The effect of Proteinase K treatment on GeoMx digital spatial profiling data quality from formalin-fixed, paraffin-embedded tissue.

The emergence of spatial profiling technologies in recent years has accelerated opportunities to profile in detail the molecular attributes of a wide range of tissue pathologies using archival specimens. However, tissue treatment for fixation and storage does not always support generation of high-quality genomic data. The purpose of this study was to investigate the impacts of Proteinase K (ProtK) treatment, as a way to increase target transcript exposure, on downstream sequencing data quality metrics for spatial transcriptomic data using formalin-fixed, paraffin-embedded samples. In a series of four independent assessments using different tissue types (nasal mucosa, tonsil, pancreas), varying concentrations of ProtK (ranging from 0.1 to 1 µg/mL) were used as part of the sample processing workflow to generate transcriptomic data using the Nanostring GeoMx DSP and Illumina NextSeq 2000 platforms. Use of higher concentrations of ProtK was generally found to increase total reads (2-4-fold). However, negative probe counts also tended to be increased (2-12-fold), resulting in reductions in the signal-to-noise ratio (10-70% lower) and the number of genes detected above background (50-80% lower). These effects were not seen in all tissues and impacts of tissue handling and processing, beyond ProtK treatment, on data quality metrics, also require consideration. Regardless, these observations highlight the need for careful consideration of a range of sample processing factors and benefits that may be achieved through the optimisation of sample processing workflows for specific tissues as a way to maximise the generation of quality data using spatial transcriptomic approaches.

Pharmacokinetics of a topical application of moxidectin in bare-nosed wombats (Vombatus ursinus).

Sarcoptic mange is a debilitating disease that affects bare-nosed wombats (Vombatus ursinus). One of the drugs currently used for treatment is moxidectin, as it has a relatively high efficacy against endo and ectoparasites and side effects are uncommon in domestic species, thus it is considered a relatively safe drug to use at the recommended doses. Developing further understanding of the pharmacokinetics of moxidectin will aid in developing treatment regimens for sarcoptic mange in wombats. Here we analyzed the pharmacokinetic parameters of using 100 ml of moxidectin (5 g/l) applied topically. We found that mean peak plasma concentration was 0.50 ng/ml and half-life was 8 days. Moxidectin was excreted in scats with the mean peak concentration of 2461.43 ng/g (on a dry matter basis). Our study has provided the pharmacokinetic parameters of a commonly used treatment for sarcoptic mange in wombats. There were no adverse side effects recorded in the wombats after applying moxidectin topically. This study replicated real-world conditions using topical application on free-living wombats. The relatively low plasma concentration suggests the drug is not accumulating in the blood stream and is excreted via scats.

Molecular analysis of human tick-bitten skin yields signatures associated with distinct spatial and temporal trajectories - A proof-of-concept study.

Tick-associated diseases present challenges due to tridirectional interactions among host-specific responses, tick toxins and salivary proteins as well as microbes. We aimed to uncover molecular mechanisms in tick-bitten skin samples (cases) and contralateral skin samples (controls) collected simultaneously from the same participants, using spatial transcriptomics. Cases and controls analysed using NanoString GeoMx Digital Spatial Profiler identified 274 upregulated and 840 downregulated differentially expressed genes (DEGs), revealing perturbations in keratinization and immune system regulation. Samples of skin biopsies taken within 72 h post tick-bite DEGs had changes in protein metabolism and viral infection pathways as compared to samples taken 3 months post tick-bite, which instead displayed significant perturbations in several epigenetic regulatory pathways, highlighting the temporal nature of the host response following tick bites. Within-individual signatures distinguished tick-bitten samples from controls and identified between-individual signatures, offering promise for future biomarker discovery to guide prognosis and therapy.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Current trends in the role of neuroinflammation & α-synuclein in alcohol use disorder: A systematic quantitative literature review.

The neurodegenerative effects alcohol use disorder (AUD) have been well characterized and are likely due to the long-term effects of alcohol on the brain. The molecular events that underlie regional neuronal loss are a focus of current research. Chronic inflammation in the central nervous system, termed neuroinflammation, contributes to the progressive loss of neurons in the brain. Using data from genome-wide association studies and genetic and gene expression data, α-synuclein was identified as a gene of interest for AUD almost 10 years ago. Despite this and the well-recognized role of α-synuclein in mediating neuroinflammation in other neurodegenerative diseases, its role in alcohol-induced brain damage and AUD is yet to be elucidated. This systematic literature review quantifies and analyzes relationships between AUD, α-synuclein, and neuroinflammation. The review identified fewer studies focused on the role in AUD of α-synuclein (30) than on neuroinflammation (177), with published studies heavily centered on the myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor 4 (TLR4) pathway. The systematic review revealed that no original literature investigates the roles of α-synuclein and neuroinflammation in AUD and that there are significantly fewer published articles on the role of α-synuclein in AUD than in other neuroinflammatory conditions. Studies of the role of neuroinflammation in AUD are largely centered on the TLR4 signaling cascade, followed by TLR2 and TLR3, and soluble cytokines such as IL-10, IL-1ß, and TNF-α. Key research themes identified in other neurodegenerative disorders provide new insights for further investigation in AUD.

Gut Microbiome and Metabolic and Immune Indices in Males with or without Evidence of Metabolic Dysregulation.

Background: The contributions of the gut microbiota to obesity and metabolic disease represent a potentially modifiable factor that may explain variation in risk between individuals. This study aimed to explore relationships among microbial composition and imputed functional attributes, a range of soluble metabolic and immune indices, and gene expression markers in males with or without evidence of metabolic dysregulation (MetDys). Methods: This case-control study included healthy males (n=15; 41.9±11.7 years; body mass index [BMI], 22.9±1.2 kg/m2) and males with evidence of MetDys (n=14; 46.6±10.0 years; BMI, 35.1±3.3 kg/m2) who provided blood and faecal samples for assessment of a range of metabolic and immune markers and microbial composition using 16S rRNA gene sequencing. Metagenomic functions were imputed from microbial sequence data for analysis. Results: In addition to elevated values in a range of traditional metabolic, adipokine and inflammatory indices in the MetDys group, 23 immunomodulatory genes were significantly altered in the MetDys group. Overall microbial diversity did not differ between groups; however, a trend for a higher relative abundance of the Bacteroidetes (P=0.06) and a lower relative abundance of the Verrucomicrobia (P=0.09) phyla was noted in the MetDys group. Using both family- and genera-level classifications, a partial least square discriminant analysis revealed unique microbial signatures between the groups. Conclusion: These findings confirm the need for ongoing investigations in human clinical cohorts to further resolve the relationships between the gut microbiota and metabolic and immune markers and risk for metabolic disease.

Lack of Informed and Affirming Healthcare for Sexual Minority Men: A Call for Patient-Centered Care.

BACKGROUND: Sexual minority men (SMM) face severe health inequities alongside negative experiences that drive avoidance of medical care. Understanding how SMM experience healthcare is paramount to improving this population's health. Patient-centered care, which emphasizes mutual respect and collaboration between patients and providers, may alleviate the disparaging effects of the homophobia that SMM face in healthcare settings. OBJECTIVE: To explore how SMM perceive their experiences with healthcare providers and how care can most effectively meet their needs. DESIGN: Semi-structured qualitative interviews focused on healthcare experiences, pre-exposure prophylaxis (PrEP), and HIV-related beliefs were conducted between July and November 2018. PARTICIPANTS: The study included a sample of 43 young adult SMM (ages 25-27), representing diverse socioeconomic, racial, and ethnic backgrounds, in New York City. APPROACH: Researchers utilized a multiphase, systematic coding method to identify salient themes in the interview transcripts. KEY RESULTS: Analyses revealed three main themes: (1) SMM perceived that their clinicians often lack adequate skills and knowledge required to provide care that considers participants' identities and behaviors; (2) SMM desired patient-centered care as a way to regain agency and actively participate in making decisions about their health; and (3) SMM felt that patient-centered care was more common with providers who were LGBTQ-affirming, including many who felt that this was especially true for LGBTQ-identified providers. CONCLUSIONS: SMM expressed a clear and strong desire for patient-centered approaches to care, often informed by experiences with healthcare providers who were unable to adequately meet their needs. However, widespread adoption of patient-centered care will require improving education and training for clinicians, with a focus on LGBTQ-specific clinical care and cultural humility. Through centering patients' preferences and experiences in the construction of care, patient-centered care can reduce health inequities among SMM and empower healthcare utilization in a population burdened by historic and ongoing stigmatization.

Influence of sex and liver cirrhosis on the expression of miR-146a-5p and its target genes, IRAK1 and TRAF6.

Long-term alcohol misuse triggers cellular adaptions in susceptible regions of the human brain, resulting in neurodegeneration, neuroinflammation and altered gene expression. Previous studies have identified ∼35 miRNAs, including miR-146a-5p, which are up-regulated in the frontal cortex of males with alcohol use disorder (AUD), but the influence of liver cirrhosis and sex is unknown. The expression of miR-146a-5p, IRAK1, and TRAF6 was measured in the prefrontal cortex of controls and individuals with AUD with and without cirrhosis of the liver. Further, individuals were genotyped for two SNPs, rs2910164 and rs57095329. The expression of miR-146a-5p was significantly different between sexes. In males the expression of miR-146a-5p was increased in individuals with AUD with and without liver cirrhosis compared with controls. In females miR-146a-5p expression was significantly lower in individuals with AUD compared with both controls and those with AUD and cirrhosis, suggesting that both the severity of alcohol misuse and the sex of the individual influences the expression of miR-146a-5p. The expression of TRAF6 was significantly lower in individuals with uncomplicated AUD compared with those with AUD and cirrhosis. The expression of IRAK1 did not differ between groups or sexes. There was no influence of genotype on expression. Increased expression of miR-146a-5p did not correlate with decreased IRAK1 or TRAF6 expression suggesting a loss of regulatory control of the TLR4 pathway. Understanding sex-specific differences in the regulation of gene expression in AUD is key to determine which inflammatory pathways could be targeted for therapeutic intervention.

A Pilot Randomized Trial Evaluating the Cool 2 Be Safe Junior Playground Safety Program for Preschool Children.

OBJECTIVE: Unintentional injury is the leading cause of death to children worldwide, and playgrounds pose a significant risk of injury. Those aged 5 and 6 years are particularly vulnerable to playground injuries. Previous interventions have typically targeted environmental modifications or increased supervision to reduce playground injuries; however, minimal research has focused on implementing behavioral interventions that seek to change children's safety knowledge and risk-taking behaviors on the playground. The current randomized trial addressed these gaps in the literature and sought to increase preschool children's hazard awareness skills and knowledge of unsafe playground behaviors and decrease their intentions to engage in risk-taking behaviors on the playground. METHOD: A total of 77 children aged 5 and 6 years were tested at a laboratory on a university campus, using a parallel group design, with 38 randomized to the playground intervention group and 39 randomly assigned to a control group. The intervention was manualized and delivered to small groups of children (3-5) over several sessions. RESULTS: Statistical analyses revealed significant group differences. Intervention experience led to significant increases in hazard awareness skills and knowledge of unsafe playground behaviors, and significant decreases in children's risk-taking behavioral intentions, whereas those in the control group showed no significant changes in these scores. Attention scores for those in the intervention group highlighted that the program content was successful in maintaining children's attention and engagement, and memory scores indicated excellent retention of lesson content. CONCLUSION: The program shows promise in addressing the issue of unintentional playground injuries in young children.

Precautionary Allergen Labeling: Avoidance for All?

PURPOSE OF REVIEW: Precautionary allergen labeling (PAL) suggests the risk of unintended allergen presence (UAP) in food but is unregulated in most countries and inconsistently applied by food manufacturers. This review evaluates the current use of PAL, its relevance to allergic consumers, and weighs possible advantages and disadvantages of avoiding products with PAL. RECENT FINDINGS: In most countries, manufacturers are free to decide whether, when, and how to apply PAL resulting in inconsistencies and consumer confusion. Patients with food allergy often interpret PAL incorrectly and without guidance from their health care providers. Health care providers are also prone to misinterpreting PAL, indicating a need for better education. Consumers desire guidance on whether to avoid products with PAL or not. Until further regulatory guidance is available, shared decision-making between patient and provider is required to offer individualized, rather than one-size-fits-all, approaches to PAL.

Extraction and analysis of moxidectin in wombat plasma and faeces.

Sarcoptic mange in wombats results from a skin infestation by Sarcoptes mites and if untreated, results in a slow and painful death. Moxidectin is a pesticide used to treat internal and external parasites in cattle, but has shown to effectively treat other animals, including wombats. Two methods were developed to analyse wombat plasma, and methods were also developed to analyse faeces and fur. Moxidectin-D3 was used as an internal standard and behaved almost identically to moxidectin, resulting in recoveries of 95-105 % across the three matrices, even when matrix interferences caused signal suppression as high 20 %, or when moxidectin loss was high. This was presumably due to the high binding efficiency of plasma for MOX and MOX-D3. Moxidectin limits of detection were 0.01 ng/mL in plasma, 0.3 ng/g dry weight equivalent for faeces and 0.5 ng/g for fur. This study also developed a method to isolate plasma macromolecules, allowing the extraction of bound moxidectin for quantitative purposes, with an LoQ of 0.05 ng/mL. This method was subsequently used to determine that moxidectin was 97-99.4 % bound to lipoproteins in wombat plasma and 98-99 % bound in sheep, cow and horse plasma. The method reported for plasma was quick, cheap, and conducive to large sample batches, while providing high sensitivity. While faecal samples required additional cleanup steps to reduce the matrix effect, co-extracted matrix components such as undigested chlorophyll continued to result in ionisation suppression in the MS/MS. The methods reported here were used to monitor moxidectin in wombats treated with a single pour-on treatment, and confirmed that the moxidectin concentration in wombat plasma had decreased by more than 90 % by 28 days after application, while providing protection against sarcoptic mites over the majority of their life cycle. Clearance of moxidectin occurred via faecal elimination over the four week period and while moxidectin accumulated on fur due to application as a pour-on, concentrations declined rapidly by the four week period as fur fell out and was replaced by fresh fur that did not contain moxidectin.

Challenges to Providing Integrated HIV Prevention in Substance Use Treatment Settings: Frontline Staff Perspectives on HIV and Sex-Related, Education, Communication and Stigma.

INTRODUCTION: Substance use treatment settings can play a critical role in ending the HIV epidemic. Community-based methadone clinics are potentially useful sites to offer biomedical HIV prevention, but little is known about how clinicians and other clinic staff communicate with patients about sexual behavior and HIV-related topics. METHODS: Thirty semi-structured interviews were conducted at two methadone clinics in Northern New Jersey. Participants included medical providers (physicians, RNs, DNPs), methadone counselors, intake coordinators, lab technicians, and other auxiliary staff members. Results: Three major themes were identified: (1) HIV education is primarily provided by external organizations, (2) there is limited staff-patient communication around HIV and sexual behaviors, and (3) HIV stigma is prevalent among staff and patients. CONCLUSION: To implement PrEP in methadone treatment settings, clinic staff must be able to engage in non-judgmental communication about HIV and sex with patients. Additionally, federal and state funding for HIV prevention in substance use treatment settings must be prioritized to enable clinics to access the necessary training and resources.

Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples.

OBJECTIVE: Growth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34-69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods. RESULTS: Diversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.

Medical Mistrust Among a Racially and Ethnically Diverse Sample of Sexual Minority Men.

Purpose: Medical mistrust is a barrier to health care utilization and is associated with suboptimal health outcomes. Research on mistrust among sexual minority men (SMM) is limited and largely focuses on Black SMM and HIV, with few studies assessing mistrust among SMM of other race/ethnicities. The purpose of this study was to examine differences in medical mistrust among SMM by race. Methods: From February 2018 to February 2019, a mixed-methods study examined the health-related beliefs and experiences of young SMM in New York City. The Group-Based Medical Mistrust Scale (GBMMS) was used to measure medical mistrust related to race, and a modified version of the scale assessed mistrust related to one's "sexual/gender minority" status (Group-Based Medical Mistrust Scale-Sexual/Gender Minority [GBMMS-SGM]). With an analytic sample of 183 cisgender SMM, a one-way multivariate analysis of variance was used to examine differences in GBMMS and GBMMS-SGM scores by race/ethnicity [Black, Latinx, White, "Another Racial Group(s)"]. Results: There were significantly different GBMMS scores by race, with participants of color reporting higher levels of race-based medical mistrust than White participants. This finding is supported by effect sizes ranging from moderate to large. Differences in GBMMS-SGM scores by race were borderline; however, the effect size for Black and White participants' GBMMS-SGM scores was moderate, indicating that higher GBMMS-SGM scores among Black participants is meaningful. Conclusion: Multilevel strategies should be used to earn the trust of minoritized populations, such as addressing both historical and ongoing discrimination, moving beyond implicit bias trainings, and strengthening the recruitment and retention of minoritized health care professionals.

Assessment of a Multispecies Probiotic Supplement for Relief of Seasonal Allergic Rhinitis: A Randomized Double-Blind Placebo-Controlled Trial.

Background: Early phase clinical research provided initial support for the use of a multispecies probiotic supplement to improve quality of life (QoL) in adults with seasonal allergic rhinitis (AR) and reduce the use of AR symptom relieving medication. This study aimed to confirm these early phase findings in a double-blind randomized placebo-controlled trial. Methods: Individuals, aged 18-65 years, with a minimum 2-year history of AR, moderate-to-severe AR symptoms, and a positive radio-allergosorbent test to Bermuda (Couch) Grass were randomized to receive either a multispecies probiotic supplement (total colony-forming units 4 × 109/day) or placebo twice daily for 8 weeks. A mini-rhinoconjunctivitis quality of life questionnaire (mRQLQ) scale was administered at screening, days 0, 28, and 56. The proportion of participants with a >0.7 improvement in mRQLQ was the primary outcome. Participants also completed a daily symptom and medication diary during the supplementation period. Results: There were 165 participants randomized, with 142 included in the primary outcome analysis. The percentage of participants meeting the threshold for a clinically meaningful reduction in the mRQLQ from days 0 to 56 was not significantly different between groups (61% vs. 62%, p = 0.90). However, 76 participants had a clinically meaningful improvement in QoL (decrease in mRQLQ >0.7) prior to the start of supplementation (screening to day 0). Conclusion: Changes in self-reported QoL and other disease severity metrics between screening and the start of supplementation limited the ability to discern an effect of supplementation and highlight the need for adaptive clinical trial designs in allergy research. Clinical Trial Registration: The trial was registered with the Australia and New Zealand Clinical Trials Registry (ACTRN12619001319167).

Using SBIRT (Screen, Brief Intervention, and Referral Treatment) Training to Reduce the Stigmatization of Substance Use Disorders Among Students and Practitioners.

Negative attitudes and stigmatization of substance-using patients lead to treatment avoidance and poor physical and health outcomes. Research suggests that training in substance use disorders is a vital tool to abate negative attitudes among health workers. The present longitudinal study trained students and experienced practitioners from various disciplines on the evidence-based Screening, Brief Intervention, and Referral to Treatment (SBIRT) model. The study found significant improvements in the attitudes of students-but not practitioners-who were trained during the program. The paper discusses policy and implementation implications to support and complement sustained impact of training on models such as SBIRT.