Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias. © 2018 Cheng et al

Hepatitis C virus attenuates mitochondrial lipid β-oxidation by downregulating mitochondrial trifunctional-protein expression

The course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid β-oxidation. However, little is known about the effects of HCV on lipid β-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and β-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid β-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid β-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-offunction study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver

Mapping Histories of Art Education In New South Wales

This article examines the historical discourse relating to the field of art education in New South Wales from the nineteenth through to the late twentieth century. While discussing antecedent historiography relating to the history of art education in Australia, it argues that the tasks associated with that history are incomplete. By focusing on the system of art education in New South Wales, the paper refers to the differences between specific state-based systems of art education in Australia. It indicates the advantages of recognizing the effect of sexual, ideological, psychological, and other social and educational practices when writing historical material about art education. It introduces evidence that some of the most common perceptions of the history of Australian art education require more detailed scrutiny.l’éducation en arts en Nouvelles Galles du Sud, depuis le XIXe siècle jusqu’à la fin du XXe siècle. Faisant état de l’historiographie touchant l’histoire de l’éducation en arts en Australie, l’auteur soutient que cette historiographie présente des lacunes. En mettant l’accent sur le système d’éducation en arts en Nouvelles Galles du Sud, le texte fait référence aux différences entre des systèmes étatiques particuliers d’éducation en arts australiens. Il souligne les avantages à reconnaître l’influence des pratiques d’ordre sexuel, idéologique, psychologique ainsi que social et éducatif lorsqu’il s’agit d’écrire l’histoire de l’éducation en arts. Arguments à l’appui, il montre que certaines des perceptions les plus répandues de l’histoire de l’éducation en arts en Australie requièrent une analyse plus fouillée

VennPlex--a novel Venn diagram program for comparing and visualizing datasets with differentially regulated datapoints.

With the development of increasingly large and complex genomic and proteomic data sets, an enhancement in the complexity of available Venn diagram analytical programs is becoming increasingly important. Current freely available Venn diagram programs often fail to represent extra complexity among datasets, such as regulation pattern differences between different groups. Here we describe the development of VennPlex, a program that illustrates the often diverse numerical interactions among multiple, high-complexity datasets, using up to four data sets. VennPlex includes versatile output features, where grouped data points in specific regions can be easily exported into a spreadsheet. This program is able to facilitate the analysis of two to four gene sets and their corresponding expression values in a user-friendly manner. To demonstrate its unique experimental utility we applied VennPlex to a complex paradigm, i.e. a comparison of the effect of multiple oxygen tension environments (1–20% ambient oxygen) upon gene transcription of primary rat astrocytes. VennPlex accurately dissects complex data sets reliably into easily identifiable groups for straightforward analysis and data output. This program, which is an improvement over currently available Venn diagram programs, is able to rapidly extract important datasets that represent the variety of expression patterns available within the data sets, showing potential applications in fields like genomics, proteomics, and bioinformatics

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide–induced proarrhythmic early afterdepolarizations

Exposure to carbon monoxide (CO) causes early afterdepolarization arrhythmias. Previous studies in rats indicated arrhythmias arose due to augmentation of the late Na+ current. The purpose of the present study was to examine the basis of CO-induced arrhythmias in guinea pig myocytes in which action potentials more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes and also from HEK293 cells expressing wild-type or a C723S mutant form of Kv11.1 (ERG). We also monitored formation of peroxynitrite (ONOO-) in HEK293 cells fluorimetrically. CO, applied as the CO releasing molecule, CORM-2, prolonged action potentials and induced early after-depolarizations (EADs) in guinea pig myocytes. In HEK293 cells CO inhibited wild-type but not C723S mutant Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors or inhibition of nitric oxide formation. CO also raised ONOO- levels, an effect reversed by the ONOO- scavenger, FeTPPS which also prevented CO inhibition of Kv11.1 currents, and abolished the effects of CO on Kv11.1 tail currents and action potentials in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via ONOO--mediated inhibition of Kv11.1 K+ channel

Regulation of the T-type Ca²⁺ channel Cav3.2 by hydrogen sulfide: Emerging controversies concerning the role of H₂S in nociception

Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H₂S is a modulator of low voltage-activated T-type Ca²⁺ channels, and discriminates between the different subtypes of T-type Ca²⁺ channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H₂S augments Cav3.2 currents, an observation which has led to the suggestion that H₂S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H₂S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H₂S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H₂S modulation of T-type Ca²⁺ channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca²⁺ channel modulation by H₂S might be exploited as a novel approach to pain management

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically

The thienopyridine A-769662 and benzimidazole 991 inhibit human TASK-3 potassium channels in an AMPK-independent manner

Heteromeric Tandem pore domain Acid Sensitive (TASK)-1/3 channels are critical to oxygen-sensing by carotid body type 1 cells, where hypoxia-induced inhibition of TASK-3 and/or TASK-1/3 potassium currents leads to voltage-gated calcium entry, exocytotic transmitter release and increases in carotid body afferent input responses that initiate corrective changes in breathing patterns. It was proposed that, in response to hypoxia, the AMP–activated protein kinase (AMPK) might directly phosphorylate and inhibit TASK channels, in particular TASK–3, but studies on rat type I cells questioned this view. However, sequence alignment identified a putative AMPK recognition motif in human (h) TASK-3, but not hTASK–1, with Ser55 representing a potential phosphorylation site. We therefore studied the effects of five different AMPK activators on recombinant hTASK–3 potassium channels expressed in human embryonic kidney (HEK)–293 cells. Two structurally unrelated AMPK activators, the thienopyridine A–769662 (100–500 µM) and the benzimidazole 991 (3–30 µM) inhibited hTASK–3 currents in a concentration–dependent manner, while the 4-azabenzimidazole MK–8722 (3–30 µM) partially inhibited hTASK–3 at concentrations above those required for maximal AMPK activation. By contrast, the 4-azabenzimidazole, BI-9774 (10–100 µM; a closely related analogue of MK8722) and the pro-drug AICA-riboside (1 mM; metabolised to ZMP, an AMP-mimetic) had no significant effect on hTASK–3 currents at concentrations sufficient to maximally activate AMPK. Importantly, A–769662 (300 µM) also inhibited hTASK–3 channel currents in HEK–293 cells that stably over-expressed an AMPK–β1 subunit mutant (S108A) that renders AMPK insensitive to activators that bind to the Allosteric Drug and Metabolite site, such as A–769662. We therefore identify A–769662 and 991 as novel hTASK–3 channel inhibitors and provide conclusive evidence that AMPK does not regulate hTASK–3 channel current

Evolutionary genomics of a cold-adapted diatom: Fragilariopsis cylindrus

The Southern Ocean houses a diverse and productive community of organisms1, 2. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice3, 4, 5, 6, 7. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus8, 9, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO2. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean