CoV-2 associated proteins, the presence or absence of viral RNA, and a unifying pathophysiology. In cooperation with the COVID Human Genome Effort, our “COVID toes” biobank was established to identify both the genetic and immunologic basis and provide clinically relevant insights into targeted therapeutics. As of March 2021, we have enrolled 96 patients, creating a prospective biorepository with clinical data, saliva, serial blood collection, and skin biopsies. Here we aim to comprehensively investigate the conflicting findings, detail the inflammatory response, and identify the source of interferon signaling with multiplex immunofluorescence (IFA) and the RNAscope fluorescent assay to detect viral mRNA. Median patient age was 17 (range 2 – 72) and 44/96 (46%) were male. Preliminary IFA results demonstrate detection of SARS-CoV-2 components, robust MxA detection and plasmacytoid dendritic cell (pDC) colocalization, identifying PDCs as the likely primary source of IFN-I production and implicates an excessive localized IFN-I response in affected patients.

The goal of this study was to test the efficacy of transforming growth factor beta 3 (TGFβ3) in reducing α- smooth muscle actin (SMA) expression in two models—an ex vivo organ culture and an in vitro 3D cell construct—both of which closely mimic an in vivo environment. For the ex vivo organ culture system, a central 6.0 mm corneal keratectomy was performed on freshly excised rabbit globes The corneas were then excised, segregated into groups treated with 1.0 ng/ml TGFβ1 or β3 (T1 or T3, respectively), and cultured for 2 weeks. The corneas were assessed for levels of haze and analyzed for SMA mRNA levels. For the 3D in vitro model, rabbit corneal fibroblasts (RbCFs) were cultured for 4 weeks on poly-transwell membranes in Eagle’s minimum essential media (EMEM) + 10% FBS + 0.5 mM vitamin C ± 0.1 ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for analysis by indirect-immunofluorescence (IF) and RT-qPCR. The RT-qPCR data showed that SMA mRNA expression in T3 samples for both models was significantly lower (p < 0.05) than T1 treatment (around 3-fold in ex vivo and 2-fold in constructs). T3 also reduced the amount of scarring in ex vivo corneas as compared with the T1 samples. IF data from RbCF constructs confirmed that T3-treated samples had up to 4-fold (p < 0.05) lower levels of SMA protein expression than samples treated with T1. These results show that T3 when compared to T1 decreases the expression of SMA in both ex vivo organ culture and in vitro 3D cell construct models. Understanding the mechanism of T3’s action in these systems and how they differ from simple cell culture models, may potentially help in developing T3 as an anti-scarring therapy.

The oxidation of tetrahydro-β-carbolines to β-carbolines using silver carbonate was developed as an alternative to current methods. The oxidation is extremely mild and provides the products in modest to good yields after purification. A number of functional groups are tolerated by this methodology, including reduction-sensitive groups which are often cleaved with other methods. Though the mechanism is not fully understood, the reaction proceeds in an open flask, is not sensitive to light or moisture, and provides a viable synthetic route to compounds that are not easily prepared via other methodologies.

Background: Individuals with chronic pain may have hyperactive microglia, which stimulate neurons to send a pain signal with little or no stimuli. Microglia are therefore a potential drug target to treat chronic pain, but drug discovery has been stymied by differences between human and animal neurobiology, and lack of healthy human CNS microglia. We cultured peripheral blood derived monocytes to develop characteristics of CNS derived microglia, termed peripheral blood derived microglia like cells (PB-MLC). We found that lipopolysaccharide (LPS) treated PB-MLCs from patients with chronic pain, from sickle cell disease (SCD) or chronic headaches, secreted more pro-inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) than PB-MLCs from normal donors, suggesting that patient pain phenotype was preserved in culture; PB-MLC from individuals with chronic pain were hyperactive in vitro as their microglia are in vivo. We hypothesize that PB-MLCs can be developed as a cell-based assay to screen compounds to treat chronic pain. To validate our model system, we compared cultured PB-MLCs to CNS derived microglia cells, using Sprague-Dawley rats, and treated human PB-MLC with microglia activation inhibitors shown to work in vivo in murine models. Methods: We isolated rat brain derived microglia (BDM) and rat peripheral blood monocytes; both were cultured with murine IL-34 (100 ng/ml) and GM-CSF (10 ng/ml). BDM and rPB-MLC were morphologically analyzed by fluorescence imaging microscopy, combined with machine learning, phenotyped by RT-qPCR and indirect immunofluorescence with anti-TMEM119, CD68, and Iba1 antibodies. Cells were treated with LPS for 24 hours, and TNF-alpha, IL-1beta, and IL-6 secretion measured by ELISA. For human PB-MLC studies, monocytes were cultured with GM-CSF (10 ng/ml) and IL-34 (100 ng/ml) for 7 days. PB-MLC morphology was analyzed as above; phenotyped with anti-CX3CR1, TMEM119, CD68, and Iba1 antibodies. PB-MLCs were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM), for 24 h; TNF-alpha and IL-1beta secretion measured by ELISA. Results: We found that rPB-MLC resemble BDM morphologically, express the same microglia specific markers (TMEM119, P2RY12) and can be activated by LPS (Figure 1). Monocytes not cultured with IL-34 and GM-CSF did not express microglia specific genes (Figure 2A). To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the following microglial inhibitors shown to be active in murine models in vivo: minocycline, MRS2395, and clopidogrel. MRS2395 and clopidogrel significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLCs in a dose-dependent manner (Figure 2B); minocycline did not. Conclusions: We validated our model system by comparing CNS derived microglia to rPB-MLCs and found they share morphology, similar cytokine secretion in response to LPS, and expression of microglia-specific genes. We confirmed that human PB-MLC expressed microglia specific genes while the original monocytes did not. Since P2Y12 is implicated in chronic pain, we tested two P2Y12 receptor agonists, clopidogrel and MRS2395, in our human PB-MLC system. When challenged with LPS, clopidogrel and MRS2395 inhibited LPS-induced PB-MLC activation in vitro as it had in vivo in a murine chronic pain model. We propose to use our human PB-MLC to screen for compounds that reduce microglia hyperactivity, to identify pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Proteus mirabilis as a nosocomial pathogen is often the cause of urinary tract infections. This announcement describes the complete genome sequence of a P. mirabilis myophage named Myduc. Phage Myduc is related to Enterobacteria phage phiEcoM-GJ1, which belongs to a group of myophages with small genome sizes (52,000 to 56,000 bp) possessing a T7-like RNA polymerase.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyte Jak2 (JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyte Jak2 (JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are liver manifestations of the metabolic syndrome and can progress to hepatocellular carcinoma (HCC). Loss of Growth Hormone (GH) signaling is reported to predispose to NAFLD and NASH through direct actions on the liver. Here, we report that aged mice lacking hepatocyteJak2(JAK2L), an obligate transducer of Growth Hormone (GH) signaling, spontaneously develop the full spectrum of phenotypes found in patients with metabolic liver disease, beginning with insulin resistance and lipodystrophy and manifesting as NAFLD, NASH and even HCC, independent of dietary intervention. Remarkably, insulin resistance, metabolic liver disease, and carcinogenesis are prevented in JAK2L mice via concomitant deletion of adipocyteJak2(JAK2LA). Further, we demonstrate that GH increases hepatic lipid burden but does so indirectly via signaling through adipocyte JAK2. Collectively, these data establish adipocytes as the mediator of GH-induced metabolic liver disease and carcinogenesis. In addition, we report a new spontaneous model of NAFLD, NASH, and HCC that recapitulates the natural sequelae of human insulin resistance-associated disease progression. The work presented here suggests a attention be paid towards inhibition of adipocyte GH signaling as a therapeutic target of metabolic liver disease.