Quantifying Exocytosis by Combination of Membrane Capacitance Measurements and Total Internal Reflection Fluorescence Microscopy in Chromaffin Cells

Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

Docking of LDCVs Is Modulated by Lower Intracellular [Ca2+] than Priming

Many regulatory steps precede final membrane fusion in neuroendocrine cells. Some parts of this preparatory cascade, including fusion and priming, are dependent on the intracellular Ca2+ concentration ([Ca2+]i). However, the functional implications of [Ca2+]i in the regulation of docking remain elusive and controversial due to an inability to determine the modulatory effect of [Ca2+]i. Using a combination of TIRF-microscopy and electrophysiology we followed the movement of large dense core vesicles (LDCVs) close to the plasma membrane, simultaneously measuring membrane capacitance and [Ca2+]i. We found that a free [Ca2+]i of 700 nM maximized the immediately releasable pool and minimized the lateral mobility of vesicles, which is consistent with a maximal increase of the pool size of primed LDCVs. The parameters that reflect docking, i.e. axial mobility and the fraction of LDCVs residing at the plasma membrane for less than 5 seconds, were strongly decreased at a free [Ca2+]i of 500 nM. These results provide the first evidence that docking and priming occur at different free intracellular Ca2+ concentrations, with docking efficiency being the most robust at 500 nM

From Cleanroom to Desktop: Emerging Micro-Nanofabrication Technology for Biomedical Applications

This review is motivated by the growing demand for low-cost, easy-to-use, compact-size yet powerful micro-nanofabrication technology to address emerging challenges of fundamental biology and translational medicine in regular laboratory settings. Recent advancements in the field benefit considerably from rapidly expanding material selections, ranging from inorganics to organics and from nanoparticles to self-assembled molecules. Meanwhile a great number of novel methodologies, employing off-the-shelf consumer electronics, intriguing interfacial phenomena, bottom-up self-assembly principles, etc., have been implemented to transit micro-nanofabrication from a cleanroom environment to a desktop setup. Furthermore, the latest application of micro-nanofabrication to emerging biomedical research will be presented in detail, which includes point-of-care diagnostics, on-chip cell culture as well as bio-manipulation. While significant progresses have been made in the rapidly growing field, both apparent and unrevealed roadblocks will need to be addressed in the future. We conclude this review by offering our perspectives on the current technical challenges and future research opportunities

Novel Blood Pressure Locus and Gene Discovery Using Genome-Wide Association Study and Expression Data Sets From Blood and the Kidney

Elevated blood pressure is a major risk factor for cardiovascular disease and has a substantial genetic contribution. Genetic variation influencing blood pressure has the potential to identify new pharmacological targets for the treatment of hypertension. To discover additional novel blood pressure loci, we used 1000 Genomes Project-based imputation in 150 134 European ancestry individuals and sought significant evidence for independent replication in a further 228 245 individuals. We report 6 new signals of association in or near HSPB7, TNXB, LRP12, LOC283335, SEPT9, and AKT2, and provide new replication evidence for a further 2 signals in EBF2 and NFKBIA. Combining large whole-blood gene expression resources totaling 12 607 individuals, we investigated all novel and previously reported signals and identified 48 genes with evidence for involvement in blood pressure regulation that are significant in multiple resources. Three novel kidney-specific signals were also detected. These robustly implicated genes may provide new leads for therapeutic innovation

Genome-wide association analysis identifies novel blood pressure loci and offers biological insights into cardiovascular risk

Elevated blood pressure is the leading heritable risk factor for cardiovascular disease worldwide. We report genetic association of blood pressure (systolic, diastolic, pulse pressure) among UK Biobank participants of European ancestry with independent replication in other cohorts, and robust validation of 107 independent loci. We also identify new independent variants at 11 previously reported blood pressure loci. In combination with results from a range of in silico functional analyses and wet bench experiments, our findings highlight new biological pathways for blood pressure regulation enriched for genes expressed in vascular tissues and identify potential therapeutic targets for hypertension. Results from genetic risk score models raise the possibility of a precision medicine approach through early lifestyle intervention to offset the impact of blood pressure-raising genetic variants on future cardiovascular disease risk

The genetics of blood pressure regulation and its target organs from association studies in 342,415 individuals

To dissect the genetic architecture of blood pressure and assess effects on target organ damage, we analyzed 128,272 SNPs from targeted and genome-wide arrays in 201,529 individuals of European ancestry, and genotypes from an additional 140,886 individuals were used for validation. We identified 66 blood pressure–associated loci, of which 17 were new; 15 harbored multiple distinct association signals. The 66 index SNPs were enriched for cis-regulatory elements, particularly in vascular endothelial cells, consistent with a primary role in blood pressure control through modulation of vascular tone across multiple tissues. The 66 index SNPs combined in a risk score showed comparable effects in 64,421 individuals of non-European descent. The 66-SNP blood pressure risk score was significantly associated with target organ damage in multiple tissues but with minor effects in the kidney. Our findings expand current knowledge of blood pressure–related pathways and highlight tissues beyond the classical renal system in blood pressure regulation

Lack of cardiac fibrosis in a new model of high prorenin hyperaldosteronism

The aim of the present study was to test the hypothesis that elevation of prorenin in plasma is sufficient to induce cardiac fibrosis. Normotensive cyp1a1ren-2 transgenic rats with normal plasma prorenin and aldosterone levels were given 0.125% indole-3-carbinol (I3C) orally for a period of 12 wk. Plasma prorenin and aldosterone levels were determined in 4-wk intervals, and cardiac marker enzymes for hypertrophy, fibrosis, and oxidative stress as well as cardiac pathology were investigated. In I3C-treated cyp1a1 ren-2 transgenic rats, plasma prorenin concentrations were >100-fold elevated (≥7.1 ± 2.6 μg ANG I·ml−1·h−1 vs. ≤0.07 ± 0.1; P < 0.001), whereas active renin levels were suppressed (0.09 ± 0.02 vs. 0.2 ± 0.1; P < 0.05). Aldosterone concentrations were elevated three- to fourfold for a period of >4 wk (574 ± 51 vs. 160 ± 68 pg/ml; P < 0.01). After 12 wk of I3C, rats exhibited moderate cardiac hypertrophy (heart weight/body weight 2.5 ± 0.04 vs. 3.1 ± 0.1 mg/g; P < 0.01). There was a slight increase in mRNA contents of endothelin 1 (1.21 ± 0.08 vs. 0.75 ± 0.007; P < 0.001), NADP oxidase-2 (1.03 ± 0.006 vs. 0.76 ± 0.04; P < 0.001), transforming growth factor-β (0.99 ± 0.06 vs. 0.84 ± 0.04; P < 0.05), collagen type I (1.32 ± 0.32 vs. 0.94 ± 0.18; P < 0.05), and intercellular adhesion molecule-1 (1.12 ± 0.12 vs. 0.84 ± 0.08; P < 0.05). These genes are known to be stimulated by the renin-angiotensin system. There were no histological signs of fibrosis in the heart. We found that prorenin and aldosterone alone are not sufficient to induce considerable cardiac fibrosis in the absence of sodium load

Predictors of outcome of non-displaced fractures of the base of the fifth metatarsal

The purpose of this study was to identify those factors that influence the outcome after conservative treatment of undisplaced fractures of the fifth metatarsal. This was done with univariate analyses and, for the first time, with regression analyses of day-to-day clinical practice. Thirty-eight patients were treated with plaster and periods of no weight bearing (NWB). Their mean age was 48 years. They were evaluated using the Olerud ankle score, with analogue scales for pain and comfort, and with questions about cosmesis and wearing of shoes. Six patients sustained a Jones fracture and 32 a tuberosity avulsion fracture. The mean period of NWB was 17 days and of casting was 38 days. Three Jones fractures and all the avulsion fractures were healed at the end of treatment. After a mean of 490 days, the global ankle score was 82/100. Ten patients reported problems with shoes and nine reported cosmetic problems. The linear analogue scale for pain was 2.11/10 and for comfort 8.42/10. Gender, age, and fracture type did not affect outcome. The most significant predictor of poor functional outcome was longer NWB, which was strongly associated with worse global outcome, discomfort, and reported stiffness. NWB should be kept to a minimum for acute avulsions of the tuberosity of the fifth metatarsal