Vfr Directly Activates exsA Transcription To Regulate Expression of the Pseudomonas aeruginosa Type III Secretion System

ABSTRACT The Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in a vfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA . ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (P exsA ) located immediately upstream of exsA . P exsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a P exsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and P exsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the P exsA promoter, which is internal to the previously characterized exsCEBA operon. IMPORTANCE Vfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa . Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA

Formation of environmentally persistent free radicals (EPFRs) on ZnO at room temperature: Implications for the fundamental model of EPFR generation.

Environmentally persistent free radicals (EPFRs) have significant environmental and public health impacts. In this study, we demonstrate that EPFRs formed on ZnO nanoparticles provide two significant surprises. First, EPR spectroscopy shows that phenoxy radicals form readily on ZnO nanoparticles at room temperature, yielding EPR signals similar to those previously measured after 250°C exposures. Vibrational spectroscopy supports the conclusion that phenoxy-derived species chemisorb to ZnO nanoparticles under both exposure temperatures. Second, DFT calculations indicate that electrons are transferred from ZnO to the adsorbed organic (oxidizing the Zn), the opposite direction proposed by previous descriptions of EPFR formation on metal oxides

Medical students\u27 knowledge of HPV, HPV vaccine, and HPV-associated head and neck cancer

On the basis of their training, medical students are considered the best case scenario among university students in knowledge of the human papillomavirus (HPV). We evaluated differences in knowledge of HPV, HPV vaccine, and head and neck cancer (HNC) among medical students. A previously validated questionnaire was completed by 247 medical students at a Midwestern university. Outcomes of interest were knowledge score for HPV and HPV vaccine, and HNC, derived from combining questionnaire items to form HPV knowledge and HNC scores, and analyzed using multivariate linear regression. Mean scores for HPV knowledge were 19.4 out of 26, and 7.2 out of 12 for HNC knowledge. In the final multivariate linear regression model, sex, race, and year of study were independently associated with HPV and HPV vaccine knowledge. Males had significantly lower HPV vaccine knowledge than females (β = -1.53; 95% CI: -2.53, -0.52), as did nonwhite students (β = -1.05; 95% CI: -2.07, -0.03). There was a gradient in HPV vaccine knowledge based on the year of study, highest among fourth year students (β = 6.75; 95% CI: 5.17, 8.33). Results were similar for factors associated with HNC knowledge, except for sex. HNC knowledge similarly increased based on year of study, highest for fourth year students (β = 2.50; 95% CI: 1.72, 3.29). Among medical students, gaps remain in knowledge of HPV, HPV vaccine, and HPV-linked HNC. Male medical students have significantly lower knowledge of HPV. This highlights the need to increase medical student knowledge of HPV and HPV-linked HNC

A Cluster Randomized Controlled Trial for a Multi-Level, Clinic-Based Smoking Cessation Program with Women in Appalachian Communities: Study Protocol for the Break Free Program

BACKGROUND: The cervical cancer burden is high among women living in Appalachia. Cigarette smoking, a cervical cancer risk factor, is also highly prevalent in this population. This project aims to increase smoking cessation among women living in Appalachia by embedding a smoking cessation program within a larger, integrated cervical cancer prevention program. METHODS: The broader program, the Take CARE study, is a multi-site research collaborative designed to address three risk factors for cervical cancer incidence and mortality: tobacco use, human papillomavirus (HPV) infection, and cervical cancer screening. Break Free is a primary care clinic-based implementation program that aims to promote smoking cessation among female smokers in Appalachia by standardizing clinical practice protocols. Break Free includes: (1) implementation of a tobacco user identification system in the Electronic Health Record, (2) clinic staff and provider training on the Ask, Advise and Refer (AAR) model, (3) provider implementation of AAR to identify and treat women who want to quit smoking within the next 6 months, (4) facilitated access to cessation phone counseling plus pharmacotherapy, and (5) the bundling of Break Free tobacco cessation with HPV vaccination and cervical cancer screening interventions in an integrated approach to cervical cancer prevention. The study spans 35 Appalachian health clinics across 10 healthcare systems. We aim to enroll 51 adult female smokers per health system (total N = 510). Baseline and follow-up data will be obtained from participant (provider and patient) surveys. The primary outcome is self-reported 12-month point prevalence abstinence among enrolled patients. All randomized patients are asked to complete follow-up surveys, regardless of whether they participated in tobacco treatment. Data analysis of the primary aims will follow intent-to-treat methodology. Secondary outcomes will assess program implementation and cost effectiveness. DISCUSSION: Addressing high tobacco use rates is critical for reducing cervical cancer morbidity and mortality among women living in Appalachia. This study evaluates the implementation and effectiveness of a smoking cessation program in increasing smoking cessation among female smokers. If results demonstrate effectiveness and sustainability, implementation of this program into other health care clinics could reduce both rates of smoking and cervical cancer. Trial registration NCT04340531 (April 9, 2020)

Blocking airway mucous cell metaplasia by inhibiting EGFR antiapoptosis and IL-13 transdifferentiation signals

Epithelial hyperplasia and metaplasia are common features of inflammatory and neoplastic disease, but the basis for the altered epithelial phenotype is often uncertain. Here we show that long-term ciliated cell hyperplasia coincides with mucous (goblet) cell metaplasia after respiratory viral clearance in mouse airways. This chronic switch in epithelial behavior exhibits genetic susceptibility and depends on persistent activation of EGFR signaling to PI3K that prevents apoptosis of ciliated cells and on IL-13 signaling that promotes transdifferentiation of ciliated to goblet cells. Thus, EGFR blockade (using an irreversible EGFR kinase inhibitor designated EKB-569) prevents virus-induced increases in ciliated and goblet cells whereas IL-13 blockade (using s-IL-13Rα2-Fc) exacerbates ciliated cell hyperplasia but still inhibits goblet cell metaplasia. The distinct effects of EGFR and IL-13 inhibitors after viral reprogramming suggest that these combined therapeutic strategies may also correct epithelial architecture in the setting of airway inflammatory disorders characterized by a similar pattern of chronic EGFR activation, IL-13 expression, and ciliated-to-goblet cell metaplasia

Lack of MEF2A Δ7aa mutation in Irish families with early onset ischaemic heart disease, a family based study

BACKGROUND: Ischaemic heart disease (IHD) is a complex disease due to the combination of environmental and genetic factors. Mutations in the MEF2A gene have recently been reported in patients with IHD. In particular, a 21 base pair deletion (Δ7aa) in the MEF2A gene was identified in a family with an autosomal dominant pattern of inheritance of IHD. We investigated this region of the MEF2A gene using an Irish family-based study, where affected individuals had early-onset IHD. METHODS: A total of 1494 individuals from 580 families were included (800 discordant sib-pairs and 64 parent-child trios). The Δ7aa region of the MEF2A gene was investigated based on amplicon size. RESULTS: The Δ7aa mutation was not detected in any individual. Variation in the number of CAG (glutamate) and CCG (proline) residues was detected in a nearby region. However, this was not found to be associated with IHD. CONCLUSION: The Δ7aa mutation was not detected in any individual within the study population and is unlikely to play a significant role in the development of IHD in Ireland. Using family-based tests of association the number of tri-nucleotide repeats in a nearby region of the MEF2A gene was not associated with IHD in our study group