Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication.

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2α, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

Community-Based Cluster-Randomized Trial to Reduce Opioid Overdose Deaths.

BACKGROUND: Evidence-based practices for reducing opioid-related overdose deaths include overdose education and naloxone distribution, the use of medications for the treatment of opioid use disorder, and prescription opioid safety. Data are needed on the effectiveness of a community-engaged intervention to reduce opioid-related overdose deaths through enhanced uptake of these practices. METHODS: In this community-level, cluster-randomized trial, we randomly assigned 67 communities in Kentucky, Massachusetts, New York, and Ohio to receive the intervention (34 communities) or a wait-list control (33 communities), stratified according to state. The trial was conducted within the context of both the coronavirus disease 2019 (Covid-19) pandemic and a national surge in the number of fentanyl-related overdose deaths. The trial groups were balanced within states according to urban or rural classification, previous overdose rate, and community population. The primary outcome was the number of opioid-related overdose deaths among community adults. RESULTS: During the comparison period from July 2021 through June 2022, the population-averaged rates of opioid-related overdose deaths were similar in the intervention group and the control group (47.2 deaths per 100,000 population vs. 51.7 per 100,000 population), for an adjusted rate ratio of 0.91 (95% confidence interval, 0.76 to 1.09; P = 0.30). The effect of the intervention on the rate of opioid-related overdose deaths did not differ appreciably according to state, urban or rural category, age, sex, or race or ethnic group. Intervention communities implemented 615 evidence-based practice strategies from the 806 strategies selected by communities (254 involving overdose education and naloxone distribution, 256 involving the use of medications for opioid use disorder, and 105 involving prescription opioid safety). Of these evidence-based practice strategies, only 235 (38%) had been initiated by the start of the comparison year. CONCLUSIONS: In this 12-month multimodal intervention trial involving community coalitions in the deployment of evidence-based practices to reduce opioid overdose deaths, death rates were similar in the intervention group and the control group in the context of the Covid-19 pandemic and the fentanyl-related overdose epidemic. (Funded by the National Institutes of Health; HCS ClinicalTrials.gov number, NCT04111939.).

Reducing perceived barriers to scaling up overdose education and naloxone distribution and medications for opioid use disorder in the United States in the HEALing (helping end addiction long-term®) communities study.

BACKGROUND: Scaling up overdose education and naloxone distribution (OEND) and medications for opioid use disorder (MOUD) is needed to reduce opioid overdose deaths, but barriers are pervasive. This study examines whether the Communities That HEAL (CTH) intervention reduced perceived barriers to expanding OEND and MOUD in healthcare/behavioral health, criminal-legal, and other/non-traditional venues. METHODS: The HEALing (Helping End Addiction Long-Term®) Communities Study is a parallel, wait-list, cluster randomized trial testing the CTH intervention in 67 communities in the United States. Surveys administered to coalition members and key stakeholders measured the magnitude of perceived barriers to scaling up OEND and MOUD in November 2019-January 2020, May-June 2021, and May-June 2022. Multilevel linear mixed models compared Wave 1 (intervention) and Wave 2 (wait-list control) respondents. Interactions by rural/urban status and research site were tested. RESULTS: Wave 1 respondents reported significantly greater reductions in mean scores for three outcomes: perceived barriers to scaling up OEND in Healthcare/Behavioral Health Venues (-0.26, 95% confidence interval, CI: -0.48, -0.05, p = 0.015), OEND in Other/Non-traditional Venues (-0.53, 95% CI: - 0.84, -0.22, p = 0.001) and MOUD in Other/Non-traditional Venues (-0.34, 95% CI: -0.62, -0.05, p = 0.020). There were significant interactions by research site for perceived barriers to scaling up OEND and MOUD in Criminal-Legal Venues. There were no significant interactions by rural/urban status. DISCUSSION: The CTH Intervention reduced perceived barriers to scaling up OEND and MOUD in certain venues, with no difference in effectiveness between rural and urban communities. More research is needed to understand facilitators and barriers in different venues.

'Yeah, they suck. It's like they don't care about our health.' Medical mistrust among Black women under community supervision in New York city.

Black women in the USA experience some of the poorest health outcomes and this is especially true for those involved in the carceral system who are at elevated risks for HIV/STIs, reproductive health, and chronic diseases. This study aimed to investigate Black women's experience accessing healthcare services. We conducted semi-structured interviews with 43 women from Project EWORTH under community supervision in New York City. We analysed responses focusing on barriers to healthcare engagement. All interviews were recorded, and data analysis was conducted using NVivo. Themes influencing Black women's ability to engage with healthcare providers and systems included: 1) disclosed provider mistrust/judgement; 2) feeling disrespected by providers and the medical system; 3) mistrust of medical providers/system/hospital/government; 4) lack of health communication; 5) low health literacy; 6) provider gender preference. Findings highlight the need to improve trust and collaboration between healthcare providers and Black women. This study addresses the critical gap in understanding perceptions of discrimination, stigma, and barriers to attaining health care. Funders and accreditation agencies must hold providers and organisations accountable for acquiring and making available diversity, equity and inclusion training for providers, demonstrating increasingly equitable medical relationships through responsiveness to patient feedback, and increasing the number of Black providers.

The Influence of Housing Status and Food Insecurity on a Behavioral HIV/STI Prevention Intervention for Black Women under Community Supervision in New York City: A Moderation Analysis.

Black women in community supervision programs (CSPs) are disproportionately affected by HIV and other sexually transmitted infections (STIs). A randomized controlled trial of a group intervention titled Empowering African-American Women on the Road to Health (E-WORTH) demonstrated effectiveness in reducing sexual risk behaviors and STI incidence among Black women in CSPs. This secondary analysis aimed to assess the moderating effects of housing status and food security on E-WORTH effectiveness in reducing sexual risk behaviors and cumulative incidence of STIs over a 12-month period which were found significant in the original trial among a sample of 351 Black women in CSPs in New York City who use drugs and/or engage in binge drinking who reported engaging in HIV risk behaviors or testing positive for HIV. We examined the moderating effects of housing stability, housing independence, and food insecurity on reducing cumulative STI incidence and number of unprotected sex acts using mixed-effects negative binomial regression and logistic regression models that controlled for age, high school education, employment status, and marital status. Findings indicate that the intervention effect was moderated by housing stability, but not housing independence or food security. Compared to the control group, E-WORTH participants who were housing insecure had 63% fewer acts of condomless sex. Our findings highlight the importance of interventions designed for women in CSPs that account for upstream determinants of health and include service linkages to basic needs provisions. Further research is needed to unpack the cumulative impacts of multiple experiences of poverty faced by this population.

Effects and avoidance of photoconversion-induced artifacts in confocal and STED microscopy.

Fluorescence microscopy is limited by photoconversion due to continuous illumination, which results in not only photobleaching but also conversion of fluorescent molecules into species of different spectral properties through photoblueing. Here, we determined different fluorescence parameters of photoconverted products for various fluorophores under standard confocal and stimulated emission depletion (STED) microscopy conditions. We observed changes in both fluorescence spectra and lifetimes that can cause artifacts in quantitative measurements, which can be avoided by using exchangeable dyes.

Effect of the Communities that HEAL intervention on receipt of behavioral therapies for opioid use disorder: A cluster randomized wait-list controlled trial.

BACKGROUND: The U.S. opioid overdose crisis persists. Outpatient behavioral health services (BHS) are essential components of a comprehensive response to opioid use disorder and overdose fatalities. The Helping to End Addiction Long-Term® (HEALing) Communities Study developed the Communities That HEAL (CTH) intervention to reduce opioid overdose deaths in 67 communities in Kentucky, Ohio, New York, and Massachusetts through the implementation of evidence-based practices (EBPs), including BHS. This paper compares the rate of individuals receiving outpatient BHS in Wave 1 intervention communities (n = 34) to waitlisted Wave 2 communities (n = 33). METHODS: Medicaid data included individuals ≥18 years of age receiving any of five BHS categories: intensive outpatient, outpatient, case management, peer support, and case management or peer support. Negative binomial regression models estimated the rate of receiving each BHS for Wave 1 and Wave 2. Effect modification analyses evaluated changes in the effect of the CTH intervention between Wave 1 and Wave 2 by research site, rurality, age, sex, and race/ethnicity. RESULTS: No significant differences were detected between intervention and waitlisted communities in the rate of individuals receiving any of the five BHS categories. None of the interaction effects used to test the effect modification were significant. CONCLUSIONS: Several factors should be considered when interpreting results-no significant intervention effects were observed through Medicaid claims data, the best available data source but limited in terms of capturing individuals reached by the intervention. Also, the 12-month evaluation window may have been too brief to see improved outcomes considering the time required to stand-up BHS. TRIAL REGISTRATION: Clinical Trials.gov http://www. CLINICALTRIALS: gov: Identifier: NCT04111939.

Characterizing Multisystem Barriers to Women's Residential SUD Treatment: A Multisite Qualitative Analysis in Los Angeles.

Residential substance use disorder (SUD) treatment programs are challenged by the differing values of the problem-solving court (PSC) and child welfare (CW) systems, along with communication barriers between staff. This study aimed to understand, from the viewpoints of SUD treatment providers, how divergent values and communication barriers adversely affect women's residential SUD treatment. We conducted qualitative semistructured interviews with 18 SUD treatment clinicians and six directors from four women's residential SUD treatment programs. Using a thematic analysis framework, we identified salient themes across specified codes. Analysis revealed six main themes, suggesting differing values and communication barriers across the SUD, PSC, and CW systems adversely affect the provision of SUD treatment. For differing values, three main themes emerged: (a) unaddressed trauma and fear of mental health treatment seeking; (b) perceptions of mothers with a SUD; and (c) the Adoption and Safe Families Act (ASFA) timeline as a barrier to SUD treatment provision. For communication barriers, three themes emerged: (a) inadequate communication and responsiveness with PSC and CW systems adversely affect treatment coordination, induce patient stress, and treatment disengagement; (b) lack of PSC and CW communication regarding child visitation planning adversely affects treatment motivation and retention; and (c) competing ASFA, PSC, and CW priorities and inadequate cross-system communication adversely affect treatment planning. Treatment providers face significant barriers in providing effective treatment to women simultaneously involved in the CW and PSC systems. Aligning values and addressing communication barriers, changes in policy, and enhanced cross-system training are crucial. Additionally, it is essential to reevaluate the ASFA timeline to align with the long-term treatment needs of mothers with a SUD. Further research should explore the viewpoints of patients, CW, and PSC staff to gain deeper insights into these SUD treatment barriers.

Cellular HSF1 expression is induced during HIV-1 infection by activation of its promoter mediated through the cooperative interaction of HSF1 and viral Nef protein.

The Human Immunodeficiency Virus-1 (HIV-1) tends to activate cellular promoters driving expression of pro-viral genes by complex host-virus interactions for productive infection. We have previously demonstrated that expression of such a positive host factor HSF1 (heat shock factor 1) is elevated during HIV-1 infection; however, the mechanism remains to be elucidated. In the present study, we therefore examined whether HSF1 promoter is induced during HIV-1 infection leading to up-regulation of hsf1 gene expression. We mapped the putative transcription start site (TSS) predicted by Eukaryotic promoter database and deletion constructs of the predicted promoter region were tested through luciferase assay to identify the active promoter. The 347 bp upstream to 153 bp downstream region around the putative TSS displayed the highest activity and both Sp1 (stimulating protein 1) and HSF1 itself were identified to be important for its basal activation. Activity of HSF1 promoter was further stimulated during HIV-1 infection in CD4+ T cells, where interestingly the HSF1-site itself seems to play a major role. In addition, HIV-1 protein Nef (negative factor) was also observed to be responsible for the virus-mediated induction of hsf1 gene expression. Chromatin-immunoprecipitation assays further demonstrate that Nef and HSF1 are co-recruited to the HSF1-binding site and cooperatively act on this promoter. The interplay between host HSF1 and viral Nef on HSF1 promoter eventually leads to increase in HSF1 expression during HIV-1 infection. Understanding the mechanism of HSF1 up-regulation during HIV-1 infection might contribute to future antiviral strategies as HSF1 is a positive regulator of virus replication.

Self-Degrading Multifunctional PEG-Based Hydrogels-Tailormade Substrates for Cell Culture.

The use of PEG-based hydrogels as cell culture matrix to mimic the natural extracellular matrix (ECM) has been realized using a range of well-defined, tunable, and dynamic scaffolds, although they require cell adhesion ligands such as RGDS-peptide (Arg-Gly-Asp-Ser) to promote cell adhesion. Herein the synthesis of ionic and degradable hydrogels is demonstrated for cell culture by crosslinking [PEG-SH]4 with the zwitterionic crosslinker N,N-bis(acryloxyethyl)-N-methyl-N-(3-sulfopropyl) ammonium betaine (BMSAB) and the cationic crosslinker N,N-bis(acryloxyethyl)-N,N-dimethyl-1-ammonium iodide (BDMAI). Depending on the amount of ionic crosslinker used in gel formation, the hydrogels show tunable gelation time and stiffness. At the same time, the ionic groups act as catalysts for hydrolytic degradation, thereby allowing to define a stability window. The latter could be tailored in a straightforward manner by introducing the non-degradable crosslinker tri(ethylene glycol) divinyl ether. In addition, both ionic crosslinkers favor cell attachment in comparison to the pristine PEG hydrogels. The degradation is examined by swelling behavior, rheology, and fluorescence correlation spectroscopy indicating degradation kinetics depending on diffusion of incorporated fluorescent molecules.

Intervention Adaptation and Implementation Method for Real-World Constraints and Using New Technologies.

Purpose: Rigorous adaptation methods are needed to revise existing evidence-based behavioral interventions for implementation for new target populations, revised/updated outcomes, new delivery modalities, recent advances, and new technologies. We describe an adaptation method designed to overcome the real-world challenges of having very limited existing expertise, resources, and time. Method: This adaptation method and accompanying visualization tool ("Deconstruction/Reconstruction Matrix") preserves theoretical mechanisms of behavior change, accounts for challenges in utilizing new technologies, and strengthens clinical processes, with an emphasis on safety. Results: The adaptation of an in-person HIV behavioral intervention for sexual and gender diverse men in Kazakhstan to one delivered remotely via telecommunication and social media technologies exemplifies the process and strengths of the method, concomitantly resulting in recommendations for adaptation and implementation of mobile health (mHealth) and digital health interventions. Discussion: This method allows researchers and clinicians to adapt interventions rapidly and rigorously and to benefit from new technologies.

Intervening on the Intersecting Issues of Intimate Partner Violence, Substance Use, and HIV: A Review of Social Intervention Group's (SIG) Syndemic-Focused Interventions for Women.

Intimate partner violence (IPV), HIV, and substance use are serious intersecting public health issues. This paper aims to describe the Social Intervention Group (SIG)'s syndemic-focused interventions for women that address the co-occurrence of IPV, HIV, and substance use, referred to as the SAVA syndemic. We reviewed SIG intervention studies from 2000 to 2020 that evaluated the effectiveness of syndemic-focused interventions which addressed two or more outcomes related to reducing IPV, HIV, and substance use among different populations of women who use drugs. This review identified five interventions that co-targeted SAVA outcomes. Of the five interventions, four showed a significant reduction in risks for two or more outcomes related to IPV, substance use, and HIV. The significant effects of SIG's interventions on IPV, substance use, and HIV outcomes among different populations of women demonstrate the potential of using syndemic theory and methods in guiding effective SAVA-focused interventions.

Traumatic Life Events and Stressors: Associations With Substance Use Problems of Men Under Community Supervision.

This study aims to determine associations between sources of stress and substance use problems among men under community supervision programs. We used ordinary least squares regression models to test the hypotheses that the traumatic life events and stressors of men under community supervision and their partners, respectively, were associated with the substance use problems of men under community supervision. The sample included 230 men involved in community supervision programs in New York City and 230 women who were their intimate partners. We found that the traumatic events and the depression and anxiety of men under community supervision and partner's substance use problems were associated with the substance use problems of the men. These findings underscore the importance of addressing trauma in substance use treatment for men under community supervision. Future research can explore how stressful events impact substance use, sources of coping for couples, and the association between racism, traumatic events, stress, and substance use. Clinical Trial Registration: NCT01690494.

Excited-State Dynamics in Borylated Arylisoquinoline Complexes in Solution and in cellulo.

Two four-coordinate organoboron N,C-chelate complexes with different functional terminals on the PEG chains are studied with respect to their photophysical properties within human MCF-7 cells. Their excited-state properties are characterized by time-resolved pump-probe spectroscopy and fluorescence lifetime microscopy. The excited-state relaxation dynamics of the two complexes are similar when studied in DMSO. Aggregation of the complexes with the carboxylate terminal group is observed in water. When studying the light-driven excited-state dynamics of both complexes in cellulo, i. e., after being taken up into human MCF-7 cells, both complexes show different features depending on the nature of the anchoring PEG chains. The lifetime of a characteristic intramolecular charge-transfer state is significantly shorter when studied in cellulo (360±170 ps) as compared to in DMSO (∼960 ps) at 600 nm for the complexes with an amino group. However, the kinetics of the complexes with the carboxylate group are in line with those recorded in DMSO. On the other hand, the lifetimes of the fluorescent state are almost identical for both complexes in cellulo. These findings underline the importance to evaluate the excited-state properties of fluorophores in a complex biological environment in order to fully account for intra- and intermolecular effects governing the light-induced processes in functional dyes.

Embedding Big Qual and Team Science into Qualitative Research: Lessons from a Large-scale, Cross-site Research Study.

Background: A major part of the HEALing Communities Study (HCS), launched in 2019 to address the growing opioid epidemic, is evaluating the study's intervention implementation process through an implementation science (IS) approach. One component of the IS approach involves teams with more than 20 researchers collaborating across four research sites to conduct in-depth qualitative interviews with over 300 participants at four time points. After completion of the first two rounds of data collection, we reflect upon our qualitative data collection and analysis approach. We aim to share our lessons learned about designing and applying qualitative methods within an implementation science framework. Methods: The HCS evaluation is based on the RE-AIM/PRISM framework and incorporates interviews at four timepoints. At each timepoint, the core qualitative team of the Intervention Work Group drafts an interview guide based on the framework and insights from previous round(s) of data collection. Researchers then conduct interviews with key informants and coalition members within their respective states. Data analysis involves drafting, iteratively refining, and finalizing a codebook in a cross-site and within-site consensus processes. Interview transcripts are then individually coded by researchers within their respective states. Results: Successes in the evaluation process includes having structured procedures for communication, data collection, and analysis, all of which are critical for ensuring consistent data collection and for achieving consensus during data analysis. Challenges include recognizing and accommodating the diversity of training and knowledge between researchers, and establishing reliable ways to securely store, manage, and share the large volumes of data. Conclusion: Qualitative methods using a team science approach have been limited in their application in large, multi-site randomized controlled trials of health interventions. Our experience provides practical guidance for future studies with large, experientially and disciplinarily diverse teams, and teams seeking to incorporate qualitative or mixed-methods components for their evaluations.

Risk factors for miscarriage in Syrian refugee women living in non-camp settings in Jordan: results from the Women ASPIRE cross-sectional study.

BACKGROUND: Syrian refugee women face health care disparities and experience worse pregnancy outcomes, including miscarriage. We investigated risk factors for miscarriage in Syrian refugee women living in non-camp settings in Jordan to identify targets for interventions. METHODS: We analyzed data from Women ASPIRE, a cross-sectional study of gendered physical and mental health concerns of 507 Syrian refugee women (≥ 18 years old) living in non-camp settings in Jordan. We recruited women using systematic clinic-based sampling from four clinics. We limited our analyses to women who had a history of pregnancy and whose most recent pregnancy was single, took place in Jordan, and ended in term live birth or miscarriage (N = 307). We grouped the women by the primary outcome (term live birth or miscarriage) and compared the sociodemographic and clinical characteristics of the two groups. We used Pearson's χ2 test or the Mann-Whitney U test to obtain unadjusted estimates and multivariable binomial logistic regression to obtain adjusted estimates. RESULTS: The most recent pregnancies of 262 women (85%) ended in term live birth and another 45 (15%) ended in miscarriage. Since crossing into Jordan, 11 women (4%) had not received reproductive health services. Of 35 women who were ≥ 35 years old, not pregnant, and did not want a (or another) child, nine (26%) did not use contraception. Of nine women who were ≥ 35 years old and pregnant, seven (78%) did not plan the pregnancy. The adjusted odds of miscarriage were higher in women who had been diagnosed with thyroid disease (aOR, 5.54; 95% CI, 1.56-19.07), had been of advanced maternal age (aOR, 5.83; 95% CI, 2.02-16.91), and had not received prenatal care (aOR, 36.33; 95% CI, 12.04-129.71). Each additional previous miscarriage predicted an increase in the adjusted odds of miscarriage by a factor of 1.94 (1.22-3.09). CONCLUSIONS: We identified several risk factors for miscarriage in Syrian refugee women living in non-camp settings in Jordan. The risk factors may be amenable to preconception and prenatal care.

Erratum: Long-term STED imaging of membrane packing and dynamics by exchangeable polarity-sensitive dyes.

[This corrects the article DOI: 10.1016/j.bpr.2021.100023.].

Secondary Traumatic Stress, Depression, and Anxiety Symptoms Among Service Providers Working with Syrian Refugees in Istanbul, Turkey.

This paper examines the mental health of service providers working with Syrian refugees. Using the Professional Quality of Life framework, we hypothesize greater stress/less support from the work, person, client environment is associated with symptoms of STS, depression, and anxiety. We surveyed a sample of 104 service providers throughout Istanbul late 2018. Multivariable logistic regression examined associations between work (organizational support, caseload, supervision), person (perceived social support), client environment (trauma disclosure, percent Syrian refugees) on STS, depression, and anxiety. We found rates of moderate-to-severe STS to be 27.88%; depression 40.38%; and anxiety 29.81%. Our hypothesis was partially supported. Lower organizational support was associated with moderate-to-severe STS (aOR 0.91, 95% CI 0.84, 0.99) while lower social support with anxiety (aOR 0.89, 95% CI 0.81, 0.99). Caseload, supervision, trauma disclosure, percent refugees did not show significant associations. Organizations working with Syrian refugees may benefit from enhancing organizational support and promoting social support for staff.

Community coalition and key stakeholder perceptions of the community opioid epidemic before an intensive community-level intervention.

INTRODUCTION: Opioid overdoses are a major public health emergency in the United States. Despite effective treatments that can save lives, access to and utilization of such treatments are limited. Community context plays an important role in addressing treatment barriers and increasing access. The HEALing Communities Study (HCS) is a multisite community-level cluster-randomized trial that will study implementation and outcomes of a community coalition-based intervention (Communities that HEAL [CTH]) that implements evidence-based practices (EBPs) to reduce opioid overdose deaths in four states. To examine contextual factors critical to understanding implementation, we assessed the perspectives of community members about their communities, current substance use-related services, and other important issues that could impact intervention implementation. METHODS: Researchers conducted 382 semi-structured qualitative interviews in the HCS communities. Interviews were audio-recorded and transcribed; researchers subsequently analyzed data using directed content analysis based on the constructs of the RE-AIM/PRISM implementation science framework to identify key themes within the external community context. RESULTS: Despite the diversity in states and communities, four similar themes related to the external community context emerged across communities: These themes included the importance of understanding: 1) community risk perceptions, 2) levels of stigma, 3) the health services environment and the availability of substance use services, and 4) funding for substance use services. CONCLUSION: Understanding and addressing the external community context in which the CTH intervention and EBPs are implemented are crucial for successful health services-related and community engaged interventions. While implementing EBPs is a challenging undertaking, doing so will help us to understand if and how a community-based intervention can successfully reduce opioid overdose deaths and influence both community beliefs and the community treatment landscape.