A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl \u3csup\u3e–\u3c/sup\u3e from acidic organelles in amacrine cells

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission

COVID-19 Clinical Guidance For the Cardiovascular Care Team

COVID-19 is a quickly evolving public health emergency. The guidance provided in this document is based on the best available published information and expert evaluation. This document is intended to supplement, not supersede, relevant guidance from the Centers for Disease Control and Prevention, state and local health authorities, and your institution’s infectious disease containment, mitigation, and response plan

Imaging X-Ray Polarimeter Explorer Systems Engineering Approach and Implementation

The Imaging X-ray Polarimetry Explorer (IXPE) is a NASA Small Explorer x-ray astrophysics mission being implemented by a geographically dispersed team. Each IXPE partner provides unique capabilities and experience which are utilized to design, build and launch the IXPE observator. A rigorous and iterative systems engineering approach is essential to ensuring the successful realization of reliable and cost effective IXPE mission system. The IXPE collaboration and observatory complexity provide both unique challenges and advantages for project systems engineering. The project uses established and tailored systems engineering (SE) methods and teaming approaches to achieve the IXPE mission goals. The IXPE systems engineering team spans all partner organizations. Currently, the project is in system integration and test working through structural environmental testing–vibration testing is just starting. Systems work is now focused on requirements management and maturity assessments, requirements verification and validation via sell-off packages (SOP) and interface control document (ICD) verification while supporting environmental test planning and execution. IXPE verification, validation and characterization (V&V) starts at the component/unit level and rolls up to appropriate higher levels where V&V compliance is assured by collaborative development by the cross-organizational V&V Team. This paper provides a technical summary of the IXPE concept of operations and mission-system (payload, spacecraft, observatory, ground system, launch vehicle), overviews the IXPE systems engineering approach (communications, project reviews, requirements analysis and management, baseline design and design trade studies, interfaces definition and documentation, resource management), describes the verification, validation and characterization activities (requirements validation, models and simulations validation, systems integration and test (I&T), system validation), discusses risk and opportunities philosophy and implementation, outlines COVID 19 accommodations, itemizes some key challenges and lessons-learned followed by the path to launch and conclusions

Distinct Functional Roles of β-Tubulin Isotypes in Microtubule Arrays of Tetrahymena thermophila, a Model Single-Celled Organism

<div><h3>Background</h3><p>The multi-tubulin hypothesis proposes that each tubulin isotype performs a unique role, or subset of roles, in the universe of microtubule function(s). To test this hypothesis, we are investigating the functions of the recently discovered, noncanonical β-like tubulins (BLTs) of the ciliate, <em>Tetrahymena thermophila</em>. <em>Tetrahymena</em> forms 17 distinct microtubular structures whose assembly had been thought to be based on single α- and β-isotypes. However, completion of the macronuclear genome sequence of <em>Tetrahymena</em> demonstrated that this ciliate possessed a β-tubulin multigene family: two synonymous genes (<em>BTU1</em> and <em>BTU2</em>) encode the canonical β-tubulin, BTU2, and six genes (<em>BLT1-6</em>) yield five divergent β-tubulin isotypes. In this report, we examine the structural features and functions of two of the BLTs (BLT1 and BLT4) and compare them to those of BTU2.</p> <h3>Methodology/Principal Findings</h3><p>With respect to BTU2, BLT1 and BLT4 had multiple sequence substitutions in their GTP-binding sites, in their interaction surfaces, and in their microtubule-targeting motifs, which together suggest that they have specialized functions. To assess the roles of these tubulins <em>in vivo</em>, we transformed <em>Tetrahymena</em> with expression vectors that direct the synthesis of GFP-tagged versions of the isotypes. We show that GFP-BLT1 and GFP-BLT4 were not detectable in somatic cilia and basal bodies, whereas GFP-BTU2 strongly labeled these structures. During cell division, GFP-BLT1 and GFP-BLT4, but not GFP-BTU2, were incorporated into the microtubule arrays of the macronucleus and into the mitotic apparatus of the micronucleus. GFP-BLT1 also participated in formation of the microtubules of the meiotic apparatus of the micronucleus during conjugation. Partitioning of the isotypes between nuclear and ciliary microtubules was confirmed biochemically.</p> <h3>Conclusion/Significance</h3><p>We conclude that <em>Tetrahymena</em> uses a family of distinct β-tubulin isotypes to construct subsets of functionally different microtubules, a result that provides strong support for the multi-tubulin hypothesis.</p> </div

Genome-Wide Analysis of the World's Sheep Breeds Reveals High Levels of Historic Mixture and Strong Recent Selection

Genomic structure in a global collection of domesticated sheep reveals a history of artificial selection for horn loss and traits relating to pigmentation, reproduction, and body size

A role for the cystic fibrosis transmembrane conductance regulator in the nitric oxide-dependent release of Cl −

© 2017 the American Physiological Society. γ-Amino butyric acid (GABA) and glycine typically mediate synaptic inhibition because their ligandgated ion channels support the influx of Cl – . However, the electrochemical gradient for Cl – across the postsynaptic plasma membrane determines the voltage response of the postsynaptic cell. Typically, low cytosolic Cl – levels support inhibition, whereas higher levels of cytosolic Cl – can suppress inhibition or promote depolarization. We previously reported that nitric oxide (NO) releases Cl – from acidic organelles and transiently elevates cytosolic Cl – , making the response to GABA and glycine excitatory. In this study, we test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in the NO-dependent efflux of organellar Cl – . We first establish the mRNA and protein expression of CFTR in our model system, cultured chick retinal amacrine cells. Using whole cell voltage- clamp recordings of currents through GABA-gated Cl – channels, we examine the effects of pharmacological inhibition of CFTR on the NO-dependent release of internal Cl – . To interfere with the expression of CFTR, we used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. We find that both pharmacological inhibition and CRISPR/Cas9-mediated knockdown of CFTR block the ability of NO to release Cl – from internal stores. These results demonstrate that CFTR is required for the NO-dependent efflux of Cl – from acidic organelles. NEW & NOTEWORTHY Although CFTR function has been studied extensively in the context of epithelia, relatively little is known about its function in neurons. We show that CFTR is involved in an NO-dependent release of Cl – from acidic organelles. This internal function of CFTR is particularly relevant to neuronal physiology because postsynaptic cytosolic Cl – levels determine the outcome of GABA- and glycinergic synaptic signaling. Thus the CFTR may play a role in regulating synaptic transmission