A laboratory methodology for dual RNA-sequencing of bacteria and their host cells in vitro

© 2017 Marsh, Humphrys and Myers. Dual RNA-Sequencing leverages established next-generation sequencing (NGS)-enabled RNA-Seq approaches to measure genome-wide transcriptional changes of both an infecting bacteria and host cells. By simultaneously investigating both organisms from the same biological sample, dual RNA-Seq can provide unique insight into bacterial infection processes and reciprocal host responses at once. However, the difficulties involved in handling both prokaryotic and eukaryotic material require distinct, optimized procedures. We previously developed and applied dual RNA-Seq to measure prokaryotic and eukaryotic expression profiles of human cells infected with bacteria, using in vitro Chlamydia-infected epithelial cells as proof of principle. Here we provide a detailed laboratory protocol for in vitro dual RNA-Seq that is readily adaptable to any host-bacteria system of interest

Chromatin accessibility dynamics of Chlamydia-infected epithelial cells.

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied formaldehyde-assisted isolation of regulatory elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions include temporally-enriched sets of transcription factors, which may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signalling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues

Clinician Perspectives on Factors Affecting Shared Decision Making about Lung Cancer Screening

Background/Objective. In 2015, the Centers for Medicare and Medicaid Services (CMS) announced coverage for annual lung cancer screening (LCS) with low dose computed tomography (LDCT) for individuals who are 55 to 77 years of age, have \u3e 30 pack years of smoking history, and undergo shared decision making (SDM) prior to screening. Most referrals for LCS are initiated in primary care. Currently, little is known about how primary care physicians view SDM and barriers in practice to SDM about LCS. This study aimed to gather information to help fill these knowledge gaps. Methods. I worked with senior leadership in the Department of Medicine to identify a set of internal medicine physicians at Thomas Jefferson University (TJU) and contacted them via email requesting their participation in an interview about SDM in LCS. I developed an interview guide that included questions about the following: understanding of SDM, perceptions about SDM in LCS, and receptivity to use of an online decision support intervention (DSI). I completed in-person, audio recorded interviews, which were transcribed for analysis. I then analyzed the interview transcripts using NVivo qualitative analysis software. Results. Nine physicians were interviewed from a pool of twenty-three physicians over a period of three weeks. With regards to understanding of SDM, physicians were in agreement that SDM is a joint decision based on a discussion about the risks and benefits of an intervention that considers patient values and medical status. Physician perceptions of SDM in LCS was influenced by patient comorbidities, LCS controversies and complexity, and limited office time. Receptivity to using an online DSI was generally positive and particularly favored its patient education component and easing of physician workload. Conclusions. Observations from this study highlight a common general understanding of SDM, yet mixed approaches to SDM in LCS. Strong support also exists for a DSI that educates patients about LCS and saves physicians time. Future steps include interviewing a set of family medicine physicians to investigate potential differences in viewpoints compared to internal medicine physicians

Prevalence and correlates of atypical patterns of drug use progression: findings from the South African Stress and Health Study

Objective: Atypical sequences of drug use progression are thought to have important implications for the development of substance dependence. The extent to which this assumption holds for South African populations is unknown. This paper attempts to address this gap by examining the prevalence and correlates of atypical patterns of drug progression among South Africans. Method: Data on substance use and other mental health disorders from a nationally representative sample of 4351 South Africans were analysed. Weighted cross tabulations were used to estimate prevalence and correlates of atypical patterns of drug use progression. Results: Overall, 12.2% of the sample reported atypical patterns of drug use progression. The most common violation was the use of extra-medical drugs prior to alcohol and tobacco. Gender was significantly associated with atypical patterns of drug use with the risk pattern varying by the type of drug. None of the anxiety or mood disorders were associated with atypical patterns of use. Atypical patterns of drug use were not associated with increased risk for a lifetime substance use disorder. Conclusion: Atypical patterns of drug use initiation seem more prevalent in South Africa compared to other countries. The early use of extra-medical drugs is common, especially among young women. Drug availability and social environmental factors may influence patterns of drug use. The findings have important implications for prevention initiatives and future research.Key words: Substance use; Gateway violations; Mental disorders; South Afric

Epithelial–mesenchymal transition is driven by transcriptional and post transcriptional modulations in copd: Implications for disease progression and new therapeutics

© 2019 Eapen et al. COPD is a common and highly destructive disease with huge impacts on people and health services throughout the world. It is mainly caused by cigarette smoking though environmental pollution is also significant. There are no current treatments that affect the overall course of COPD; current drugs focus on symptomatic relief and to some extent reducing exacerbation rates. There is an urgent need for in-depth studies of the fundamental pathogenic mechanisms that underpin COPD. This is vital, given the fact that nearly 40%– 60% of the small airway and alveolar damage occurs in COPD well before the first measurable changes in lung function are detected. These individuals are also at a high risk of lung cancer. Current COPD research is mostly centered around late disease and/or innate immune activation within the airway lumen, but the actual damage to the airway wall has early onset. COPD is the end result of complex mechanisms, possibly triggered through initial epithelial activation. To change the disease trajectory, it is crucial to understand the mechanisms in the epithelium that are switched on early in smokers. One such mechanism we believe is the process of epithelial to mesenchymal transition. This article highlights the importance of this profound epithelial cell plasticity in COPD and also its regulation. We consider that understanding early changes in COPD will open new windows for therapy

Lyrical Music Improves 5 km Time Trial Performance Compared to Non-Lyrical Music

This study examined the effects of lyrical music compared to non-lyrical music on 5 km running performance. Thirteen subjects with an average age of 33.5 ± 8.3 years of age ran three separate 5 km time trials. The first trial acted as a familiarization trial where no music was present, followed by either a lyrical or non-lyrical music trial in a random counterbalanced order. Trial times, RPE, HR, and questionnaire information was analyzed using paired samples t-tests, ANOVA, and multivariate regression analyses. Lyrical music showed a significant improvement over non-lyrical music improving performance time compared to non-lyrical music, on average by 36 ± 41s (p=.000). While not statistically significant, a trend showing lyrical music was faster than no music, followed by the slowest trial of non-lyrical music. No change was detected in HR, or RPE at the 3.05 km mark suggesting that at the end although subjects were working harder (indicated by the faster completion times) they do not perceive themselves to be working harder. A correlation between increased mileage and the decreased effects of music as an ergogenic aid was seen (p=.044 r=.638) which supports previous research. This study suggests that lyrical music may improve 5 km running performance compared to no lyrical music

Evaluating the Impact of an Interprofessional Practice Experience Involving Pharmacy and Dental Students on Medication Histories within an Urban Academic Dental Admissions Clinic

Primary Objective: To compare interprofessional (IP) care versus standard care on medication history clarifications in dental patients. Secondary Objectives: To assess the clinical significance of these clarifications with regards to the potential impact on dental treatment plans. To describe the interventions provided by IP care to clarify discrepancies and/or resolve medication-related problems

Effect of Prior Anterior Superior Iliac Spine Compression Testing on Second Assessor Findings: Implications for Inter-Examiner Reliability Testing

BACKGROUND: Osteopathic physicians use palpation to diagnose sacroiliac joint somatic dysfunction (SD) -- including the Anterior Superior Iliac Spine (ASIS) Compression Test for dysfunctional side lateralization. (Literature suggests right-sided lateralization in 80% of asymptomatic individuals). Accurate, reliable tests are crucial however to diagnose SD and kappa (κ) analysis is a gold-standard to determine the degree of interexaminer reliability for tests. Few studies have examined the effect the palpatory examination has on subsequent diagnostic findings and therefore on κ-values