el Structural basis of cell-surface signaling by a conserved sigma regulator in Gram-negative bacteria [Molecular Biophysics] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Cell-surface signaling (CSS) in Gram-negative bacteria involves highly conserved regulatory pathways that optimize gene expression by transducing extracellular environmental signals to the cytoplasm via inner-membrane sigma regulators. The molecular details of ferric siderophore-mediated activation of the iron import machinery through a sigma regulator are unclear. Here, we present the 1.56 Å resolution structure of the periplasmic complex of the C-terminal CSS domain (CCSSD) of PupR, the sigma regulator in the Pseudomonas capeferrum pseudobactin BN7/8 transport system, and the N-terminal signaling domain (NTSD) of PupB, an outer-membrane TonB-dependent transducer. The structure revealed that the CCSSD consists of two subdomains: a juxta-membrane subdomain, which has a novel all-β-fold, followed by a secretin/TonB, short N-terminal subdomain at the C terminus of the CCSSD, a previously unobserved topological arrangement of this domain. Using affinity pulldown assays, isothermal titration calorimetry, and thermal denaturation CD spectroscopy, we show that both subdomains are required for binding the NTSD with micromolar affinity and that NTSD binding improves CCSSD stability. Our findings prompt us to present a revised model of CSS wherein the CCSSD:NTSD complex forms prior to ferric-siderophore binding. Upon siderophore binding, conformational changes in the CCSSD enable regulated intramembrane proteolysis of the sigma regulator, ultimately resulting in transcriptional regulation. Full Article
el An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1 [Enzymology] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers. Full Article
el Deletion of fatty acid transport protein 2 (FATP2) in the mouse liver changes the metabolic landscape by increasing the expression of PPAR{alpha}-regulated genes [Lipids] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney, where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased RNA-Seq to identify concomitant changes in the liver transcriptome. WT and FATP2-null (Fatp2−/−) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2−/− mice had reduced weight gain, lowered serum triglyceride, and increased serum cholesterol levels and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2−/− mice and a total of 91 in female Fatp2−/− mice. These genes mapped to the following gene ontology categories: fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-quantitative PCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator–activated receptor α (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2−/− liver revealed increases of total C16:0, C16:1, and C18:1 fatty acids; increases in lipoxin A4 and prostaglandin J2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism through its transport or activation activities. Full Article
el Noncatalytic Bruton's tyrosine kinase activates PLC{gamma}2 variants mediating ibrutinib resistance in human chronic lymphocytic leukemia cells [Membrane Biology] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs. Full Article
el Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of {alpha}-crystallin: Implications for lens aging and presbyopia [Glycobiology and Extracellular Matrices] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia. Full Article
el ER stress increases store-operated Ca2+ entry (SOCE) and augments basal insulin secretion in pancreatic beta cells [Molecular Bases of Disease] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress. Full Article
el Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B'{delta} holoenzyme [Enzymology] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge–swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge–swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal–regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity. Full Article
el Long noncoding RNA pncRNA-D reduces cyclin D1 gene expression and arrests cell cycle through RNA m6A modification [RNA] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression. Full Article
el Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V [Glycobiology and Extracellular Matrices] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis. Full Article
el Determination of globotriaosylceramide analogs in the organs of a mouse model of Fabry disease [Lipids] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Fabry disease is a heritable lipid disorder caused by the low activity of α-galactosidase A and characterized by the systemic accumulation of globotriaosylceramide (Gb3). Recent studies have reported a structural heterogeneity of Gb3 in Fabry disease, including Gb3 isoforms with different fatty acids and Gb3 analogs with modifications on the sphingosine moiety. However, Gb3 assays are often performed only on the selected Gb3 isoforms. To precisely determine the total Gb3 concentration, here we established two methods for determining both Gb3 isoforms and analogs. One was the deacylation method, involving Gb3 treatment with sphingolipid ceramide N-deacylase, followed by an assay of the deacylated products, globotriaosylsphingosine (lyso-Gb3) and its analogs, by ultra-performance LC coupled to tandem MS (UPLC-MS/MS). The other method was a direct assay established in the present study for 37 Gb3 isoforms and analogs/isoforms by UPLC-MS/MS. Gb3s from the organs of symptomatic animals of a Fabry disease mouse model were mainly Gb3 isoforms and two Gb3 analogs, such as Gb3(+18) containing the lyso-Gb3(+18) moiety and Gb3(−2) containing the lyso-Gb3(−2) moiety. The total concentrations and Gb3 analog distributions determined by the two methods were comparable. Gb3(+18) levels were high in the kidneys (24% of total Gb3) and the liver (13%), and we observed Gb3(−2) in the heart (10%) and the kidneys (5%). These results indicate organ-specific expression of Gb3 analogs, insights that may lead to a deeper understanding of the pathophysiology of Fabry disease. Full Article
el Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase [Molecular Biophysics] By www.jbc.org Published On :: 2020-04-24T06:08:45-07:00 Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases. Full Article
el Withdrawal: Distinct roles of Ape1 protein, an enzyme involved in DNA repair, in high or low linear energy transfer ionizing radiation-induced cell killing. [Withdrawals/Retractions] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 VOLUME 289 (2014) PAGES 30635–30644This article has been withdrawn by Guangnan Chen, Dongkyoo Park, Francis A. Cucinotta, David S. Yu, Xingming Deng, William S. Dynan, Paul W. Doetsch, and Ya Wang. Hongyan Wang, Xiang Wang, Xiangming Zhang, and Xiaobing Tang could not be reached. The last two lanes of the actin immunoblot in Fig. 1A were reused in the last two lanes of the actin immunoblot in Fig. 1C. In Fig. 2A, the γ-H2AX and the merge with DAPI images for no IR treatment do not match. In Fig. 3A, lanes 3 and 4 of the γ-H2AX immunoblot were reused in lanes 7 and 8, and lanes 5 and 6 of the H2A immunoblot were reused in lanes 7 and 8. In Fig. 3B, lanes 5 and 6 of the H2A immunoblot were reused in lanes 7 and 8. In Fig. 3C, lanes 5 and 6 of the γ-H2AX immunoblot were reused in lanes 7 and 8. Additionally, lanes 1 and 2 of the H2A immunoblot were reused in lanes 3 and 4. In Fig. 3D, lanes 1 and 2 of the Mre11 immunoblot from lysates were reused in lanes 4 and 5. In the γ-H2AX immunoblot, lane 3 was reused in lane 7, and lane 4 was reused in lanes 6 and 8. Also in the H2A immunoblot, lanes 1 and 2 were reused in lanes 3 and 4. In Fig. 4B, lanes 2 and 6 of the Mre11 immunoblot from Ogg1−/− cells are the same. In the Ape1... Full Article
el The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase [Glycobiology and Extracellular Matrices] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes. Full Article
el A Legionella effector kinase is activated by host inositol hexakisphosphate [Enzymology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis. Full Article
el Delineating an extracellular redox-sensitive module in T-type Ca2+ channels [Membrane Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc. Full Article
el NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling [Microbiology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell. Full Article
el S-Palmitoylation of the sodium channel Nav1.6 regulates its activity and neuronal excitability [Cell Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 S-Palmitoylation is a reversible post-translational lipid modification that dynamically regulates protein functions. Voltage-gated sodium channels are subjected to S-palmitoylation and exhibit altered functions in different S-palmitoylation states. Our aim was to investigate whether and how S-palmitoylation regulates Nav1.6 channel function and to identify S-palmitoylation sites that can potentially be pharmacologically targeted. Acyl-biotin exchange assay showed that Nav1.6 is modified by S-palmitoylation in the mouse brain and in a Nav1.6 stable HEK 293 cell line. Using whole-cell voltage clamp, we discovered that enhancing S-palmitoylation with palmitic acid increases Nav1.6 current, whereas blocking S-palmitoylation with 2-bromopalmitate reduces Nav1.6 current and shifts the steady-state inactivation in the hyperpolarizing direction. Three S-palmitoylation sites (Cys1169, Cys1170, and Cys1978) were identified. These sites differentially modulate distinct Nav1.6 properties. Interestingly, Cys1978 is exclusive to Nav1.6 among all Nav isoforms and is evolutionally conserved in Nav1.6 among most species. Cys1978 S-palmitoylation regulates current amplitude uniquely in Nav1.6. Furthermore, we showed that eliminating S-palmitoylation at specific sites alters Nav1.6-mediated excitability in dorsal root ganglion neurons. Therefore, our study reveals S-palmitoylation as a potential isoform-specific mechanism to modulate Nav activity and neuronal excitability in physiological and diseased conditions. Full Article
el Certain ortho-hydroxylated brominated ethers are promiscuous kinase inhibitors that impair neuronal signaling and neurodevelopmental processes [Cell Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 The developing nervous system is remarkably sensitive to environmental signals, including disruptive toxins, such as polybrominated diphenyl ethers (PBDEs). PBDEs are an environmentally pervasive class of brominated flame retardants whose neurodevelopmental toxicity mechanisms remain largely unclear. Using dissociated cortical neurons from embryonic Rattus norvegicus, we found here that chronic exposure to 6-OH–BDE-47, one of the most prevalent hydroxylated PBDE metabolites, suppresses both spontaneous and evoked neuronal electrical activity. On the basis of our previous work on mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) (MEK) biology and our observation that 6-OH–BDE-47 is structurally similar to kinase inhibitors, we hypothesized that certain hydroxylated PBDEs mediate neurotoxicity, at least in part, by impairing the MEK–ERK axis of MAPK signal transduction. We tested this hypothesis on three experimental platforms: 1) in silico, where modeling ligand–protein docking suggested that 6-OH–BDE-47 is a promiscuous ATP-competitive kinase inhibitor; 2) in vitro in dissociated neurons, where 6-OH–BDE-47 and another specific hydroxylated BDE metabolite similarly impaired phosphorylation of MEK/ERK1/2 and activity-induced transcription of a neuronal immediate early gene; and 3) in vivo in Drosophila melanogaster, where developmental exposures to 6-OH–BDE-47 and a MAPK inhibitor resulted in offspring displaying similarly increased frequency of mushroom-body β–lobe midline crossing, a metric of axonal guidance. Taken together, our results support that certain ortho-hydroxylated PBDE metabolites are promiscuous kinase inhibitors and can cause disruptions of critical neurodevelopmental processes, including neuronal electrical activity, pre-synaptic functions, MEK–ERK signaling, and axonal guidance. Full Article
el Kruppel-like factor 3 (KLF3) suppresses NF-{kappa}B-driven inflammation in mice [Immunology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Bacterial products such as lipopolysaccharides (or endotoxin) cause systemic inflammation, resulting in a substantial global health burden. The onset, progression, and resolution of the inflammatory response to endotoxin are usually tightly controlled to avoid chronic inflammation. Members of the NF-κB family of transcription factors are key drivers of inflammation that activate sets of genes in response to inflammatory signals. Such responses are typically short-lived and can be suppressed by proteins that act post-translationally, such as the SOCS (suppressor of cytokine signaling) family. Less is known about direct transcriptional regulation of these responses, however. Here, using a combination of in vitro approaches and in vivo animal models, we show that endotoxin treatment induced expression of the well-characterized transcriptional repressor Krüppel-like factor 3 (KLF3), which, in turn, directly repressed the expression of the NF-κB family member RELA/p65. We also observed that KLF3-deficient mice were hypersensitive to endotoxin and exhibited elevated levels of circulating Ly6C+ monocytes and macrophage-derived inflammatory cytokines. These findings reveal that KLF3 is a fundamental suppressor that operates as a feedback inhibitor of RELA/p65 and may be important in facilitating the resolution of inflammation. Full Article
el Catabolic degradation of endothelial VEGFA via autophagy [Glycobiology and Extracellular Matrices] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer. Full Article
el The hibernating 100S complex is a target of ribosome-recycling factor and elongation factor G in Staphylococcus aureus [Protein Synthesis and Degradation] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity. Full Article
el Atomic force microscopy-based characterization of the interaction of PriA helicase with stalled DNA replication forks [DNA and Chromosomes] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA–binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB. Full Article
el COQ11 deletion mitigates respiratory deficiency caused by mutations in the gene encoding the coenzyme Q chaperone protein Coq10 [Lipids] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Coenzyme Q (Qn) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1–coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6. The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because “fused” proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism. Full Article
el Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3 [Cell Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies. Full Article
el The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3 [Metabolism] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Heat shock factor 1 (HSF1) regulates cellular adaptation to challenges such as heat shock and oxidative and proteotoxic stresses. We have recently reported a previously unappreciated role for HSF1 in the regulation of energy metabolism in fat tissues; however, whether HSF1 is differentially expressed in adipose depots and how its levels are regulated in fat tissues remain unclear. Here, we show that HSF1 levels are higher in brown and subcutaneous fat tissues than in those in the visceral depot and that HSF1 is more abundant in differentiated, thermogenic adipocytes. Gene expression experiments indicated that HSF1 is transcriptionally regulated in fat by agents that modulate cAMP levels, by cold exposure, and by pharmacological stimulation of β-adrenergic signaling. An in silico promoter analysis helped identify a putative response element for activating transcription factor 3 (ATF3) at −258 to −250 base pairs from the HSF1 transcriptional start site, and electrophoretic mobility shift and ChIP assays confirmed ATF3 binding to this sequence. Furthermore, functional assays disclosed that ATF3 is necessary and sufficient for HSF1 regulation. Detailed gene expression analysis revealed that ATF3 is one of the most highly induced ATFs in thermogenic tissues of mice exposed to cold temperatures or treated with the β-adrenergic receptor agonist CL316,243 and that its expression is induced by modulators of cAMP levels in isolated adipocytes. To the best of our knowledge, our results show for the first time that HSF1 is transcriptionally controlled by ATF3 in response to classic stimuli that promote heat generation in thermogenic tissues. Full Article
el DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels [Cell Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension. Full Article
el Genetic evidence for reconfiguration of DNA polymerase {theta} active site for error-free translesion synthesis in human cells [DNA and Chromosomes] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function. Full Article
el The cytochrome P450 enzyme CYP24A1 increases proliferation of mutant KRAS-dependent lung adenocarcinoma independent of its catalytic activity [Cell Biology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle–dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high. Full Article
el 5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses [Microbiology] By www.jbc.org Published On :: 2020-05-01T00:06:09-07:00 Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA. Full Article
el Hong Kong Scholarship for Excellence Scheme opens for applications By www.info.gov.hk Published On :: Wed, 02 Oct 2019 11:32:47 Full Article
el Revamp of Committee on Self-financing Post-secondary Education announced By www.info.gov.hk Published On :: Tue, 22 Oct 2019 15:15:22 Full Article
el Life Planning Education Conference 2019 held today By www.info.gov.hk Published On :: Fri, 01 Nov 2019 15:04:41 Full Article
el Results of Primary One discretionary places to be released on Monday By www.info.gov.hk Published On :: Fri, 15 Nov 2019 15:12:55 Full Article
el Subsidy amount for Non-means-tested Subsidy Scheme for Self-financing Undergraduate Studies in Hong Kong in 2020/21 academic year announced By www.info.gov.hk Published On :: Thu, 12 Dec 2019 15:13:57 Full Article
el Hong Kong team excels at the International Junior Science Olympiad By www.info.gov.hk Published On :: Thu, 12 Dec 2019 16:06:52 Full Article
el Government proactively assists students unable to return to Taiwan to attend school By www.info.gov.hk Published On :: Tue, 11 Feb 2020 22:37:23 Full Article
el Continuous learning and development in time of epidemic By www.edb.gov.hk Published On :: Mon, 27 Apr 2020 10:49:58 Full Article
el EDB progressively disburses anti-epidemic subsidies and support grants to schools By www.info.gov.hk Published On :: Tue, 17 Mar 2020 18:30:17 Full Article
el Loan repayment by self-financing post-secondary institutions under Start-up Loan Scheme, non-profit-making international schools and student loan repayers to be deferred for two years By www.info.gov.hk Published On :: Mon, 20 Apr 2020 22:18:19 Full Article
el EDB provides more relief grants to affected sectors By www.info.gov.hk Published On :: Tue, 21 Apr 2020 23:08:33 Full Article
el New AI enables teachers to rapidly develop intelligent tutoring systems By www.eurekalert.org Published On :: Thu, 30 Apr 2020 00:00:00 EDT (Carnegie Mellon University) Intelligent tutoring systems have been shown to be effective in helping to teach certain subjects, such as algebra or grammar, but creating these computerized systems is difficult and laborious. Now, researchers at Carnegie Mellon University have shown they can rapidly build them by, in effect, teaching the computer to teach. Full Article
el NIH invests in rapid innovation and development for COVID-19 testing By www.eurekalert.org Published On :: Fri, 01 May 2020 00:00:00 EDT (University of Massachusetts Lowell) UMass Medical School and UMass Lowell will perform a key role in a new National Institutes of Health initiative aimed at speeding innovation, development and commercialization of COVID-19 testing technologies via their Center for Advancing Point of Care Technologies collaboration. Full Article
el New guidelines for treating the sickest COVID-19 patients By www.eurekalert.org Published On :: Mon, 04 May 2020 00:00:00 EDT (University of Houston) A new set of recommendations for health care workers on the front lines, to help them make decisions on how to treat the most critical COVID-19 patients, those with severe lung or heart failure, has been published. Full Article
el Electricity relief details announced By www.news.gov.hk Published On :: Tue, 21 Apr 2020 00:00:00 +0800 Non-residential and residential electricity accounts are benefitting from government relief measures, the Environmental Bureau announced today. Under the measures, nearly 90% of non-residential electricity bills obtained a 75% subsidy for electricity charges in March, while 40% of residential electricity accounts enjoyed zero electricity charges in the first quarter of the year. The Government announced about $2.3 billion in provisions last December to provide an electricity charge subsidy to each eligible non-residential electricity account holder to cover 75% of their monthly electricity charges for four months, subject to a monthly cap of $5,000. The Budget further provided $2.9 billion to extend the subsidy period to eight months. According to the bills issued by the two power companies in March, 360,000 non-residential bills obtained a 75% subsidy. This is close to 90% of the total eligible non-residential tariff bills. To balance the impact on people's livelihood of the recent transition to cleaner electricity generating systems in Hong Kong, the bureau implemented the electricity charges relief scheme in January 2019. A monthly electricity charge relief of $50 has been granted to each eligible residential electricity account for 60 months. To help the public cope with the challenging economic environment, the Government implemented a new round of one-off electricity charge subsidy schemes in January. A subsidy of $160 will be credited to each residential electricity account from January to November, while $240 will be credited in December. From early this year, over 2.7 million households have been benefitting from both the electricity charges relief measures and the electricity charges subsidy. The bills of the two power companies indicated that 40% of residential electricity accounts, representing 1 million households, enjoyed zero electricity charges. The bureau called on the community to cherish environmental resources, including saving energy and electricity to mitigate climate change and improve air quality. Full Article
el Shoreline clean-up conducted By www.news.gov.hk Published On :: Mon, 27 Apr 2020 00:00:00 +0800 Various government departments, together with volunteers today conducted a shoreline clean-up along the remote rocky beach in Kung Pui Wan, Tap Mun to remove two tonnes of refuse. The Environmental Protection Department, the Food & Environmental Hygiene Department (FEHD) and the Marine Department participated in the operation. The Inter-departmental Working Group on Marine Environmental Management said the beach, facing the windy and wavy sea, is not easily accessible by working vessels and the rough terrain connecting the rocky beach also increases the difficulty of routine cleaning work. FEHD cleaners along with the volunteers packed the refuse and delivered it on foot to a nearby pier for temporary storage. FEHD staff then took the refuse to the Marine Department's collection vessel in batches for onward delivery to a rubbish collection point for centralised handling. To minimise the risk of spreading COVID-19, the operation was carried out in groups of no more than four participants each. They maintained an appropriate distance from each other and paid heed to personal protection, including wearing masks. The working group thanked the volunteers for taking part and called on the public to keep the countryside and shoreline clean during outings. For information on clean shorelines, visit the Clean Shorelines facebook and Instagram pages. Full Article
el Rental aid boost helps park tenants By www.news.gov.hk Published On :: Tue, 28 Apr 2020 00:00:00 +0800 Tenants of the EcoPark, country park refreshment kiosks and the Hong Kong Wetland Park will benefit from increased rental concessions for government premises. To help tenants cope with the economic impact of the COVID-19 epidemic, rental concessions of government premises from April to September will be increased from 50% to 75%. The Environment Bureau said tenants of the EcoPark, country park refreshment kiosks and the Hong Kong Wetland Park will receive additional rental concessions of about $3.6 million. Together with the 50% rental concessions provided by the Government from last October to March, these tenants will receive concessions of more than $17 million within 12 months, the bureau said. Meanwhile, tenants of the Hong Kong Wetland Park will receive a full rental waiver during the park’s closure. Full Article
el Meteorological dev't plan welcomed By www.news.gov.hk Published On :: Wed, 29 Apr 2020 00:00:00 +0800 The Hong Kong Special Administrative Region Government today welcomed the promulgation of the Meteorological Development Plan for the Guangdong-Hong Kong-Macao Greater Bay Area (2020-2035) by the China Meteorological Administration. In a statement, the Hong Kong SAR Government said the promulgation of the plan would further strengthen meteorological co-operation among Guangdong, Hong Kong and Macau in areas such as data sharing, scientific research and innovation, and training of meteorological personnel. Such co-operation would in turn enhance the meteorological services in the three places, it added. The relevant co-operation initiatives will benefit the Hong Kong Observatory (HKO) in its development of fine-scale meteorological monitoring, warning and forecasting services, and will further enhance Hong Kong's capability in forecasting extreme weather events, the statement said, adding the HKO will take forward the initiatives in collaboration with the relevant authorities in Guangdong and Macau. The full text of the meteorological plan is available on the China Meteorological Administration's website. It can also be accessed via the Hong Kong SAR Government's Greater Bay Area and HKO websites. Full Article
el Celebrating a Cultural Centre star By www.news.gov.hk Published On :: Sun, 03 Nov 2019 00:00:00 +0800 Mozart once called the pipe organ the “king of instruments” and Hong Kong has musical royalty sitting in the heart of Tsim Sha Tsui. It is hard to miss the pipe organ when you are inside the Cultural Centre Concert Hall. With its four manuals, 93 stops and 8,000 pipes, it can produce an exceptional array of timbre and volume. “The pipe organ is the ‘pearl’ of a concert hall,” explained pipe organist Chiu Siu-ling. “Its enormous range of sounds, coupled with a huge variety of tone colours, creates a remarkable musical experience equivalent to that of an orchestra. It is the king of instruments.” Star performerThe four-storey tall pipe organ was hand-made by famed Austrian manufacturer Rieger Orgelbau and installed when the Cultural Centre was built 30 years ago. It has attracted several world-renowned performers to Hong Kong over the years. “When we built it in 1989, it was the first, huge mechanically operated organ in Asia. I think it is still our biggest organ we have ever built in Asia,” Rieger pipe organ maintenance technician Gerhard Pohl said. He added that the instrument is also remarkably versatile.“There are many organs they are built just for one musical style. On this organ, you can play everything, even jazz music.” Mr Pohl comes to Hong Kong once a year to give the 30-year-old organ its annual health check. In between visits by the manufacturer’s technicians, William Wen ensures the Cultural Centre’s star performer remains in tip-top condition. Top tunerMr Wen is Hong Kong’s only pipe organ maintenance technician. He carries out routine checks on the instrument and tunes it every month. “Keeping it in tune is my responsibility. I take care of it.” Mr Wen was one of the first in Hong Kong to learn the instrument in the 1980s. He became interested in pipe organ maintenance after a visit to an organ manufacturing company in Europe. He recalled that in the ‘80s, organists in Hong Kong were few and far between. He was called upon to test the Cultural Centre organ before the grand opening, making him one of the first organists to play it. “It was dark in the hall, so at first, I only noticed the organ’s keys. When I got closer, I was astounded by just how big the instrument was.” Mr Wen takes up to five hours per maintenance visit to thoroughly check the pipes and tune the organ. He explained that the organ is regularly updated and was fitted with an electrical coupler system, providing greater flexibility in switching between lighter and stronger key actions as desired by the player. “It has been updated a few times. In 2010, the organ was installed with an electrical coupler system. I used to get someone to press the keys for me while I would tune it from inside. But now, I just need a mobile phone on which I can tune it by myself.” Musical legacyTo promote organ music and nurture local organists, the Cultural Centre has been organising the “King of the Instruments” Pipe Organ Education Series annually since 1999. Now the programme’s principal instructor, Ms Chiu has been teaching from the outset. “People are not allowed to touch the instrument in a lot of other concert halls, and they seldom hold pipe organ concerts. This is actually not good for the organ. It has to be played to keep it in good condition or the keys and parts will become hard and old. This inspires me teach and let more and more musicians learn and play the instrument,” Ms Chiu explained. More than 300 organists have so far been trained on the Cultural Centre’s organ. But it has also played something other than music - it has played Cupid too. Like-minded musiciansPipe organist Simon Chan met his wife, Shirley Cheng, through the programme. “I joined the Pipe Organ Education Series in 2000. After that, I continued learning the organ with Ms Chiu and eventually became one of the course tutors. I met my wife in 2003 on the course.” Ms Cheng was also a student on the course. She switched majors from information technology to music and that fateful decision led to meeting her husband. “He asked me to watch him play. He was playing a big piece which was powerful and loud, and that was when I fell for his charms.” The couple decided to embark on a life journey together and now hope to pass on their love of music and the pipe organ to their children. “I wish for my kids to learn music too and to find out more about the pipe organ. This instrument brought their father and mother together and I want them to know our story,” said Ms Cheng. The organist-music teacher duo also hopes to share the joy of the pipe organ with the rest of the community. Full Article
el SAS Notes for SAS®9 - 65925: Clicking a URL for a stored process in Excel brings up the SAS Stored Process Web Application Welcome page By feedproxy.google.com Published On :: Tue, 5 May 2020 17:23:17 EST After you submit a stored process from the SAS Stored Process Web Application, the generated URL is placed into a cell in a Microsoft Excel worksheet. When you click this URL, you expect that the stored process is sub Full Article BISRVTIER+BI+Server+Tier
el New Research: Crisis of Confidence over COVID-19 Could Delay Economic Recovery for a Decade By www8.gsb.columbia.edu Published On :: Wed, 29 Apr 2020 15:42:22 +0000 Business Economics and Public Policy Strategy Wednesday, April 29, 2020 - 11:45 Working Paper from Columbia Business School Quantifies Impact of “Belief Scarring” on Economic Recovery, Finds Crisis Could Result in over 180% loss of annual GDP Full Article