Two-Photon Uncaging of Glutamate

Two-photon microscopy produces the excited singlet state of a chromophore with wavelengths approximately double that used for normal excitation. Two photons are absorbed almost simultaneously, via a virtual state, and this makes the excitation technique inherently non-linear. It requires ultra-fast lasers to deliver the high flux density needed to access intrinsically very short lived intermediates, and in combination with lenses of high numerical aperture, this confines axial excitation highly. Since the two-photon excitation volume is similar to a large spine head, the technique has been widely used to study glutamatergic transmission in brain slices. Here I describe the principles of two-photon uncaging of glutamate and provide a practical guide to its application

Local recovery of cardiac calcium‐induced calcium release interrogated by ultra‐effective, two‐photon uncaging of calcium

KEY POINTS: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two‐photon photolysis of a new ultra‐effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types. ABSTRACT: In cardiac myocytes Ca(2+)‐induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca(2+) release occurs via subcellular Ca(2+) signalling events, Ca(2+) sparks. Local recovery of Ca(2+) release depends on both SR refilling and restoration of Ca(2+) sensitivity of the RyRs. We used two‐photon (2P) photolysis of the ultra‐effective caged Ca(2+) compound BIST‐2EGTA and laser‐scanning confocal Ca(2+) imaging to probe refractoriness of local Ca(2+) release in control conditions and in the presence of cAMP or low‐dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST‐2EGTA and rhod‐2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca(2+) release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self‐learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyR(R420Q) mutation were used for further validation and revealed a higher Ca(2+) sensitivity of CICR. This new 2P approach provides composite information of Ca(2+) release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca(2+) sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias

Elliptical Galaxies and Bulges of Disk Galaxies: Summary of Progress and Outstanding Issues

This is the summary chapter of a review book on galaxy bulges. Bulge properties and formation histories are more varied than those of ellipticals. I emphasize two advances: 1 - "Classical bulges" are observationally indistinguishable from ellipticals, and like them, are thought to form by major galaxy mergers. "Disky pseudobulges" are diskier and more actively star-forming (except in S0s) than are ellipticals. Theys are products of the slow ("secular") evolution of galaxy disks: bars and other nonaxisymmetries move disk gas toward the center, where it starbursts and builds relatively flat, rapidly rotating components. This secular evolution is a new area of galaxy evolution work that complements hierarchical clustering. 2 - Disks of high-redshift galaxies are unstable to the formation of mass clumps that sink to the center and merge - an alternative channel for the formation of classical bulges. I review successes and unsolved problems in the formation of bulges+ellipticals and their coevolution (or not) with supermassive black holes. I present an observer's perspective on simulations of dark matter galaxy formation including baryons. I review how our picture of the quenching of star formation is becoming general and secure at redshifts z < 1. The biggest challenge is to produce realistic bulges+ellipticals and disks that overlap over a factor of 10**3 in mass but that differ from each other as observed over that whole range. Second, how does hierarchical clustering make so many giant, bulgeless galaxies in field but not cluster environments? I argue that we rely too much on AGN and star-formation feedback to solve these challenges.Comment: 46 pages, 10 postscript figures, accepted for publication in Galactic Bulges, ed. E. Laurikainen, R. F. Peletier, & D. A. Gadotti (New York: Springer), in press (2015

A review of elliptical and disc galaxy structure, and modern scaling laws

A century ago, in 1911 and 1913, Plummer and then Reynolds introduced their models to describe the radial distribution of stars in `nebulae'. This article reviews the progress since then, providing both an historical perspective and a contemporary review of the stellar structure of bulges, discs and elliptical galaxies. The quantification of galaxy nuclei, such as central mass deficits and excess nuclear light, plus the structure of dark matter halos and cD galaxy envelopes, are discussed. Issues pertaining to spiral galaxies including dust, bulge-to-disc ratios, bulgeless galaxies, bars and the identification of pseudobulges are also reviewed. An array of modern scaling relations involving sizes, luminosities, surface brightnesses and stellar concentrations are presented, many of which are shown to be curved. These 'redshift zero' relations not only quantify the behavior and nature of galaxies in the Universe today, but are the modern benchmark for evolutionary studies of galaxies, whether based on observations, N-body-simulations or semi-analytical modelling. For example, it is shown that some of the recently discovered compact elliptical galaxies at 1.5 < z < 2.5 may be the bulges of modern disc galaxies.Comment: Condensed version (due to Contract) of an invited review article to appear in "Planets, Stars and Stellar Systems"(www.springer.com/astronomy/book/978-90-481-8818-5). 500+ references incl. many somewhat forgotten, pioneer papers. Original submission to Springer: 07-June-201

An experimental study exploring the impact of vignette gender on the quality of university students’ mental health first aid for peers with symptoms of depression

Background University students have high rates of depression, and friends are often the most commonly-used source of support for emotional distress in this population. This study aimed to explore students’ ability to provide effective support for their peers with depressive symptoms and the factors influencing the quality of their mental health first aid (MHFA) skills, including students’ gender, course of study, and gender of student experiencing depression. Methods Via an online survey, students at two British universities (N = 483) were quasi-randomly allocated to view a video vignette of either a male or female student depicting symptoms of depression. An open-ended question probed MHFA actions they would take to help the vignette character, which were rated using a standardised scoring scheme based on MHFA guidelines. Results Students reported low MHFA scores (mean 2.89, out of possible 12). The most commonly reported action was provision of support and information, but only eight (1.6 %) students stated an intention to assess risk of harm. Those studying clinically non-relevant degrees with limited mental health content reported poorer MHFA (p = <0.001) and were less confident about their ability to support a friend with depression (p = 0.04). There was no main effect of vignette gender, but within the group of students on non-relevant courses the male vignette received significantly poorer MHFA than the female vignette (p = 0.02). A significant three-way interaction found that male participants studying non-relevant degrees who viewed a male vignette had poorer MHFA compared to females studying non-relevant degrees who viewed the female vignette (p = 0.005). Conclusions Most students lack the necessary MHFA skills to support friends suffering from symptoms of depression, or to help them get appropriate support and prevent risk of harm. Students on courses which do not include mental health related content are particularly ill-equipped to support male students, with male students receiving the poorest quality MHFA from fellow male students on these courses. MHFA training has the potential to improve outcomes for students with depression, and could have a valuable role in reducing the excess risk of harm seen in male students

Rapid identification of bovine MHCI haplotypes in genetically divergent cattle populations Using Next-Generation Sequencing

The major histocompatibility complex (MHC) region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Our current knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire

Cold atoms in space: community workshop summary and proposed road-map

We summarise the discussions at a virtual Community Workshop on Cold Atoms in Space concerning the status of cold atom technologies, the prospective scientific and societal opportunities offered by their deployment in space, and the developments needed before cold atoms could be operated in space. The cold atom technologies discussed include atomic clocks, quantum gravimeters and accelerometers, and atom interferometers. Prospective applications include metrology, geodesy and measurement of terrestrial mass change due to, e.g., climate change, and fundamental science experiments such as tests of the equivalence principle, searches for dark matter, measurements of gravitational waves and tests of quantum mechanics. We review the current status of cold atom technologies and outline the requirements for their space qualification, including the development paths and the corresponding technical milestones, and identifying possible pathfinder missions to pave the way for missions to exploit the full potential of cold atoms in space. Finally, we present a first draft of a possible road-map for achieving these goals, that we propose for discussion by the interested cold atom, Earth Observation, fundamental physics and other prospective scientific user communities, together with the European Space Agency (ESA) and national space and research funding agencies

Whole-genome sequencing reveals host factors underlying critical COVID-19

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2–4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes—including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)—in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease

Interpretative and predictive modelling of Joint European Torus collisionality scans

Transport modelling of Joint European Torus (JET) dimensionless collisionality scaling experiments in various operational scenarios is presented. Interpretative simulations at a fixed radial position are combined with predictive JETTO simulations of temperatures and densities, using the TGLF transport model. The model includes electromagnetic effects and collisions as well as □(→┬E ) X □(→┬B ) shear in Miller geometry. Focus is on particle transport and the role of the neutral beam injection (NBI) particle source for the density peaking. The experimental 3-point collisionality scans include L-mode, and H-mode (D and H and higher beta D plasma) plasmas in a total of 12 discharges. Experimental results presented in (Tala et al 2017 44th EPS Conf.) indicate that for the H-mode scans, the NBI particle source plays an important role for the density peaking, whereas for the L-mode scan, the influence of the particle source is small. In general, both the interpretative and predictive transport simulations support the experimental conclusions on the role of the NBI particle source for the 12 JET discharges

Hot gas flows on global and nuclear galactic scales

Since its discovery as an X-ray source with the Einstein Observatory, the hot X-ray emitting interstellar medium of early-type galaxies has been studied intensively, with observations of improving quality, and with extensive modeling by means of numerical simulations. The main features of the hot gas evolution are outlined here, focussing on the mass and energy input rates, the relationship between the hot gas flow and the main properties characterizing its host galaxy, the flow behavior on the nuclear and global galactic scales, and the sensitivity of the flow to the shape of the stellar mass distribution and the mean rotation velocity of the stars.Comment: 22 pages. Abbreviated version of chapter 2 of the book "Hot Interstellar Matter in Elliptical Galaxies", Springer 201