Systematic alteration of ATAC-seq for profiling open chromatin in cryopreserved nuclei preparations from livestock tissues.

The use of Assay for Transposase-Accessible Chromatin (ATAC-seq) to profile chromatin accessibility has surged over the past years, but its applicability to tissues has been very limited. With the intent of preserving nuclear architecture during long-term storage, cryopreserved nuclei preparations from chicken lung were used to optimize ATAC-seq. Sequencing data were compared with existing DNase-seq, ChIP-seq, and RNA-seq data to evaluate library quality, ultimately resulting in a modified ATAC-seq method capable of generating high quality chromatin accessibility data from cryopreserved nuclei preparations. Using this method, nucleosome-free regions (NFR) identified in chicken lung overlapped half of DNase-I hypersensitive sites, coincided with active histone modifications, and specifically marked actively expressed genes. Notably, sequencing only the subnucleosomal fraction dramatically improved signal, while separation of subnucleosomal reads post-sequencing did not improve signal or peak calling. The broader applicability of this modified ATAC-seq technique was tested using cryopreserved nuclei preparations from pig tissues, resulting in NFR that were highly consistent among biological replicates. Furthermore, tissue-specific NFR were enriched for binding motifs of transcription factors related to tissue-specific functions, and marked genes functionally enriched for tissue-specific processes. Overall, these results provide insights into the optimization of ATAC-seq and a platform for profiling open chromatin in animal tissues

In vivo manipulation of the extracellular matrix induces vascular regression in a basal chordate.

We investigated the physical role of the extracellular matrix (ECM) in vascular homeostasis in the basal chordate Botryllus schlosseri, which has a large, transparent, extracorporeal vascular network encompassing an area >100 cm2 We found that the collagen cross-linking enzyme lysyl oxidase is expressed in all vascular cells and that in vivo inhibition using β-aminopropionitrile (BAPN) caused a rapid, global regression of the entire network, with some vessels regressing >10 mm within 16 h. BAPN treatment changed the ultrastructure of collagen fibers in the vessel basement membrane, and the kinetics of regression were dose dependent. Pharmacological inhibition of both focal adhesion kinase (FAK) and Raf also induced regression, and levels of phosphorylated FAK in vascular cells decreased during BAPN treatment and FAK inhibition but not Raf inhibition, suggesting that physical changes in the vessel ECM are detected via canonical integrin signaling pathways. Regression is driven by apoptosis and extrusion of cells through the basal lamina, which are then engulfed by blood-borne phagocytes. Extrusion and regression occurred in a coordinated manner that maintained vessel integrity, with no loss of barrier function. This suggests the presence of regulatory mechanisms linking physical changes to a homeostatic, tissue-level response

Qualitative protocol for understanding the contribution of Australian policy in the urban planning, justice, energy and environment sectors to promoting health and health equity

Introduction: A well-established body of literature demonstrates that health and equity are strongly influenced by the consequences of governments’ policy and resultant actions (or inactions) outside the health sector. Consequently, the United Nations, and its agency the WHO, have called for national leadership and whole-of-government action to understand and address the health impacts of policies in all sectors. This research responds to that call by investigating how policymaking in four sectors—urban planning, justice, energy and environment—may influence the social determinants of health and health equity (SDH/HE). Methods and analysis: The research design is informed by a critical qualitative approach. Three successive stages are included in the design. The first involves analysing all strategic policy documents and selected legislative documents from the four sectors (n=583). The document analysis is based on a coding framework developed to identify alignment between the documents and the SDH/HE. Two policies that demonstrate good practice in regard to SDH/HE will be selected from each sector during the second stage for embedded case study analysis (total n=8). This is intended to illuminate which factors have supported recognition and action on SDH/HE in the selected policies. The third stage involves progressive theoretical integration and development to understand political and institutional facilitators and barriers to action on SDH/HE, both within and between sectors. Ethics and dissemination: The research will provide much needed evidence about how coherent whole-of-government action on SDH/HE can be advanced and contribute knowledge about how health-enhancing policy activity in the four sectors may be optimised. Learnings from the research will be shared via a project advisory group, policy briefings, academic papers, conference presentations and research symposia. Ethics approval has been secured for the embedded case studies, which involve research participants

Physical Activity Level and Physical Functionality in Nonagenarians Compared to Individuals Aged 60–74 Years

Background: Functional dependence and the risks of disability increase with age. The loss of independence is thought to be partially due to a decrease in physical activity. However, in populations, accurate measurement of physical activity is challenging and may not provide information on functional impairment. Methods: This study therefore assessed physical functionality and physical activity level in a group of nonagenarians (11 men/11 women; 93+/-1 years, 66.6+/-2.4 kg, body mass index [BMI]=24+/-1 kg/m2) and a group of participants aged 60-74 years (17 men/15 women; 70+/-1 years, 83.3+/-3.0 kg, BMI=29+/-1 kg/m2) from the Louisiana Healthy Aging Study. Physical activity level was calculated from total energy expenditure (TEE) and resting metabolic rate (RMR). Physical functionality was assessed using the Reduced Continuous Scale Physical Functional Performance Test (CS-PFP10). Results: Nonagenarians had lower absolute (p Conclusions: When compared to individuals aged 60-74 years, 73% of the reduction in TEE in nonagenarians can be attributed to a reduction in physical activity level, the remaining being accounted for by a reduction in RMR. The reduced physical activity in nonagenarians is associated with less physical functionality. This study provides the first objective comparison of physical functionality and actual levels of physical activity in older individuals

Practicing physiotherapy in Danish private practice: an ethical perspective.

Despite an increasingly growth of professional guidelines, textbooks and research about ethics in health care, awareness about ethics in Danish physiotherapy private practice seen vague. This article explores how physiotherapists in Danish private practice, from an ethical perspective, perceive to practice physiotherapy. The empirical data consists of interviews with twenty-one physiotherapists. The interviews are analysed from a hermeneutic approach, inspired by Ricoeur's textual interpretation of distanciation. The analysis follows three phases: naïve reading, structural analysis and comprehensive analysis. Four main themes are constructed: Beneficence as the driving force; Disciplining the patient through the course of physiotherapy; Balancing between being a trustworthy professional and a businessperson; The dream of a code of practice. Private practice physiotherapy is embedded in a structural frame directed by both political and economical conditions that shape the conditions for practicing physiotherapy. It means that beneficence in practice is a balance between the patient, the physiotherapists themselves and the business. Beneficence towards the patient is expressed as an implicit demand. Physiotherapeutic practice is expressed as being an integration of professionalism and personality which implies that the physiotherapists also have to benefit themselves. Private practice seems to be driven by a paternalistic approach towards the patient, where disciplining the patient is a crucial element of practice, in order to optimise profit. Physiotherapists wish for a more beneficent practice in the future by aiming at bridging 'to be' and 'ought to be'

A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter

Cost-effectiveness of tenofovir gel in urban South Africa: model projections of HIV impact and threshold product prices.

BACKGROUND: There is urgent need for effective HIV prevention methods that women can initiate. The CAPRISA 004 trial showed that a tenofovir-based vaginal microbicide had significant impact on HIV incidence among women. This study uses the trial findings to estimate the population-level impact of the gel on HIV and HSV-2 transmission, and price thresholds at which widespread product introduction would be as cost-effective as male circumcision in urban South Africa. METHODS: The estimated 'per sex-act' HIV and HSV-2 efficacies were imputed from CAPRISA 004. A dynamic HIV/STI transmission model, parameterised and fitted to Gauteng (HIV prevalence of 16.9% in 2008), South Africa, was used to estimate the impact of gel use over 15 years. Uptake was assumed to increase linearly to 30% over 10 years, with gel use in 72% of sex-acts. Full economic programme and averted HIV treatment costs were modelled. Cost per DALY averted is estimated and a microbicide price that equalises its cost-effectiveness to that of male circumcision is estimated. RESULTS: Using plausible assumptions about product introduction, we predict that tenofovir gel use could lead to a 12.5% and 4.9% reduction in HIV and HSV-2 incidence respectively, by year 15. Microbicide introduction is predicted to be highly cost-effective (under $300 per DALY averted), though the dose price would need to be just$0.12 to be equally cost-effective as male circumcision. A single dose or highly effective (83% HIV efficacy per sex-act) regimen would allow for more realistic threshold prices ($0.25 and$0.33 per dose, respectively). CONCLUSIONS: These findings show that an effective coitally-dependent microbicide could reduce HIV incidence by 12.5% in this setting, if current condom use is maintained. For microbicides to be in the range of the most cost-effective HIV prevention interventions, product costs will need to decrease substantially

A longitudinal analysis of the vaginal microbiota and vaginal immune mediators in women from sub-Saharan Africa

In cross-sectional studies increased vaginal bacterial diversity has been associated with vaginal inflammation which can be detrimental for health. We describe longitudinal changes at 5 visits over 8 weeks in vaginal microbiota and immune mediators in African women. Women (N = 40) with a normal Nugent score at all visits had a stable lactobacilli dominated microbiota with prevailing Lactobacillus iners. Presence of prostate-specific antigen (proxy for recent sex) and being amenorrhoeic (due to progestin-injectable use), but not recent vaginal cleansing, were significantly associated with microbiota diversity and inflammation (controlled for menstrual cycle and other confounders). Women (N = 40) with incident bacterial vaginosis (Nugent 7-10) had significantly lower concentrations of lactobacilli and higher concentrations of Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia, at the incident visit and when concentrations of proinflammatory cytokines (IL-1β, IL-12p70) were increased and IP-10 and elafin were decreased. A higher 'composite-qPCR vaginal-health-score' was directly associated with decreased concentrations of proinflammatory cytokines (IL-1α, IL-8, IL-12(p70)) and increased IP-10. This longitudinal study confirms the inflammatory nature of vaginal dysbiosis and its association with recent vaginal sex and progestin-injectable use. A potential role for proinflammatory mediators and IP-10 in combination with the vaginal-health-score as predictive biomarkers for vaginal dysbiosis merits further investigation

Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

BACKGROUND: S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma. METHODS: Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings. RESULTS: N6022 attenuated AHR (ED(50) of 0.015 ± 0.002 mg/kg; Mean ± SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh. CONCLUSIONS: The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease