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

Disrupting the one-loop renormalization group invariant M/alpha in supersymmetry

It is well known that in low energy supersymmetry the ratio of the gaugino mass to the gauge coupling squared, M/alpha, is renormalization group invariant to one-loop. We present a systematic analysis of the corrections to this ratio, including standard two-loop corrections from gauge and Yukawa couplings, corrections due to an additional U(1)' gaugino, threshold corrections, superoblique corrections, corrections due to extra matter, GUT and Planck scale corrections, and ``corrections'' from messenger sectors with supersymmetry breaking communicated via gauge-mediation. We show that many of these effects induce corrections at the level of a few to tens of percent, but some could give much larger corrections, drastically disrupting the renormalization group extrapolation of the ratio to higher scales. Our analysis is essentially model-independent, and therefore can be used to determine the ambiguities in extrapolating the ratio in any given model between the weak scale and higher scales.Comment: 43 pages, LaTeX, uses epsf.sty, axodraw.sty, 12 eps figures. Minor typos corrected. To appear in Nucl. Phys.

Aromatase inhibition remodels the clonal architecture of estrogen-receptor-positive breast cancers

Resistance to oestrogen-deprivation therapy is common in oestrogen-receptor-positive (ER+) breast cancer. To better understand the contributions of tumour heterogeneity and evolution to resistance, here we perform comprehensive genomic characterization of 22 primary tumours sampled before and after 4 months of neoadjuvant aromatase inhibitor (NAI) treatment. Comparing whole-genome sequencing of tumour/normal pairs from the two time points, with coincident tumour RNA sequencing, reveals widespread spatial and temporal heterogeneity, with marked remodelling of the clonal landscape in response to NAI. Two cases have genomic evidence of two independent tumours, most obviously an ER− ‘collision tumour', which was only detected after NAI treatment of baseline ER+ disease. Many mutations are newly detected or enriched post treatment, including two ligand-binding domain mutations in ESR1. The observed clonal complexity of the ER+ breast cancer genome suggests that precision medicine approaches based on genomic analysis of a single specimen are likely insufficient to capture all clinically significant information

The Next-to-Minimal Supersymmetric Standard Model

We review the theoretical and phenomenological aspects of the Next-to-Minimal Supersymmetric Standard Model: the Higgs sector including radiative corrections and the 2-loop beta-functions for all parameters of the general NMSSM; the tadpole and domain wall problems, baryogenesis; NMSSM phenomenology at colliders, B physics and dark matter; specific scenarios as the constrained NMSSM, Gauge Mediated Supersymmetry Breaking, U(1)'-extensions, CP and R-parity violation.Comment: 144 pages, 11 figures, corrections in Eqs.(2.2), (2.21), (B.9

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

Growth and cellular differentiation: a physical-biochemical conundrum? The example of the hand

Currently, the predominant hypothesis explains cellular differentiation as an essentially genetic intracellular process. The goal of this paper is to suggest that cell growth and differentiation may be, simply, the result of physical and chemical constraints.Bone growth occurs at the level of cartilage conjunction (growth plate) in a zone of lesser constrain. It appears that this growth also induces muscle, tendon, nerve and skin elongation. This cartilage growth by itself seems to explain the elongation of the hand. Growth stops at puberty likely because of feed-back from an increasing muscle load. The ossification (that is differentiation of cartilage into bone) appears to result from the shear stress induced. The study of bone age, obtained by X-ray picture of the hand, shows that ossification of epiphyses is very precise both in time and space. Computer modelization suggests that this ossification occurs where shear stress is greatest. The cartilage which does not ossify (joint, nose, larynx, ear, bronchus, etc.) is not exposed to high shear.Shear stress induces the secretion of extracellular matrix and a change of the biochemical environment of the cell. Precipitation of calcium phosphate, as in ossification, seems related to the alkalosis induced by shear stress.To speak in more general terms, loss of cellular differentiation, as occurs with cancer, can result from a change in the physical–chemical environments

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

Retinoid Metabolism and Diabetes Mellitus

Retinoid acid is a metabolite of vitamin A and functions as an important factor in cell survival, differentiation and death. Most previous studies on retinoid metabolism have focused on its association with cancer, hematologic and dermatologic disorders. Given the special concern over the recent increase in the prevalence of diabetes worldwide, the role of retinoid metabolism on glucose metabolism and insulin resistance in the human body is of marked importance. Therefore, in this issue, we review the literature on the association of retinoid metabolism with glucose tolerance, with regard to insulin secretion, pancreatic autoimmunity, insulin sensitivity and lipid metabolism. Further, we tried to assess the possibility of using retinoids as a novel therapeutic strategy for diabetes