Identification of a genetic region linked to tolerance to MRSA infection using Collaborative Cross mice.

Staphylococcus aureus (S. aureus) colonizes humans asymptomatically but can also cause opportunistic infections, ranging from mild skin infections to severe life-threatening conditions. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. In previous work, we established that collaborative cross (CC) mouse line CC061 is highly susceptible to Methicillin-resistant S. aureus infection (MRSA, USA300), while CC024 is tolerant. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice to generate F1 and F2 populations. Survival after MRSA infection in the F1 and F2 generations was 65% and 55% and followed a complex dominant inheritance pattern for the CC024 increased survival phenotype. Colonization in F2 animals was more extreme than in their parents, suggesting successful segregation of genetic factors. We identified a Quantitative Trait Locus (QTL) peak on chromosome 7 for survival and weight change after infection. In this QTL, the WSB/EiJ (WSB) allele was present in CC024 mice and contributed to their MRSA tolerant phenotype. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. C5ar1 and C5ar2 are receptors for the complement factor C5a, an anaphylatoxin that can trigger a massive immune response by binding to these receptors. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival.

Estimating the potential number of cases prevented by infant/ toddler immunisation with a MenACWY vaccine.

INTRODUCTION: Meningococcal disease control in the UK relies on various vaccines, with the discontinuation of the Hib/MenC combination vaccine Menitorix® in 2018 necessitating reassessment of the immunisation strategy. The quadrivalent MenACWY vaccine emerges as a promising long-term solution, already integrated into the teenage immunisation regimen. While indirect control of group W and C cases is anticipated through existing programs, the high incidence of meningococcal disease in infancy underscores the potential benefits of infant/toddler vaccination. METHODS: Utilizing data from two UK studies, we recalibrated age-specific carriage prevalence curves and estimated the proportion of meningococcal carriage attributed to ACWY and non-ACWY strains. Employing a dynamic transmission model, we evaluated the combined indirect effects of the teenage MenACWY vaccination initiative and the direct impact of administering MenACWY vaccine at either 3 or 12 months, alongside ongoing 4CMenB vaccination efforts. Given the pandemic-induced decline in cases and alterations in social contact patterns reported in prior research, we also simulated the transmission model to reflect periods of COVID-19 lockdown. RESULTS: Our projections indicate effective control of carriage and disease associated with groups A, C, W, and Y through the teenage vaccination campaign. Assuming sustained high uptake of teenage vaccines amid pandemic scenario, we forecast MenACWY carriage prevalence to be below 1% by 2025. Across all scenarios, the impact of an infant/toddler MenACWY program on case reduction remains modest. Notably, administering the MenACWY dose at 3 months yields a greater number of prevented cases compared to administration at 12 months. With sustained uptake of teenage vaccination, our estimates suggest that between 3 and 22 cases could be averted in a 2025 birth cohort through a 3-month MenACWY dose. CONCLUSIONS: Provided teenage uptake remains high and the infant 4CMenB programme is maintained, we suggest that few cases will be prevented from an infant/ toddler MenACWY dose.

Strategies used to reduce harms associated with fentanyl exposure among rural people who use drugs: multi-site qualitative findings from the rural opioid initiative.

AIM: Illicitly manufactured fentanyl and its analogs are the primary drivers of opioid overdose deaths in the United States (U.S.). People who use drugs may be exposed to fentanyl or its analogs intentionally or unintentionally. This study sought to identify strategies used by rural people who use drugs to reduce harms associated with unintentional fentanyl exposure. METHODS: This analysis focused on 349 semi-structured qualitative interviews across 10 states and 58 rural counties in the U.S conducted between 2018 and 2020. Interview guides were collaboratively standardized across sites and included questions about drug use history (including drugs currently used, frequency of use, mode of administration) and questions specific to fentanyl. Deductive coding was used to code all data, then inductive coding of overdose and fentanyl codes was conducted by an interdisciplinary writing team. RESULTS: Participants described being concerned that fentanyl had saturated the drug market, in both stimulant and opioid supplies. Participants utilized strategies including: (1) avoiding drugs that were perceived to contain fentanyl, (2) buying drugs from trusted sources, (3) using fentanyl test strips, 4) using small doses and non-injection routes, (5) using with other people, (6) tasting, smelling, and looking at drugs before use, and (7) carrying and using naloxone. Most people who used drugs used a combination of these strategies as there was an overwhelming fear of fatal overdose. CONCLUSION: People who use drugs living in rural areas of the U.S. are aware that fentanyl is in their drug supply and use several strategies to prevent associated harms, including fatal overdose. Increasing access to harm reduction tools (e.g., fentanyl test strips, naloxone) and services (e.g., community drug checking, syringe services programs, overdose prevention centers) should be prioritized to address the polysubstance-involved overdose crisis. These efforts should target persons who use opioids and other drugs that may contain fentanyl.

An Automated Text Messaging Intervention to Reduce Substance Use Self-Stigma (Project RESTART): Protocol for a Feasibility and Acceptability Pilot Study.

BACKGROUND: Stigma is a barrier to treatment and harm reduction seeking in people who use drugs. Most stigma reduction interventions offer psychotherapy or psychoeducation in group-based clinical settings, failing to reach people who are not in treatment. SMS text messaging is an effective and acceptable modality for delivering health information to people who use drugs and may be a suitable conduit for providing information and advice to understand and cope with stigma. OBJECTIVE: This paper presents the protocol for a study that aims to determine the feasibility, acceptability, and preliminary effectiveness of a 4-week automated SMS text message intervention to increase stigma resistance and reduce self-stigma in people who use drugs. METHODS: We designed a novel automated SMS text message intervention to address the four personal-level constructs of stigma resistance: (1) not believing stigma and catching and challenging stigmatizing thoughts, (2) empowering oneself through learning about substance use and one's recovery, (3) maintaining one's recovery and proving stigma wrong, and (4) developing a meaningful identity and purpose apart from one's substance use. Theory-based messages were developed and pilot-tested in qualitative elicitation interviews with 22 people who use drugs, resulting in a library of 56 messages. In a single-group, within-subjects, community-based pilot trial, we will enroll 30 participants in the Resisting Stigma and Revaluating Your Thoughts (RESTART) intervention. Participants will receive 2 daily SMS text messages for 4 weeks. Implementation feasibility will be assessed through recruitment, enrollment, retention, and message delivery statistics. User feasibility and acceptability will be assessed at follow-up using 23 survey items informed by the Theoretical Framework of Acceptability. Primary effectiveness outcomes are changes in self-stigma (Substance Abuse Self-Stigma Scale) and stigma resistance (Stigma Resistance Scale) from baseline to follow-up measured via a self-administered survey. Secondary outcomes are changes in hope (Adult Dispositional Hope Scale) and self-esteem (Rosenberg Self-Esteem Scale). Feasibility and acceptability will be assessed with descriptive statistics; effectiveness outcomes will be assessed with paired 2-tailed t tests, and group differences will be explored using ANOVA. Overall, 12 participants will also be selected to complete acceptability interviews. RESULTS: This pilot study was funded by the National Institute on Drug Abuse in April 2023 and received regulatory approval in January 2024 by the University of North Carolina-Chapel Hill Institutional Review Board. Recruitment and enrollment began in March 2024. Follow-up visits are expected to conclude by May 2024. Results will be disseminated in relevant peer-reviewed journals. CONCLUSIONS: To the best of our knowledge, this is the first study to address substance use stigma via a self-help SMS text messaging program. Results will add to the nascent literature on stigma reduction in people who use drugs. This protocol may interest researchers who are considering text messaging to address psychosocial needs in hard-to-reach populations. TRIAL REGISTRATION: ClinicalTrials.gov NCT06281548; https://clinicaltrials.gov/ct2/show/NCT06281548. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/59224.

Late-life dietary folate restriction reduces biosynthesis without compromising healthspan in mice.

Folate is a vitamin required for cell growth and is present in fortified foods in the form of folic acid to prevent congenital abnormalities. The impact of low-folate status on life-long health is poorly understood. We found that limiting folate levels with the folate antagonist methotrexate increased the lifespan of yeast and worms. We then restricted folate intake in aged mice and measured various health metrics, metabolites, and gene expression signatures. Limiting folate intake decreased anabolic biosynthetic processes in mice and enhanced metabolic plasticity. Despite reduced serum folate levels in mice with limited folic acid intake, these animals maintained their weight and adiposity late in life, and we did not observe adverse health outcomes. These results argue that the effectiveness of folate dietary interventions may vary depending on an individual's age and sex. A higher folate intake is advantageous during the early stages of life to support cell divisions needed for proper development. However, a lower folate intake later in life may result in healthier aging.

Overdose responses among rural people who use drugs: A multi-regional qualitative study.

BACKGROUND: Efforts to distribute naloxone have equipped more people with the ability to reverse opioid overdoses but people who use drugs are often reluctant to call 911 due to concerns for legal repercussions. Rural communities face unique challenges in reducing overdose deaths compared to urban communities, including limited access to harm reduction services as well as greater concerns about stigma and privacy. METHODS: The Rural Opioid Initiative was funded in 2017 to better understand the health-related harms associated with the opioid crisis in rural US communities and consists of eight studies spanning ten states and 65 counties. Each study conducted semi-structured qualitative interviews with people who use drugs to understand contextual factors influencing drug use and health behaviors. We analyzed qualitative data from seven studies with data available at the time of analysis to understand peer response to overdose. RESULTS: Of the 304 participants interviewed, 55% were men, 70% were white, 80% reported current injection drug use, and 60% reported methamphetamine use. Similar to what has been found in studies focused on urban settings, people who use drugs in rural communities use a range of strategies to reverse overdoses, including non-evidence-based approaches. Several reported that multiple doses of naloxone are needed to reverse overdose. Three themes emerged around the willingness to call 911, including (1) hesitancy to call 911 for fear of legal consequences, (2) negative perceptions or experiences with law enforcement officers, and (3) efforts to obtain medical intervention while avoiding identification/law enforcement involvement. CONCLUSION: People who use drugs employ multiple strategies to attempt overdose reversal, including non-evidence-based approaches. Greater education about the most effective and least harmful strategies is needed. Reluctance to call 911 is rooted in concerns about potential legal consequences as well as perceptions about law enforcement officers, which may be heightened in rural communities where people who use drugs are more easily identified by law enforcement. People who use drugs will go to great strides to connect their peers to needed medical services, suggesting that comprehensive interventions to reduce interactions with law enforcement officers and eliminate legal consequences for reporting overdoses are critical.

Case example of a female solo sailor competing in the Vendée Globe reveals extraordinary biological demands.

The Vendée Globe is a non-stop, unassisted, single-handed round the world sailing race. It is regarded as the toughest sailing race, requiring high cognitive functioning and constant alertness. Little is known about the amount of sleep restriction and nutritional deficit experienced at sea and effects that fatigue have on sailors' performance. This report aimed to investigate these aspects by monitoring one of the female participants of the latest Vendée Globe. Sleep, food intake and stress were self-reported daily using specific app. Cognitive assessments were digitally completed. Heart rate and activity intensity were measured using a wrist-worn wearable device. Mean self-report sleep duration per 24 h was 3 hours 40 minutes. By the end of the 95 race days, the sailor reached a caloric deficit of 27,900 kcal. On average, the sailor spent 50 minutes per day in moderate-to-vigorous activity. Cognitive assessments did not show any effect of fatigue or stress on completion time or performance. Recent technological and communication advancement for offshore sailors, enabled continuous data to be monitored in near real time, even from the Southern Ocean. Moving forward this will enable greater understanding of when sailors will be at risk of poor decision making, illness or injury.

Collaborative Cross mice have diverse phenotypic responses to infection with Methicillin-resistant Staphylococcus aureus USA300.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen causing diseases ranging from mild skin infections to life threatening conditions, including endocarditis, pneumonia, and sepsis. To identify host genes modulating this host-pathogen interaction, we infected 25 Collaborative Cross (CC) mouse strains with methicillin-resistant S. aureus (MRSA) and monitored disease progression for seven days using a surgically implanted telemetry system. CC strains varied widely in their response to intravenous MRSA infection. We identified eight 'susceptible' CC strains with high bacterial load, tissue damage, and reduced survival. Among the surviving strains, six with minimal colonization were classified as 'resistant', while the remaining six tolerated higher organ colonization ('tolerant'). The kidney was the most heavily colonized organ, but liver, spleen and lung colonization were better correlated with reduced survival. Resistant strains had higher pre-infection circulating neutrophils and lower post-infection tissue damage compared to susceptible and tolerant strains. We identified four CC strains with sexual dimorphism: all females survived the study period while all males met our euthanasia criteria earlier. In these CC strains, males had more baseline circulating monocytes and red blood cells. We identified several CC strains that may be useful as new models for endocarditis, myocarditis, pneumonia, and resistance to MRSA infection. Quantitative Trait Locus (QTL) analysis identified two significant loci, on Chromosomes 18 and 3, involved in early susceptibility and late survival after infection. We prioritized Npc1 and Ifi44l genes as the strongest candidates influencing survival using variant analysis and mRNA expression data from kidneys within these intervals.

The relationship between felt stigma and non-fatal overdose among rural people who use drugs.

BACKGROUND: Drug overdose deaths in the United States exceeded 100,000 in 2021 and 2022. Substance use stigma is a major barrier to treatment and harm reduction utilization and is a priority target in ending the overdose epidemic. However, little is known about the relationship between stigma and overdose, especially in rural areas. We aimed to characterize the association between felt stigma and non-fatal overdose in a multi-state sample of rural-dwelling people who use drugs. METHODS: Between January 2018 and March 2020, 2,608 people reporting past 30-day opioid use were recruited via modified chain-referral sampling in rural areas across 10 states. Participants completed a computer-assisted survey of substance use and substance-related attitudes, behaviors, and experiences. We used multivariable logistic regression with generalized estimating equations to test the association between felt stigma and recent non-fatal overdose. RESULTS: 6.6% of participants (n = 173) reported an overdose in the past 30 days. Recent non-fatal overdose was significantly associated with felt stigma after adjusting for demographic and substance use-related covariates (aOR: 1.47, 95% CI: 1.20-1.81). The association remained significant in sensitivity analyses on component fear of enacted stigma items (aOR: 1.48, 95% CI: 1.20-1.83) and an internalized stigma item (aOR: 1.51, 95% CI: 1.07-2.14). CONCLUSIONS: Felt stigma related to substance use is associated with higher risk of non-fatal overdose in rural-dwelling people who use drugs. Stigma reduction interventions and tailored services for those experiencing high stigma are underutilized approaches that may mitigate overdose risk.

Late-life dietary folate restriction reduces biosynthetic processes without compromising healthspan in mice.

Folate is a vitamin required for cell growth and is present in fortified foods in the form of folic acid to prevent congenital abnormalities. The impact of low folate status on life-long health is poorly understood. We found that limiting folate levels with the folate antagonist methotrexate increased the lifespan of yeast and worms. We then restricted folate intake in aged mice and measured various health metrics, metabolites, and gene expression signatures. Limiting folate intake decreased anabolic biosynthetic processes in mice and enhanced metabolic plasticity. Despite reduced serum folate levels in mice with limited folic acid intake, these animals maintained their weight and adiposity late in life, and we did not observe adverse health outcomes. These results argue that the effectiveness of folate dietary interventions may vary depending on an individual's age and sex. A higher folate intake is advantageous during the early stages of life to support cell divisions needed for proper development. However, a lower folate intake later in life may result in healthier aging.

Interventions to reduce self-stigma in people who use drugs: A systematic review.

BACKGROUND: Substance use stigma is a key barrier to treatment and harm reduction engagement among people who use drugs (PWUD). Previous systematic reviews have focused on interventions to reduce stigma in healthcare providers and the public; less is known about interventions to address self-stigma among PWUD. The purpose of this review is to evaluate the evidence for substance use self-stigma reduction interventions. METHODS: We reviewed English-language studies published between 2011 and 2023 using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (PROSPERO #CRD42022321305). We searched seven bibliographic databases (PubMed; SCOPUS; APA PsycInfo; CINAHL; Social Work Abstracts; Sociological Abstracts; ProQuest Dissertations & Theses). This review included studies if 1) they evaluated the effectiveness of a psychosocial intervention, 2) participants were PWUD, 3) authors reported self-stigma as a primary outcome, 4) the study design was experimental or quasi-experimental. We reviewed, interpreted and reported intervention characteristics and effectiveness using narrative synthesis. We assessed study quality with the Downs & Black checklist. RESULTS: Among 1195 screened studies, 15 met the inclusion criteria (N = 2280 PWUD). We categorized the interventions according to three approaches: psychotherapeutic (n = 8), psychoeducational (n = 5), and multimodal (n = 2). Most interventions were delivered in clinical settings (n = 11) and in a group format (n = 13). Study quality was fair-to-good and included nine randomized controlled trials (RCTs) and six quasi-experiments. Measurement heterogeneity was high, with 11 different stigma-related scales used across the 15 studies. Eleven studies showed significant favorable effects in at least one stigma measure. Six of these demonstrated positive effects in all stigma measures. Evidence was mixed for all three intervention categories; however, Acceptance and Commitment Therapy, a form of group psychotherapy, demonstrated effectiveness in four of five RCTs incorporating this approach. CONCLUSIONS: Overall, there is promising evidence for the effectiveness of substance use self-stigma interventions, although more studies are needed to determine which approaches are most effective. Consistent conceptualization and measurement of self-stigma across studies will improve comparability in future intervention trials. Current offerings are largely limited to clinical settings and group-based formats; self-help interventions, available for other stigmatized conditions, could be developed to serve the majority of PWUD not engaged in treatment.

Precision pharmacological reversal of strain-specific diet-induced metabolic syndrome in mice informed by epigenetic and transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

"I would do anything but that": Attitudes towards sex work among rural people who use drugs.

BACKGROUND: Stigma towards people who use drugs and those who engage in sex work is well-documented, leading to consequences such as reduced access to health services and support, especially in rural milieus. Stigma reduction has been recognized as a priority in the opioid overdose crisis, but little attention has been paid to within-group attitudes and beliefs. This study aimed to explore how people who use drugs in rural counties across the United States appraise sex work by themselves or other community members. METHODS: Qualitative interview data came from the Rural Opioid Initiative (ROI), a project coordinated by research teams across 65 rural counties in 10 states. Interviews were individual and conducted from 2018 to 2020. All participants reported past 30-day opioid use and/or any injection drug use. A working group coded the data, then used an iterative inductive-deductive approach to organize data into themes of stigma among people who use drugs, focusing on stigma towards sex work. RESULTS: Across sites, 355 interviews were conducted. Mean participant age was 36, 55 % of participants were male, and 93 % were white. Participants expressed negative attitudes towards sex work as a function of its criminal-legal repercussions or framed sex work as morally transgressive. Many appraisals were gendered, with the behavior conveyed as being "easier" for women who were often described as "whores," with more neutral terms used to describe men. Some viewed sex work as an implicit "exchange" for drugs. Several participants noted a lack of agency as a feature leading to involvement in sex work, with partner power dynamics influencing an individual's behavior. Finally, a few participants acknowledged the circumstances under which they would newly engage in sex work. CONCLUSION: We identified several patterns by which people who use drugs evaluate community members who sell sex. These included gendered and morally-charged forms of stigma, which may represent barriers to community acceptance and support among this subgroup.

Precision pharmacological reversal of genotype-specific diet-induced metabolic syndrome in mice informed by transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genomic DNA methylation analyses and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects reveals a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the Farnesoid X receptor pathway, and found that GW4064 exerts genotype-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis, as well as increased inflammatory-related gene expression changes in NOD. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention, and a mouse platform for guiding this approach.

Predictors of firearm purchasing during the coronavirus pandemic in the United States: a cross-sectional study.

OBJECTIVES: This study aimed to compare determinants of firearm purchasing related to the pandemic. STUDY DESIGN: This was a cross-sectional survey. METHODS: A total of 3853 online panel participants completed a survey between December 22, 2020, and January 2, 2021, to approximate a nationally representative sample of US adults (aged ≥18 years). Four firearm ownership groups were created: non-owners, a proxy for first-time COVID-19 owners, prepandemic owners with COVID-19 purchase, and prepandemic owners without COVID-19 purchase. Explanatory variables were in four domains: demographics, concern about the pandemic, actions taken in response to COVID-19, and emotional response to COVID-19. Multivariate analysis estimated the adjusted odds of the outcomes. RESULTS: Respondents were categorized as non-owners (n = 2440), pandemic-related purchasers with no other firearms (n = 257), pandemic-related purchasers with other firearms (n = 350), and those who did not purchase in response to the pandemic but have other firearms (n = 806). Multivariable logistic regression found that compared with non-owners, those who had firearms at home with no pandemic-related purchases are more likely to be male, live in rural settings, have higher income, and be Republican. CONCLUSIONS: The results highlight the changing profile of American firearm owners and identify that those who purchased firearms for the first time (in response to the pandemic) should be the focus of tailored public health interventions, including provision of education about recommended firearm storage to reduce firearm violence, particularly because they are more likely to have children at home, and belong to demographic groups that may have less experience with firearm safety.

Borrelia burgdorferi Engages Mammalian Type I IFN Responses via the cGAS-STING Pathway.

Borrelia burgdorferi, the etiologic agent of Lyme disease, is a spirochete that modulates numerous host pathways to cause a chronic, multisystem inflammatory disease in humans. B. burgdorferi infection can lead to Lyme carditis, neurologic complications, and arthritis because of the ability of specific borrelial strains to disseminate, invade, and drive inflammation. B. burgdorferi elicits type I IFN (IFN-I) responses in mammalian cells and tissues that are associated with the development of severe arthritis or other Lyme-related complications. However, the innate immune sensors and signaling pathways controlling IFN-I induction remain unclear. In this study, we examined whether intracellular nucleic acid sensing is required for the induction of IFN-I to B. burgdorferi. Using fluorescence microscopy, we show that B. burgdorferi associates with mouse and human cells in culture, and we document that internalized spirochetes colocalize with the pattern recognition receptor cyclic GMP-AMP synthase (cGAS). Moreover, we report that IFN-I responses in mouse macrophages and murine embryonic fibroblasts are significantly attenuated in the absence of cGAS or its adaptor stimulator of IFN genes (STING), which function to sense and respond to intracellular DNA. Longitudinal in vivo tracking of bioluminescent B. burgdorferi revealed similar dissemination kinetics and borrelial load in C57BL/6J wild-type, cGAS-deficient, or STING-deficient mice. However, infection-associated tibiotarsal joint pathology and inflammation were modestly reduced in cGAS-deficient compared with wild-type mice. Collectively, these results indicate that the cGAS-STING pathway is a critical mediator of mammalian IFN-I signaling and innate immune responses to B. burgdorferi.

"I am not a junkie": Social categorization and differentiation among people who use drugs.

BACKGROUND: Substance use stigma is a form of group-based exclusion, and delineating pathways from stigma to poor health requires a deeper understanding of the social dynamics of people who use drugs (PWUD). Outside of recovery, scant research has examined the role of social identity in addiction. Framed by Social Identity Theory/Self-Categorization Theory, this qualitative study investigated strategies of within-group categorization and differentiation among PWUD and the roles these social categories may play in shaping intragroup attitudes, perceptions, and behaviors. METHODS: Data come from the Rural Opioid Initiative, a multi-site study of the overdose epidemic in rural United States. We conducted in-depth interviews with people who reported using opioids or injecting any drug (n=355) living in 65 counties across 10 states. Interviews focused on participants' biographical histories, past and current drug use, risk behaviors, and experiences with healthcare providers and law enforcement. Social categories and dimensions along which categories were evaluated were inductively identified using reflexive thematic analysis. RESULTS: We identified seven social categories that were commonly appraised by participants along eight evaluative dimensions. Categories included drug of choice, route of administration, method of attainment, gender, age, genesis of use, and recovery approach. Categories were evaluated by participants based on ascribed characteristics of morality, destructiveness, aversiveness, control, functionality, victimhood, recklessness, and determination. Participants performed nuanced identity work during interviews, including reifying social categories, defining 'addict' prototypicality, reflexively comparing self to other, and disidentifying from the PWUD supra-category. CONCLUSION: We identify several facets of identity, both behavioral and demographic, along which people who use drugs perceive salient social boundaries. Beyond an addiction-recovery binary, identity is shaped by multiple aspects of the social self in substance use. Patterns of categorization and differentiation revealed negative intragroup attitudes, including stigma, that may hinder solidary-building and collective action in this marginalized group.

Acceptability and feasibility of long-acting injectable antiretroviral therapy for HIV-infected persons who inject drugs in Vietnam: A qualitative study.

Background: In Vietnam, HIV prevalence among people who inject drugs (PWID) is several times higher than in the general population (15% versus 0.3%). PWID also experience higher rates of HIV-related mortality, driven by poor antiretroviral therapy (ART) adherence. Long-acting injectable ART (LAI) is a compelling opportunity to improve treatment outcomes, but acceptability and feasibility among HIV-infected PWID remains unexplored. Methods: We conducted key informant in-depth interviews in Hanoi, Vietnam (February-November 2021). Participants were purposively sampled and included policymakers, ART clinic staff, and HIV-infected PWID. We applied the Consolidated Framework for Implementation Research to guide study design and analysis, using thematic coding to develop and iteratively refine a codebook and characterize barriers and facilitators to LAI implementation. Findings: We interviewed 38 key stakeholders: 19 PWID, 14 ART clinic staff, and five policymakers. Participants were enthusiastic about LAI convenience, highlighting less frequent and more discreet dosing. However, contrasting providers, several policymakers suggested LAI was not needed given perceived exceptional oral ART outcomes and rare viral failure among PWID. Policymakers also criticized strategies prioritizing PWID for LAI, emphasizing equity, whereas providers identified PWID as an ideal population for LAI given adherence challenges. LAI complexity, including storage and administration logistics, were deemed surmountable with training and resources. Finally, providers and policymakers acknowledged that adding LAI to drug formularies was key, but an onerous process. Interpretation: Although anticipated to be resource-intensive, LAI was a welcome addition for interviewed stakeholders and likely an acceptable alternative to oral ART among PWID living with HIV in Vietnam. Despite enthusiasm among PWID and providers that LAI could improve viral outcomes, some policymakers-whose buy-in is critical to LAI implementation-opposed strategies that preferentially distributed LAI to PWID, highlighting values of equity and revealing differences in perceived HIV outcomes among PWID. Results provide a vital foundation for developing LAI implementation strategies. Funding: Supported by National Institutes of Health.

Differential DNA methylation of steatosis and non-alcoholic fatty liver disease in adolescence.

BACKGROUND AND AIMS: Epigenetic modifications are associated with hepatic fat accumulation and non-alcoholic fatty liver disease (NAFLD). However, few epigenetic modifications directly implicated in such processes have been identified during adolescence, a critical developmental window where physiological changes could influence future disease trajectory. To investigate the association between DNA methylation and NAFLD in adolescence, we undertook discovery and validation of novel methylation marks, alongside replication of previously reported marks. APPROACH AND RESULTS: We performed a DNA methylation epigenome-wide association study (EWAS) on DNA from whole blood from 707 Raine Study adolescents phenotyped for steatosis score and NAFLD by ultrasound at age 17. Next, we performed pyrosequencing validation of loci within the most 100 strongly associated differentially methylated CpG sites (dmCpGs) for which ≥ 2 probes per gene remained significant across four statistical models with a nominal p value < 0.007. EWAS identified dmCpGs related to three genes (ANK1, MIR10a, PTPRN2) that met our criteria for pyrosequencing. Of the dmCpGs and surrounding loci that were pyrosequenced (ANK1 n = 6, MIR10a n = 7, PTPRN2 n = 3), three dmCpGs in ANK1 and two in MIR10a were significantly associated with NAFLD in adolescence. After adjustment for waist circumference only dmCpGs in ANK1 remained significant. These ANK1 CpGs were also associated with γ-glutamyl transferase and alanine aminotransferase concentrations. Three of twenty-two differentially methylated dmCpGs previously associated with adult NAFLD were associated with NAFLD in adolescence (all adjusted p < 2.3 × 10-3). CONCLUSIONS: We identified novel DNA methylation loci associated with NAFLD and serum liver biochemistry markers during adolescence, implicating putative dmCpG/gene regulatory pathways and providing insights for future mechanistic studies.

Taxonomic and Metagenomic Analyses Define the Development of the Microbiota in the Chick.

Chicks are ideal to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Taxonomic/metagenomic analyses captured the development of the chick microbiota in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm) during development. Taxonomic analysis suggests that colonization by the chicken microbiota takes place in several waves. The cecal microbiota stabilizes at day 12 posthatch with prominent Gammaproteobacteria and Clostridiales. Introduction of S. Typhimurium at day 4 posthatch disrupted the expected waves of intestinal colonization. Taxonomic and metagenomic shotgun sequencing analyses allowed us to identify species present in uninfected chicks. Untargeted metabolomics suggested different metabolic activities in infected chick microbiota. This analysis and gas chromatography-mass spectrometry on ingesta confirmed that lactic acid in cecal content coincides with the stable presence of enterococci in STm-infected chicks. Unique metabolites, including 2-isopropylmalic acid, an intermediate in the biosynthesis of leucine, were present only in the cecal content of STm-infected chicks. The metagenomic data suggested that the microbiota in STm-infected chicks contained a higher abundance of genes, from STm itself, involved in branched-chain amino acid synthesis. We generated an ilvC deletion mutant (STM3909) encoding ketol-acid-reductoisomerase, a gene required for the production of l-isoleucine and l-valine. ΔilvC mutants are disadvantaged for growth during competitive infection with the wild type. Providing the ilvC gene in trans restored the growth of the ΔilvC mutant. Our integrative approach identified biochemical pathways used by STm to establish a colonization niche in the chick intestine during development. IMPORTANCE Chicks are an ideal model to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Using taxonomic and metagenomic analyses, we captured the development of chick microbiota to 19 days posthatch in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm). We show that normal development of the microbiota takes place in waves and is altered in the presence of a pathogen. Metagenomics and metabolomics suggested that branched-chain amino acid biosynthesis is especially important for Salmonella growth in the infected chick intestine. Salmonella mutants unable to make l-isoleucine and l-valine colonize the chick intestine poorly. Restoration of the pathway for biosynthesis of these amino acids restored the colonizing ability of Salmonella. Integration of multiple analyses allowed us to correctly identify biochemical pathways used by Salmonella to establish a niche for colonization in the chick intestine during development.