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.

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.

"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.

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.

Elucidating Mechanisms of Tolerance to Salmonella Typhimurium across Long-Term Infections Using the Collaborative Cross.

Understanding the molecular mechanisms underlying resistance and tolerance to pathogen infection may present the opportunity to develop novel interventions. Resistance is the absence of clinical disease with a low pathogen burden, while tolerance is minimal clinical disease with a high pathogen burden. Salmonella is a worldwide health concern. We studied 18 strains of collaborative cross mice that survive acute Salmonella Typhimurium (STm) infections. We infected these strains orally and monitored them for 3 weeks. Five strains cleared STm (resistant), six strains maintained a bacterial load and survived (tolerant), while seven strains survived >7 days but succumbed to infection within the study period and were called "delayed susceptible." Tolerant strains were colonized in the Peyer's patches, mesenteric lymph node, spleen, and liver, while resistant strains had significantly reduced bacterial colonization. Tolerant strains had lower preinfection core body temperatures and had disrupted circadian patterns of body temperature postinfection sooner than other strains. Tolerant strains had higher circulating total white blood cells than resistant strains, driven by increased numbers of neutrophils. Tolerant strains had more severe tissue damage and higher circulating levels of monocyte chemoattractant protein 1 (MCP-1) and interferon gamma (IFN-γ), but lower levels of epithelial neutrophil-activating protein 78 (ENA-78) than resistant strains. Quantitative trait locus (QTL) analysis revealed one significant association and six suggestive associations. Gene expression analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals that overlapped these QTLs. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and top canonical pathways, making them the best candidate genes for differentiating tolerance and resistance. IMPORTANCE To survive a bacterial infection, an infected host can display resistance or tolerance. Resistance is indicated by a decrease in pathogen load, while for tolerance a high pathogen load is accompanied by minimal disease. We infected genetically diverse mice with Salmonella Typhimurium for 21 days and discovered new phenotypes for disease outcome (delayed susceptible, tolerant, and resistant). Tolerant strains showed the lowest preinfection core body temperatures and the most rapid disruption in circadian patterns of body temperature postinfection. Tolerant strains had higher circulating neutrophils and higher circulating levels of MCP-1 and IFN-γ, but lower levels of ENA-78 than did resistant strains, in addition to more severe tissue damage. QTL analysis revealed multiple associated regions, and gene expression analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals in these regions. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and the top canonical pathways, suggesting a role in tolerance.

Comparison of the cardiometabolic profiles of adolescents conceived through ART with those of a non-ART cohort.

STUDY QUESTION: Is the cardiometabolic health of adolescents conceived through ART worse than that of their counterparts conceived without ART? SUMMARY ANSWER: The majority of cardiometabolic and vascular health parameters of adolescents conceived through ART are similar or more favourable, than those of their counterparts of similar age and conceived without ART. WHAT IS KNOWN ALREADY: It has been proposed that the cardiometabolic health of offspring conceived with ART may be unfavourable compared to that of their counterparts conceived without ART. The literature pertaining to cardiometabolic health of offspring conceived after ART is contradictory, but generally suggests unfavourable cardiometabolic health parameters, such as an increase in blood pressure (BP), vascular dysfunction and adiposity, as well as unfavourable glucose and lipid profiles. With over 8 million children and adults born through ART worldwide, it is important to investigate whether these early signs of adverse cardiometabolic differences persist into adolescence and beyond. STUDY DESIGN, SIZE, DURATION: The Growing Up Healthy Study (GUHS) is a prospective cohort study that recruited 303 adolescents and young adults conceived after ART (aged 13-21 years) and born between 1991 and 2001 in Western Australia. Their health parameters, including cardiometabolic factors, were assessed and compared with counterparts from the Raine Study Generation 2 (Gen2). The 2868 Gen2 participants were born 1989-1992 and are representative of the Western Australian adolescent population. At ∼17 years of age (2013-2017), 163 GUHS participants replicated assessments previously completed by Gen2 at a similar age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cardiometabolic parameters were compared between a total of 163 GUHS and 1457 Gen2 adolescents. Separate male (GUHS n = 81, Gen2 n = 735) and female (GUHS n = 82, Gen2 n = 722) analyses were conducted. Assessments consisted of a detailed questionnaire including health, lifestyle and demographic parameters, anthropometric assessments (height, weight, BMI, waist circumference and skinfold thickness), fasting serum biochemistry, arterial stiffness and BP (assessed using applanation tonometry). Abdominal ultrasonography was used to assess the presence and severity of hepatic steatosis, and thickness of abdominal fat compartments. Non-alcoholic fatty liver disease (NAFLD) was diagnosed if there was sonographic fatty liver in the absence of significant alcohol consumption. Chi2, Fisher's exact and Mann-Whitney U tests, performed in SPSS V25, examined cohort differences and generalized estimating equations adjusted for the following covariates: singleton vs non-singleton pregnancy, birthweight (z-score), gestational age, BMI, smoking, alcohol consumption in the past 6 months and parent cardiovascular status. Arterial stiffness measures and waist circumference were additionally adjusted for height, and female analyses were additionally adjusted for use of oral contraceptives in the preceding 6 months. MAIN RESULTS AND THE ROLE OF CHANCE: In adjusted analyses, GUHS females had a lower BMI (22.1 vs 23.3 kg/m2, P = 0.014), and thinner skinfolds (triceps, subscapular, mid-abdominal; 16.9 vs 18.7 mm, P = 0.021, 13.4 vs 15.0 mm, P = 0.027, 19.7 vs 23.2 mm, P < 0.001, respectively), whereas males were not significantly different. Waist circumference was lower in GUHS adolescents (males: 78.1 vs 81.3 cm, P = 0.008, females: 76.7 vs 83.3 cm, P = 0.007). There were no significant differences between the two groups in glucose, insulin, homeostatic model assessment for insulin resistance, low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol (non-HDL-C), total cholesterol (TC), alanine aminotransferase and high-sensitivity C-reactive protein in both sexes. In females, serum triglycerides were lower in GUHS adolescents (1.0 vs 1.2 mmol/l, P = 0.029). GUHS males had higher serum HDL-C (1.1 vs 1.0 mmol/l, P = 0.004) and a lower TC/HDL-C ratio (3.2 vs 3.6, P = 0.036). There were no significant differences in the prevalence of NAFLD or steatosis severity scores between the cohorts in males and females. GUHS females had less subcutaneous adipose tissue (9.4 vs 17.9 mm, P < 0.001), whereas GUHS males had greater visceral adipose thickness (44.7 vs 36.3 mm, P < 0.001). There was no significant difference in pre-peritoneal adipose thickness. Pulse wave velocity was lower in GUHS males (5.8 vs 6.3 m/s, P < 0.001) and heart rate corrected augmentation index was lower in GUHS females (-8.4 vs -2.7%, P = 0.048). There were no significant differences in BP or heart rate in males or females between the two groups. LIMITATIONS, REASONS FOR CAUTION: Despite the substantial study size and the unique study design of the ART cohort, we were unable to differentiate between different types of ART, due to the low number of ICSI cycles (e.g. IVF vs ICSI), draw definite conclusions, or relate the outcomes to the cause of infertility. Considering the differences in time points when both cohorts were studied, external factors could have changed, which could not be accounted for. Given the observational nature of this study, causation cannot be proven. WIDER IMPLICATIONS OF THE FINDINGS: Contrary to our hypothesis and previous findings focussing mainly on childhood, this study reports mostly similar or favourable cardiometabolic markers in adolescents conceived with ART compared to those conceived without ART. The greater visceral adipose thickness, particularly present in males, requires further investigation. While these findings are generally reassuring, future well-designed and appropriately powered studies are required to definitively address the issue of cardiometabolic health in ART adults. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NHMRC project grant number 1042269 and R.J.H. received education grant funding support from Ferring Pharmaceuticals. R.J.H. is the Medical Director of Fertility Specialists of Western Australia and a shareholder in Western IVF. He has received educational sponsorship from MSD, Merck-Serono and Ferring Pharmaceuticals. P.B. is the Scientific Director of Concept Fertility Centre, Subiaco, Western Australia. J.L.Y. is the Medical Director of PIVET Medical Centre, Perth, Western Australia. TRIAL REGISTRATION NUMBER: N/A.

Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross.

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection.

Vaginal Inoculation of Uropathogenic Escherichia coli during Estrus Leads to Genital and Renal Colonization.

Urinary tract infection (UTI) is one of the most prevalent bacterial infections, particularly in women, children, and the elderly. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Uropathogens are directly instilled in the urinary bladder, bypassing the lower urogenital tract, in the widely used murine model of UTI. We assessed whether vaginal inoculation of UPEC led to UTI and how stages of the estrous cycle would impact bacterial colonization in mice. Mice in proestrus, estrus, metestrus, and diestrus were identified by vaginal cytology and inoculated with UPEC in the vaginal tract. Mice were euthanized 1 day after infection, and bacterial loads in the urogenital tract, liver, and spleen were enumerated. Mice in estrus exhibited the highest and most consistent UPEC burdens in all organs, except the bladder. Vaginal inoculation resulted in bladder colonization in a UPEC strain-specific manner. In contrast, transurethral inoculation of UPEC led to bladder colonization. Importantly, inoculation by both routes led to vaginal and uterine colonization and concomitant systemic dissemination to the spleen and liver. The kinetics of bacterial colonization over 2 weeks following vaginal inoculation was comparable in the urogenital tract. Tissue sections revealed the induction of vaginitis and cystitis upon the vaginal instillation of UPEC. In summary, vaginal inoculation of UPEC in mice during estrus represents a novel approach to investigate infection of the kidneys and genital tract and systemic dissemination from the urogenital tract. Our findings suggest that estrogen primes the urogenital tract to create a conducive milieu for UPEC colonization.

Implementing Digital Pathology into Veterinary Academics and Research.

The advantages of digital pathology (DP) have been recognized as early as 1963, but only within the last decade or so have the advancements of slide scanners and viewing software made the use and implementation of DP feasible in the classroom and in research. Several factors must be considered prior to undertaking the project of implementing the DP workflow in any setting, but particularly in an academic environment. Sustained and open dialogue with information technology (IT) is critical to the success of this enterprise. In addition to IT, there is a multitude of criteria to consider when determining the best hardware and software to purchase to support the project. The goals and limitations of the laboratory and the requirements of its users (students, instructors, and researchers) will ultimately direct these decisions. The objectives of this article are to provide an overview of the opportunities and challenges associated with the integration of DP in education and research, to highlight some important IT considerations, and to discuss some of the requirements and functionalities of some hardware and software options.

Copper Resistance Promotes Fitness of Methicillin-Resistant Staphylococcus aureus during Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

The Relationship Between Hepatitis C Virus Rates and Office-Based Buprenorphine Access in Ohio.

BACKGROUND: The United States is experiencing an epidemic of hepatitis C virus (HCV) infections due to injection drug use, primarily of opioids and predominantly in rural areas. Buprenorphine, a medication for opioid use disorder, may indirectly prevent HCV transmission. We assessed the relationship of HCV rates and office-based buprenorphine prescribing in Ohio. METHODS: We conducted an ecological study of the county-level (n = 88) relationship between HCV case rates and office-based buprenorphine prescribing in Ohio. We fit adjusted negative binomial models between the county-level acute and total HCV incidence rates during 2013-2017 and 1) the number of patients in each county that could be served by office-based buprenorphine (prescribing capacity) and 2) the number served by office-based buprenorphine (prescribing frequency) from January-March, 2018. RESULTS: For each 10% increase in acute HCV rate, office-based buprenorphine prescribing capacity differed by 1% (95% CI: -1%, 3%). For each 10% increase in total HCV rate, office-based buprenorphine prescribing capacity was 12% (95% CI: 7%, 17%) higher. For each 10% increase in acute HCV rate, office-based buprenorphine prescribing frequency was 1% (95% CI: -1%, 3%) higher. For each 10% increase in total HCV rate, office-based buprenorphine prescribing frequency was 14% (95% CI: 7%, 20%) higher. CONCLUSIONS: Rural counties in Ohio have less office-based buprenorphine and higher acute HCV rates versus urban counties, but a similar relationship between office-based buprenorphine prescribing and HCV case rates. To adequately prevent and control HCV rates, certain rural counties may need more office-based buprenorphine prescribing in areas with high HCV case rates.