Connexin-hemichannels are Involved in Acidosis-induced ATP Release from Skeletal Myocytes

Oral PresentationATP is an important extracellular signalling molecule which contributes to exercise vasodilation. We have previously shown that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in acidosis-induced ATP release from skeletal muscle. However, it is still unknown whether ATP is released through CFTR itself or whether CFTR regulates a separate ATPrelease channel. So we investigated: (1) the pathway responsible for CFTR activation in myocytes at low pH; (2) whether connexin (Cx) hemichannels were involved in the acidosis-induced ATP release from skeletal muscle. Lactic acid (10 mM) increased the intracellular cAMP and the extracellular ATP in L6 skeletal myocytes. Similarly, the cAMP-elevating agent, forskolin, increased extracellular ATP. The phosphodiesterase inhibitor, IBMX, increased extracellular ATP in the absence or presence of lactic acid. CFTR phosphorylation was increased by the addition of forskolin alone, and further increased by forskolin plus dibutyryl-cAMP and IBMX, but the forskolininduced increase in CFTR phosphorylation was inhibited by the PKA inhibitor, KT5720. Whereas KT5720 inhibited acidosis-induced ATP release from myocytes. These data suggest that skeletal muscle CFTR is activated through the cAMP/PKA pathway at low pH. RT-PCR indicated that cultured rat L6 skeletal myocytes expressed mRNA for both Cx40 and Cx43, but Cx40 was expressed only weakly in western blot, whereas Cx43 was strongly expressed. Co-immunoprecipitation results showed that CFTR and Cx43 were associated with each other in the cell membrane. A Cx43 over-expression model was created by transfecting myocytes with a Cx43 plasmid: Cx43 over-expression was confirmed using western blot. Cx43 over-expressing myocytes released significantly more ATP than control myocytes at pH 6.8, suggesting that Cx43 may be involved in acidosis-induced ATP release, whereas silencing Cx43 expression using siRNA inhibited the acidosis-induced ATP release. Over-expression of CFTR alone did not alter ATP release from myocytes, whereas co-over-expression of CFTR with Cx43 increased ATP release significantly more than over-expression of Cx43 alone. These data suggest that Cx43 co-localises with CFTR in the myocyte membrane, and that it may be involved in ATP release during acidosis; further investigation is required to determine whether and how CFTR interacts with Cx43 to induce ATP release.published_or_final_versio

cAMP/Protein Kinase A activates Cystic Fibrosis Transmembrane Conductance Regulator for ATP release from rat skeletal muscle during low pH or contractions

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Hypernatural Numbers as Ultrafilters

In this paper we present a use of nonstandard methods in the theory of ultrafilters and in related applications to combinatorics of numbers

Defective Fluid Secretion from Submucosal Glands of Nasal Turbinates from CFTR-/- and CFTRΔF508/ΔF508 Pigs

Cystic fibrosis (CF), caused by reduced CFTR function, includes severe sinonasal disease which may predispose to lung disease. Newly developed CF pigs provide models to study the onset of CF pathophysiology. We asked if glands from pig nasal turbinates have secretory responses similar to those of tracheal glands and if CF nasal glands show reduced fluid secretion.Unexpectedly, we found that nasal glands differed from tracheal glands in five ways, being smaller, more numerous (density per airway surface area), more sensitive to carbachol, more sensitive to forskolin, and nonresponsive to Substance P (a potent agonist for pig tracheal glands). Nasal gland fluid secretion from newborn piglets (12 CF and 12 controls) in response to agonists was measured using digital imaging of mucus bubbles formed under oil. Secretion rates were significantly reduced in all conditions tested. Fluid secretory rates (Controls vs. CF, in pl/min/gland) were as follows: 3 µM forskolin: 9.2±2.2 vs. 0.6±0.3; 1 µM carbachol: 143.5±35.5 vs. 52.2±10.3; 3 µM forskolin + 0.1 µM carbachol: 25.8±5.8 vs. CF 4.5±0.9. We also compared CF(ΔF508/ΔF508) with CFTR(-/-) piglets and found significantly greater forskolin-stimulated secretion rates in the ΔF508 vs. the null piglets (1.4±0.8, n = 4 vs. 0.2±0.1, n = 7). An unexpected age effect was also discovered: the ratio of secretion to 3 µM forskolin vs. 1 µM carbachol was ∼4 times greater in adult than in neonatal nasal glands.These findings reveal differences between nasal and tracheal glands, show defective fluid secretion in nasal glands of CF pigs, reveal some spared function in the ΔF508 vs. null piglets, and show unexpected age-dependent differences. Reduced nasal gland fluid secretion may predispose to sinonasal and lung infections

A systematic review of correlates of sedentary behaviour in adults aged 18–65 years: a socio-ecological approach

Background: Recent research shows that sedentary behaviour is associated with adverse cardio-metabolic consequences even among those considered sufficiently physically active. In order to successfully develop interventions to address this unhealthy behaviour, factors that influence sedentariness need to be identified and fully understood. The aim of this review is to identify individual, social, environmental, and policy-related determinants or correlates of sedentary behaviours among adults aged 18-65 years. Methods: PubMed, Embase, CINAHL, PsycINFO and Web of Science were searched for articles published between January 2000 and September 2015. The search strategy was based on four key elements and their synonyms: (a) sedentary behaviour (b) correlates (c) types of sedentary behaviours (d) types of correlates. Articles were included if information relating to sedentary behaviour in adults (18-65 years) was reported. Studies on samples selected by disease were excluded. The full protocol is available from PROSPERO (PROSPERO 2014:CRD42014009823). Results: 74 original studies were identified out of 4041: 71 observational, two qualitative and one experimental study. Sedentary behaviour was primarily measured as self-reported screen leisure time and total sitting time. In 15 studies, objectively measured total sedentary time was reported: accelerometry (n = 14) and heart rate (n = 1). Individual level factors such as age, physical activity levels, body mass index, socio-economic status and mood were all significantly correlated with sedentariness. A trend towards increased amounts of leisure screen time was identified in those married or cohabiting while having children resulted in less total sitting time. Several environmental correlates were identified including proximity of green space, neighbourhood walkability and safety and weather. Conclusions: Results provide further evidence relating to several already recognised individual level factors and preliminary evidence relating to social and environmental factors that should be further investigated. Most studies relied upon cross-sectional design limiting causal inference and the heterogeneity of the sedentary measures prevented direct comparison of findings. Future research necessitates longitudinal study designs, exploration of policy-related factors, further exploration of environmental factors, analysis of inter-relationships between identified factors and better classification of sedentary behaviour domains

3D Echo systematically underestimates right ventricular volumes compared to cardiovascular magnetic resonance in adult congenital heart disease patients with moderate or severe RV dilatation

<p>Abstract</p> <p>Background</p> <p>Three dimensional echo is a relatively new technique which may offer a rapid alternative for the examination of the right heart. However its role in patients with non-standard ventricular size or anatomy is unclear. This study compared volumetric measurements of the right ventricle in 25 patients with adult congenital heart disease using both cardiovascular magnetic resonance (CMR) and three dimensional echocardiography.</p> <p>Methods</p> <p>Patients were grouped by diagnosis into those expected to have normal or near-normal RV size (patients with repaired coarctation of the aorta) and patients expected to have moderate or worse RV enlargement (patients with repaired tetralogy of Fallot or transposition of the great arteries). Right ventricular end diastolic volume, end systolic volume and ejection fraction were compared using both methods with CMR regarded as the reference standard</p> <p>Results</p> <p>Bland-Altman analysis of the 25 patients demonstrated that for both RV EDV and RV ESV, there was a significant and systematic under-estimation of volume by 3D echo compared to CMR. This bias led to a mean underestimation of RV EDV by -34% (95%CI: -91% to + 23%). The degree of underestimation was more marked for RV ESV with a bias of -42% (95%CI: -117% to + 32%). There was also a tendency to overestimate RV EF by 3D echo with a bias of approximately 13% (95% CI -52% to +27%).</p> <p>Conclusions</p> <p>Statistically significant and clinically meaningful differences in volumetric measurements were observed between the two techniques. Three dimensional echocardiography does not appear ready for routine clinical use in RV assessment in congenital heart disease patients with more than mild RV dilatation at the current time.</p

Method for Quantitative Study of Airway Functional Microanatomy Using Micro-Optical Coherence Tomography

We demonstrate the use of a high resolution form of optical coherence tomography, termed micro-OCT (μOCT), for investigating the functional microanatomy of airway epithelia. μOCT captures several key parameters governing the function of the airway surface (airway surface liquid depth, periciliary liquid depth, ciliary function including beat frequency, and mucociliary transport rate) from the same series of images and without exogenous particles or labels, enabling non-invasive study of dynamic phenomena. Additionally, the high resolution of μOCT reveals distinguishable phases of the ciliary stroke pattern and glandular extrusion. Images and functional measurements from primary human bronchial epithelial cell cultures and excised tissue are presented and compared with measurements using existing gold standard methods. Active secretion from mucus glands in tissue, a key parameter of epithelial function, was also observed and quantified

African tropical rainforest net carbon dioxide fluxes in the twentieth century

The African humid tropical biome constitutes the second largest rainforest region, significantly impacts global carbon cycling and climate, and has undergone major changes in functioning owing to climate and land-use change over the past century. We assess changes and trends in CO2 fluxes from 1901 to 2010 using nine land surface models forced with common driving data, and depict the inter-model variability as the uncertainty in fluxes. The biome is estimated to be a natural (no disturbance) net carbon sink (−0.02 kg C m−2 yr−1 or −0.04 Pg C yr−1, p < 0.05) with increasing strength fourfold in the second half of the century. The models were in close agreement on net CO2 flux at the beginning of the century (σ1901 = 0.02 kg C m−2 yr−1), but diverged exponentially throughout the century (σ2010 = 0.03 kg C m−2 yr−1). The increasing uncertainty is due to differences in sensitivity to increasing atmospheric CO2, but not increasing water stress, despite a decrease in precipitation and increase in air temperature. However, the largest uncertainties were associated with the most extreme drought events of the century. These results highlight the need to constrain modelled CO2 fluxes with increasing atmospheric CO2 concentrations and extreme climatic events, as the uncertainties will only amplify in the next century

Effect of a low fat versus a low carbohydrate weight loss dietary intervention on biomarkers of long term survival in breast cancer patients ('CHOICE'): study protocol

<p>Abstract</p> <p>Background</p> <p>Weight loss in overweight or obese breast cancer patients is associated with an improved prognosis for long term survival. However, it is not clear whether the macronutrient composition of the chosen weight loss dietary plan imparts further prognostic benefit. A study protocol is presented for a dietary intervention to investigate the effects of weight loss dietary patterns that vary markedly in fat and carbohydrate contents on biomarkers of exposure to metabolic processes that may promote tumorigenesis and that are predictive of long term survival. The study will also determine how much weight must be lost for biomarkers to change in a favorable direction.</p> <p>Methods/Design</p> <p>Approximately 370 overweight or obese postmenopausal breast cancer survivors (body mass index: 25.0 to 34.9 kg/m²) will be accrued and assigned to one of two weight loss intervention programs or a non-intervention control group. The dietary intervention is implemented in a free living population to test the two extremes of popular weight loss dietary patterns: a high carbohydrate, low fat diet versus a low carbohydrate, high fat diet. The effects of these dietary patterns on biomarkers for glucose homeostasis, chronic inflammation, cellular oxidation, and steroid sex hormone metabolism will be measured. Participants will attend 3 screening and dietary education visits, and 7 monthly one-on-one dietary counseling and clinical data measurement visits in addition to 5 group visits in the intervention arms. Participants in the control arm will attend two clinical data measurement visits at baseline and 6 months. The primary outcome is high sensitivity C-reactive protein. Secondary outcomes include interleukin-6, tumor necrosis factor-α, insulin-like growth factor-1 (IGF), IGF binding protein-3, 8-isoprostane-F2-alpha, estrone, estradiol, progesterone, sex hormone binding globulin, adiponectin, and leptin.</p> <p>Discussion</p> <p>While clinical data indicate that excess weight for height is associated with poor prognosis for long term survival, little attention is paid to weight control in the clinical management of breast cancer. This study will provide information that can be used to answer important patient questions about the effects of dietary pattern and magnitude of weight loss on long term survival following breast cancer treatment.</p> <p>Clinical Trial Registration</p> <p>CA125243</p

Bacillus anthracis Lethal Toxin Disrupts TCR Signaling in CD1d-Restricted NKT Cells Leading to Functional Anergy

Exogenous CD1d-binding glycolipid (α-Galactosylceramide, α-GC) stimulates TCR signaling and activation of type-1 natural killer–like T (NKT) cells. Activated NKT cells play a central role in the regulation of adaptive and protective immune responses against pathogens and tumors. In the present study, we tested the effect of Bacillus anthracis lethal toxin (LT) on NKT cells both in vivo and in vitro. LT is a binary toxin known to suppress host immune responses during anthrax disease and intoxicates cells by protective antigen (PA)-mediated intracellular delivery of lethal factor (LF), a potent metalloprotease. We observed that NKT cells expressed anthrax toxin receptors (CMG-2 and TEM-8) and bound more PA than other immune cell types. A sub-lethal dose of LT administered in vivo in C57BL/6 mice decreased expression of the activation receptor NKG2D by NKT cells but not by NK cells. The in vivo administration of LT led to decreased TCR-induced cytokine secretion but did not affect TCR expression. Further analysis revealed LT-dependent inhibition of TCR-stimulated MAP kinase signaling in NKT cells attributable to LT cleavage of the MAP kinase kinase MEK-2. We propose that Bacillus anthracis–derived LT causes a novel form of functional anergy in NKT cells and therefore has potential for contributing to immune evasion by the pathogen