Reverse mode Na+/Ca2+ exchange mediated by STIM1 contributes to Ca2+ influx in airway smooth muscle following agonist stimulation

<p>Abstract</p> <p>Background</p> <p>Agonist stimulation of airway smooth muscle (ASM) results in IP₃mediated Ca²⁺release from the sarcoplasmic reticulum followed by the activation of store operated and receptor operated non-selective cation channels. Activation of these non-selective channels also results in a Na⁺influx. This localised increase in Na⁺levels can potentially switch the Na⁺/Ca²⁺exchanger into reverse mode and so result in a further influx of Ca²⁺. The aim of this study was to characterise the expression and physiological function of the Na⁺/Ca²⁺exchanger in cultured human bronchial smooth muscle cells and determine its contribution to agonist induced Ca²⁺influx into these cells.</p> <p>Methods</p> <p>The expression profile of NCX (which encodes the Na⁺/Ca²⁺exchanger) homologues in cultured human bronchial smooth muscle cells was determined by reverse transcriptase PCR. The functional activity of reverse mode NCX was investigated using a combination of whole cell patch clamp, intracellular Ca²⁺measurements and porcine airway contractile analyses. KB-R7943 (an antagonist for reverse mode NCX) and target specific siRNA were utilised as tools to inhibit NCX function.</p> <p>Results</p> <p>NCX1 protein was detected in cultured human bronchial smooth muscle cells (HBSMC) cells and NCX1.3 was the only mRNA transcript variant detected. A combination of intracellular Na⁺loading and addition of extracellular Ca²⁺induced an outwardly rectifying current which was augmented following stimulation with histamine. This outwardly rectifying current was inhibited by 10 μM KB-R7943 (an antagonist of reverse mode NCX1) and was reduced in cells incubated with siRNA against NCX1. Interestingly, this outwardly rectifying current was also inhibited following knockdown of STIM1, suggesting for the first time a link between store operated cation entry and NCX1 activation. In addition, 10 μM KB-R7943 inhibited agonist induced changes in cytosolic Ca²⁺and induced relaxation of porcine peripheral airways.</p> <p>Conclusions</p> <p>Taken together, these data demonstrate a potentially important role for NCX1 in control of Ca²⁺homeostasis and link store depletion via STIM1 directly with NCX activation.</p

Plasminogen Alleles Influence Susceptibility to Invasive Aspergillosis

Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection

Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo

Modelling phytoplankton adaptation to global warming based on resurrection experiments

Due to its crucial role in the ecosystem, phytoplankton is incorporated in marine ecosystem models. Most models however neglect the evolutionary potential of phytoplankton. Previous resurrection experiments with a spring bloom dinoflagellate suggest that the past century of global warming has caused an adaptive response in an important life cycle trait, the encystment rate. Here, based on this resurrection case study, we apply an advanced ecosystem model including selection and mutation, to test whether a temperature increase could induce a change in encystment. In line with the findings from resurrection experiments, our results show that in warmer waters strains with a lower encystment rate benefit over those with a higher encystment rate. The magnitude of change in encystment rate is however only reproduced, if additional factors, like eutrophication and a cyst mortality that increases with temperature, are considered. By using this ecosystem model including selection and mutation, we demonstrate that ecosystem modeling represents a powerful approach to investigate the adaptive potential of phytoplankton. © 2019 Elsevier B.V

Modeling the role of pH on Baltic Sea Cyanobacteria

We simulate pH-dependent growth of cyanobacteria with an ecosystem model for the central Baltic Sea. Four model components—a life cycle model of cyanobacteria, a biogeochemical model, a carbonate chemistry model and a water column model—are coupled via the framework for aquatic biogeochemical models. The coupled model is forced by the output of a regional climate model, based on the A1B emission scenario. With this coupled model, we perform simulations for the period 1968–2098. Our simulation experiments suggest that in the future, cyanobacteria growth is hardly affected by the projected pH decrease. However, in the simulation phase prior to 1980, cyanobacteria growth and N2-fixation are limited by the relatively high pH. The observed absence of cyanobacteria before the 1960s may thus be explained not only by lower eutrophication levels, but also by a higher alkalinity

Additive manufacturing of WC-13%Co by selective electron beam melting: Achievements and challenges

Additive manufacturing is a powerful tool for rapid prototyping and fabricating metal articles having a complicated geometry. This method is known to be used almost solely for the manufacture of articles consisting of pure metals and alloys. In the present work the possibility of obtaining dense carbide articles by a single-step process of additive manufacturing based on selective electron beam melting was evaluated. A new technology for fabricating cemented carbide granules suitable for selective electron beam melting was developed. It includes conventional granulating WC-Co powders followed by solid-state pre-sintering and preliminary screening of the granules. After that their liquid-phase sintering and final screening are carried out to obtain a desired fraction needed for the additive manufacturing process. Results of experiments on selective electron beam melting at different scan rates and current values indicated that it was possible to obtain non-porous carbide articles of complex geometry from WC-Co granules initially containing 13 wt% Co. The selective electron beam melting process led to the evaporation of some liquid Co and very intense local WC grain growth resulting in peculiar microstructures of the cemented carbide articles comprising layers with medium-coarse and abnormally large WC grains. A near-surface layer of the cemented carbide articles obtained by additive manufacturing is characterized by a high roughness comparable with the mean size of the original WC-Co granules

Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin

BACKGROUND: Claudin-4, a tight junction (TJ) protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is overexpressed in epithelial ovarian cancer (EOC). Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4-expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular) resistance (Rb) in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin-4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies