No abstract

One major limitation of microbial community marker gene sequencing is that it does not provide direct information on the functional composition of sampled communities. Here, we present PICRUSt2 (https://github.com/picrust/picrust2), which expands the capabilities of the original PICRUSt method1 to predict the functional potential of a community based on marker gene sequencing profiles. This updated method and implementation includes several improvements over the previous algorithm: an expanded database of gene families and reference genomes, a new approach now compatible with any OTU-picking or denoising algorithm, and novel phenotype predictions. Upon evaluation, PICRUSt2 was more accurate than PICRUSt1 and other current approaches overall. PICRUSt2 is also now more flexible and allows the addition of custom reference databases. We highlight these improvements and also important caveats regarding the use of predicted metagenomes, which are related to the inherent challenges of analyzing metagenome data in general.

The human gastrointestinal microbiota plays a key homeostatic role in normal functioning of physiologic processes commonly undermined by aging. We used a previously validated 34-item frailty index (FI34) to identify changes in gut microbiota community structure associated with biological age of community-dwelling adults. Stool 16S rRNA cDNA libraries from 85 subjects ranging in age (43-79) and FI34 score (0-0.365) were deep sequenced, denoised, and clustered using DADA2. Subject biological age but not chronological age correlated with a decrease in stool microbial diversity. Specific microbial genera were differentially abundant in the lower, middle, and upper 33rd percentiles of biological age. Using Sparse Inverse Covariance Estimation for Ecological Association and Statistical Inference (SPIEC-EASI) and Weighted Gene Co-Expression Network Analysis (WGCNA), we identified modules of coabundant microbial genera that distinguished biological from chronological aging. A biological age-associated module composed of Eggerthella, Ruminococcus, and Coprobacillus genera was robust to correction for subject age, sex, body mass index, antibiotic usage, and other confounders. Subject FI34 score positively correlated with the abundance of this module, which exhibited a distinct inferred metagenome as predicted by Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). We conclude that increasing biological age in community-dwelling adults is associated with gastrointestinal dysbiosis.

Chronic alcohol consumption perturbs the normal intestinal microbial communities (dysbiosis). To investigate the relationship between alcohol-mediated dysbiosis and pulmonary host defense we developed a fecal adoptive transfer model, which allows us to investigate the impact of alcohol-induced gut dysbiosis on host immune response to an infectious challenge at a distal organ, independent of prevailing alcohol use. Male C57BL/6 mice were treated with a cocktail of antibiotics (ampicillin, gentamicin, neomycin, vancomycin, and metronidazole) via daily gavage for two weeks. A separate group of animals was fed a chronic alcohol (or isocaloric dextrose pair-fed controls) liquid diet for 10 days. Microbiota-depleted mice were recolonized with intestinal microbiota from alcohol-fed or pair-fed (control) animals. Following recolonization groups of mice were sacrificed prior to and 48 hrs. post respiratory infection with Klebsiella pneumoniae. Klebsiella lung burden, lung immunology and inflammation, as well as intestinal immunology, inflammation, and barrier damage were examined. Results showed that alcohol-associated susceptibility to K. pneumoniae is, in part, mediated by gut dysbiosis, as alcohol-naïve animals recolonized with a microbiota isolated from alcohol-fed mice had an increased respiratory burden of K. pneumoniae compared to mice recolonized with a control microbiota. The increased susceptibility in alcohol-dysbiosis recolonized animals was associated with an increase in pulmonary inflammatory cytokines, and a decrease in the number of CD4+ and CD8+ T-cells in the lung following Klebsiella infection but an increase in T-cell counts in the intestinal tract following Klebsiella infection, suggesting intestinal T-cell sequestration as a factor in impaired lung host defense. Mice recolonized with an alcohol-dysbiotic microbiota also had increased intestinal damage as measured by increased levels of serum intestinal fatty acid binding protein. Collectively, these results suggest that alterations in the intestinal immune response as a consequence of alcohol-induced dysbiosis contribute to increased host susceptibility to Klebsiella pneumonia.

BACKGROUND-The average lifespan of persons living with HIV (PLWH) on antiretroviral therapy approximates the general population. However, PLWH are susceptible to early aging and frailty. Behaviors such as alcohol consumption may contribute to frailty among PLWH. OBJECTIVE-To determine the relationships between recent and lifetime alcohol use and frailty among PLWH.DESIGN-Cross-sectional, prospective cohort study of in-care PLWH (n=365) participating in the New Orleans Alcohol Use in HIV Study. METHODS-Recent alcohol exposure was measured by the 30-day alcohol timeline follow-back (TLFB) assessment and by whole-blood-spot phosphatidylethanol (PEth) quantitation. Lifetime alcohol exposure (LAE) was estimated by a modified lifetime drinking history instrument. Frailty was assessed by a 58-item deficit index (DI58) and the phenotypic frailty index (PFI). The Veterans Aging Cohort Study Risk Index 2.0 was calculated.

Background: High frequency of alcohol use among people living with HIV (PLWH) warrants careful assessment and screening to better understand its impact on HIV disease progression and development of comorbidities. Due to the limitations of the tools used to measure alcohol use, the links to health consequences are not fully understood.Methods: We completed a cross-sectional analysis to examine the prevalence of alcohol consumption using multiple alcohol assessment tools and their correlation and consistency in a cohort of PLWH (N = 365) enrolled in the New Orleans Alcohol Use in HIV (NOAH) Study. Alcohol use was assessed with the Alcohol Use Disorders Identification Test (AUDIT), timeline followback (TLFB) Calendar, lifetime drinking history, Alcohol and Drug Addiction Severity Index, and blood levels of phosphatidylethanol (PEth). Spearman's correlations were estimated for continuous measures of alcohol consumption; Wilcoxon rank-sum tests were used to compare means; and logistic regression was used to estimate odds of alcohol use by demographic characteristics.Results: Self-report of current alcohol use varied from 58.9 to 73.7% depending on the assessment. All the self-reported alcohol measures showed statistically significant correlations with the biological marker PEth. The highest correlation was with TLFB grams (r = 0.67, p < 0.001). Using TLFB, 73.7% of the cohort reported using alcohol in the last 30 days, and 61.6% had a positive PEth value. The prevalence of risky drinkers, meeting the TLFB > 3 (women) or >4 (men) drinks/day or>7 (women) or>14 (men) drinks/week, was 49.0%. Medium-risk drinking defined as an AUDIT score ≥ 8 was reported in 40.3%, and high-risk drinkers/probable AUD (AUDIT score ≥ 16) was met by 17.0% of the cohort.Conclusions: Our results demonstrate the importance of comprehensive assessments for alcohol use, including self-report via multiple assessment tools administered by trained staff, as well as the addition of biomarkers for improved classification of subjects into different drinking categories.

BACKGROUND Inflammation persists among people living with HIV (PWH) despite effective anti-retroviral therapy and may contribute to T-cell dysfunction. Alcohol use is prevalent among PWH and promotes intestinal leak, dysbiosis, and a pro-inflammatory milieu. Whether alcohol use is associated with T-cell late-differentiation remains to be investigated. METHODS Data and samples from PWH (N=359/365) enrolled in the New Orleans Alcohol Use in HIV Study were used. Alcohol use was assessed by self-report (Alcohol Use Disorders Identification Test [AUDIT]; lifetime alcohol exposure; thirty-day Alcohol Timeline Follow Back) and phosphatidylethanol (PEth) quantitation. In a subset of participants, fecal bacterial content was assessed by ribosomal 16S marker-gene deep-sequencing and quantitative PCR. Intestinal leak was assessed by fecal-to-plasma α-1-anti-trypsin (A1AT) ELISA ratio. Peripheral T-cell populations were quantified by flow-cytometry. RESULTS AUDIT scores were positively associated with activated-senescent, exhausted, and terminal effector memory CD45RA+ CD8+ but not CD4+ T-cells (cells/μL) after confounder adjustment (p&lt;.050). PEth was positively associated with A1AT (p&lt;.050). PEth and activated-senescent CD8+ were associated with bacterial β-diversity (p&lt;.050) and positively associated with the relative abundance of co-abundant Prevotellaceae members (q&lt;.100). DISCUSSION Alcohol use among PWH is associated with CD8+ T-cell late-differentiation, intestinal leak, and dysbiosis. Alcohol-associated dysbiosis is implicated in CD8+ T-cell senescence.

Although numerous studies have examined in vivo and in vitro effects of irritants, most focused on events developing hours to days after exposure. Molecular events occurring immediately after skin contact remain incompletely defined. Characterization of early events could lead to the identification of key molecular signals necessary for the production of inflammatory mediators responsible for the signs and symptoms of irritant contact dermatitis (ICD). HaCaT cells treated with sodium lauryl sulfate (SLS), a model irritant, were used to examine early molecular events of ICD. Western analysis showed SLS-mediated induction of early growth response-1 (egr-1), a transcription factor capable of regulating hallmarks of ICD such as angiogenesis, hyperproliferation, and inflammation. Additionally, de novo egr-1 expression was commensurate with transcriptional activation of egr-1 mRNA and heteronuclear RNA. Use of pharmacological inhibitors demonstrated that SLS-induced egr-1 was dependent on MEK1/p44/42 ERK, but not on p38 or JNK signaling. The EGFR inhibitor PD168393 and the metalloprotease inhibitor TAPI-2 both inhibited SLS-induced egr-1. Finally, small interfering RNA silencing of the EGFR diminished SLS-induced egr-1 mRNA. These studies suggest a role of the EGFR in SLS signaling as well as a, to our knowledge, previously unreported association between ICD and EGFR induction of egr-1.