se Hello All - Looking For Technical Advise By forums.digitalpoint.com Published On :: Sat, 09 May 2020 22:51:13 +0000 Full Article
se eCommerce Research By forums.digitalpoint.com Published On :: Sun, 10 May 2020 00:49:31 +0000 Full Article
se Get Your Automated Software solution| Custom Programming service. By forums.digitalpoint.com Published On :: Sun, 10 May 2020 05:53:21 +0000 Full Article
se Google adsense Important Guides Lines By forums.digitalpoint.com Published On :: Sun, 10 May 2020 05:59:01 +0000 Full Article
se My Lead Gen Secret review needed By forums.digitalpoint.com Published On :: Sun, 10 May 2020 07:47:34 +0000 Full Article
se Keyword Service & Site Audit By forums.digitalpoint.com Published On :: Sun, 10 May 2020 07:55:32 +0000 Full Article
se How to choose a topic/ niche By forums.digitalpoint.com Published On :: Sun, 10 May 2020 08:00:12 +0000 Full Article
se 2020-05-06 The old man of the sea! By www.flickr.com Published On :: Sat, 09 May 2020 19:28:49 -0700 Mary Wardell posted a photo: This old lighthouse keeper stared out into the storm to see if there were any ships out there caught in the wnds and waves . Full Article
se Roosevelt the Snailien By www.flickr.com Published On :: Sat, 09 May 2020 23:46:26 -0700 ridureyu1 posted a photo: Wearing only the best hi-tech snail shell! Full Article
se Roosevelt By www.flickr.com Published On :: Sat, 09 May 2020 23:46:26 -0700 ridureyu1 posted a photo: Too bad he's named after FDR and not Teddy. Full Article
se Sean Hannity on the Obama administration's big lie By news.yahoo.com Published On :: Sat, 09 May 2020 20:18:36 -0400 Sean Hannity reacts on 'Watters' World' to the Obama administration being caught lying about Russia collusion. Full Article
se Ousted health official dismisses Trump's claim he is disgruntled: 'I am frustrated at a lack of leadership' By news.yahoo.com Published On :: Fri, 08 May 2020 22:24:26 -0400 Ousted vaccine expert Rick Bright said he is "frustrated at our inability to be heard as scientists." Full Article
se A former editor at the Observer says Kushner's claim of coronavirus 'success' stems from his inability to empathize with other people's grief By news.yahoo.com Published On :: Sat, 09 May 2020 01:00:18 -0400 Elizabeth Spiers wrote about an incident where Jared Kushner used the memorial of an employee to congratulate himself for success. Full Article
se Ahmaud Arbery is dead because Americans think black men are criminals By news.yahoo.com Published On :: Fri, 08 May 2020 10:07:46 -0400 Whenever Americans see videos of police brutality against black men and women, the first thing they do is assume they deserved their executionWhat skin color are the bad guys in America’s fantasies of vigilantism? When the proverbial “fellas” get together to drink beers and talk about their newest guns and who they’d take down, what race are the “criminals” in the theater of their minds?When Greg McMichael and his son, Travis, got the call from their neighbor that a “burglar” was running through their Brunswick, Georgia neighborhood that chilly February day, what color man do you think they imagined as they locked, loaded, and embarked on their “mission”?Ahmaud Arbery is dead today because when Americans dream of vigilante justice, black men are the villains of their imaginations.We as a nation are so comfortable with this baseline bigotry that our first assumption whenever we see videos of police brutality against or shootings of black men and women, the first thing we do is assume that the victims must have done something wrong to earn their own public execution.This assumption is both a function of white America having a completely different experience with police officers than black America as well as the hundreds of years of vilifying blackness in media and American culture.I will never forget the biggest and most uproarious applause during the theater debut of the lackluster 2007 vigilante film, Brave One, came when the protagonist Jodi Foster got her first vigilante kills of the movie – two threatening and scary black men. That theater filled with men the same age range as Greg and Travis McMichael erupted as if at that moment, all that they had ever imagined had been fulfilled on the big screen. Needless to say, I left that theater before the credits rolled.Across the country, our political leaders hold these same bigoted beliefs which inevitably lead to policies that directly assume criminality based on skin color.During his tenure as mayor of New York City, billionaire Michael Bloomberg made it explicitly clear why it was that he sent police officers into black and brown communities to “throw them” up against the wall. In his 2015 Aspen Institute speech he stated:“People say, ‘Oh my God, you are arresting kids for marijuana who are all minorities.’ Yes, that’s true. Why? Because we put all the cops in the minority neighborhoods. Yes, that’s true. Why’d we do it? Because that’s where all the crime is. And the way you should get the guns out of the kids’ hands is throw them against the wall and frisk them.”And it is for this reason that I do not distinguish between the violence committed by American citizens acting as vigilantes and the violence committed by so-called officers of the law when, in both cases, the working assumption and driving force behind that violence is the deeply bigoted and firmly American association between blackness and criminality.For Ahmaud, that association not only led to his brutal killing, but it also initially meant his killer not being arrested. It took more than two months for the father and son duo to be arrested. When explaining why they were not charged immediately the district attorney, George Barnhill, immediately stated that the victim, Ahmaud Arbery, was, in fact, the “criminal suspect”.“It appears that [Greg and Travis McMichael’s] intent was to stop and hold this criminal suspect until law enforcement arrived. Under Georgia Law [sic] this is perfectly legal.”Even after viewing the video and with no evidence beyond Ahmaud’s skin color, the top cop in the institution designed to bring equal justice under the law concluded that Ahmaud was a criminal suspect when he was simply a black man taking a jog.What are black Americans to do when justice is delayed or outright denied because of the assignment of innocence to vigilantes and police officers?What are black Americans to do when the assumption of guilt because of our skin color is as American as the guns they use to kill us?What are we to do when in our neighbors’ dreams and fantasies of cop-and-robber, the skin color of the bad guy matches our own?The very first thing we are going to do is defend ourselves as if our lives depend on it because when Americans fantasize about killing, those fantasies become our living nightmares. * Benjamin Dixon is the host of the Benjamin Dixon show. Full Article
se Top Republican Lawmaker Disclosed Stock in Chinese Company He Labeled a Nat Sec Threat By news.yahoo.com Published On :: Fri, 08 May 2020 10:25:45 -0400 Representative Michael McCaul, who has harshly criticized China in his position as the ranking member on the House Foreign Affairs Committee, disclosed that his family owns stock in a Chinese tech company he described as a threat to national security.An April 20 periodic transaction report showed that McCaul disclosed a February purchase of between $50,000 and $100,000 in shares of the Chinese firm Tencent Holdings, Politico reported.In November, several months before the shares were purchased, the Texas congressman said that Tencent Holdings is among the "Chinese companies that threaten America’s economic and national security."The tech conglomerate is "heavily involved" in the "social credit system, a dystopian system China has implemented to score its citizens’ behavior," McCaul said at the time, as well as an "integral part" of the Chinese Communist Party’s industrial policies and "one of four national champions for artificial intelligence."McCaul's lawyer, Elliot Berke, said that the shares are not owned personally by McCaul but by his wife, and the decision to invest in Tencent was made by a third party.“Congressman McCaul did not purchase any shares in China’s Tencent Holdings or any other Chinese company,” the attorney said. “Congressman McCaul’s wife has assets she solely owns and a third party manager made the purchase without her direction.”Rachel Walker, a spokeswoman for McCaul, emphasized that the revelation of the Tencent shares “highlights that many Americans unwittingly invest their money in Chinese owned companies."Federal employees are often unaware they own such investments because the federal government’s thrift savings plan program creates portfolios that include Tencent and other Chinese companies, Walker said. McCaul has argued that such retirement investment plans should not invest American dollars in such "shady" Chinese companies, often without the knowledge of the investor."Congressman McCaul has been a fierce critic of the brutal behavior of the Chinese Communist Party and will continue to fight to hold them accountable as the Chair of the China Task Force," Walker said. "This should be a wake-up call to us all that the CCP’s involvement in the U.S. economy is far more reaching than many Americans realize and that we need to change the way we do business with China, including our investments."Tencent owns the Chinese social media platform WeChat, which has more than one billion users and is suspected of monitoring the activities of users both inside and outside of China. Tencent is also associated with Chinese tech firm Huawei, which U.S. officials said can secretly access American cellular phone networks, giving it access to sensitive information.McCaul has taken a leading role in criticizing China's handling of the coronavirus pandemic as well, accusing Beijing of launching perhaps the "worst cover-up in human history."He was tapped on Thursday by House Minority Leader Kevin McCarthy as chairman of the China Task Force, the aim of which is to develop "legislative solutions to address the Chinese Communist Party’s malign global agenda."The task force will "develop new and enduring policy solutions that, among others, enhance our economic strength and create jobs, protect our national security, rethink our supply chains and grow our competitive edge in technology," McCaul said in a statement on his appointment. Full Article
se South Dakota Governor demands Sioux tribes 'immediately' remove COVID-19 checkpoints because they interfere with traffic By news.yahoo.com Published On :: Sat, 09 May 2020 17:44:40 -0400 South Dakota Governor Kristi Noem sent letters to two Sioux tribes demanding they remove COVID-19 checkpoints because they interfere with traffic. Full Article
se White House won't consider another stimulus bill in May -Kudlow By news.yahoo.com Published On :: Fri, 08 May 2020 10:58:38 -0400 The White House has halted talks with Congress over any further coronavirus stimulus package as it waits for more information about how U.S. state reopenings affect the economy, White House top economic adviser Larry Kudlow told reporters on Friday. The Senate and House of Representatives have already passed four major bills to address the novel coronavirus outbreak, including three aimed at stabilizing the economy as most Americans have sheltered in place and unemployment has soared. Full Article
se Trump donor and lawyer to represent Biden's accuser By news.yahoo.com Published On :: Fri, 08 May 2020 11:13:15 -0400 The former Senate staffer, who is accusing Joe Biden of sexually assaulting her 27 years ago, is being represented by a lawyer who is also a donor to President Trump. Full Article
se As many as 75,000 could die from "deaths of despair" as a result of the coronavirus pandemic. One expert says economic turmoil has always led to an increase in suicides. By news.yahoo.com Published On :: Sat, 09 May 2020 10:08:21 -0400 "I think it's always important to think about suicide as individual vulnerabilities and context," Eric Caine said. Full Article
se China reports first coronavirus case in Wuhan since April 3 among 14 new infections By news.yahoo.com Published On :: Sat, 09 May 2020 21:46:27 -0400 China's National Health Commission reported 14 new confirmed coronavirus cases on May 9, the highest number since April 28, including the first for more than a month in the city of Wuhan where the outbreak was first detected late last year. While China had officially designated all areas of the country as low-risk last Thursday, the new cases according to data published on Sunday represent a jump from the single case reported for the day before. The new Wuhan case, the first reported in the epicentre of China's outbreak since April 3, was previously asymptomatic, according to the health commission. Full Article
se Tripoli airport shelling hits fuel tanks, passenger plane-ministry By news.yahoo.com Published On :: Sat, 09 May 2020 06:08:57 -0400 Shelling of Tripoli's Mitiga airport early on Saturday, part of an intensified barrage of artillery fire on the capital in recent days, hit fuel tanks and damaged passenger planes, the Transport Ministry said in a statement. Mitiga is the last functioning airport in the Libyan capital, though civilian flights stopped in March because of repeated shelling even before the country imposed a lockdown over the coronavirus pandemic. Brega Petroleum Marketing Company, part of the National Oil Corporation, said its jet fuel tanks at Mitiga caught fire after coming under attack and firemen were working to control the blaze. Full Article
se Pakistanis crowd markets as virus lockdown eased By news.yahoo.com Published On :: Sat, 09 May 2020 11:16:19 -0400 Pakistanis crowded markets on Saturday after a nationwide coronavirus lockdown was eased, despite the country declaring its second highest daily infection toll. Prime Minister Imran Khan has allowed businesses to reopen in phases from the weekend, citing the economic havoc the virus restrictions have wreaked on the improvised nation. In the garrison city of Rawalpindi, thousands of shoppers were preparing for Eid, which follows the Muslim fasting month of Ramadan, with many flouting social distancing rules and advice to wear masks. Full Article
se A photographer spent two weeks flying around in helicopters to capture the parked planes at US airports during the pandemic — see his eerie and beautiful work By news.yahoo.com Published On :: Fri, 08 May 2020 15:01:07 -0400 Travel blogger and photographer Andy Luten drove 4,200 miles across six states to see the grounded jets, detailing the shocking state of aviation. Full Article
se Elon Musk says Tesla will 'immediately' leave California after coronavirus shutdowns forced the company to close its main car factory By news.yahoo.com Published On :: Sat, 09 May 2020 13:34:00 -0400 In a tweet Saturday morning, Tesla's chief executive said it would file a lawsuit against county officials over not being able to run its factory. Full Article
se Structures of the MHC-I molecule BF2*1501 disclose the preferred presentation of an H5N1 virus-derived epitope [Protein Structure and Folding] By feedproxy.google.com Published On :: 2020-04-17T00:06:05-07:00 Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens. Full Article
se NF-{kappa}B mediates lipopolysaccharide-induced alternative pre-mRNA splicing of MyD88 in mouse macrophages [Signal Transduction] By feedproxy.google.com Published On :: 2020-05-01T00:06:09-07:00 Although a robust inflammatory response is needed to combat infection, this response must ultimately be terminated to prevent chronic inflammation. One mechanism that terminates inflammatory signaling is the production of alternative mRNA splice forms in the Toll-like receptor (TLR) signaling pathway. Whereas most genes in the TLR pathway encode positive mediators of inflammatory signaling, several, including that encoding the MyD88 signaling adaptor, also produce alternative spliced mRNA isoforms that encode dominant-negative inhibitors of the response. Production of these negatively acting alternatively spliced isoforms is induced by stimulation with the TLR4 agonist lipopolysaccharide (LPS); thus, this alternative pre-mRNA splicing represents a negative feedback loop that terminates TLR signaling and prevents chronic inflammation. In the current study, we investigated the mechanisms regulating the LPS-induced alternative pre-mRNA splicing of the MyD88 transcript in murine macrophages. We found that 1) the induction of the alternatively spliced MyD88 form is due to alternative pre-mRNA splicing and not caused by another RNA regulatory mechanism, 2) MyD88 splicing is regulated by both the MyD88- and TRIF-dependent arms of the TLR signaling pathway, 3) MyD88 splicing is regulated by the NF-κB transcription factor, and 4) NF-κB likely regulates MyD88 alternative pre-mRNA splicing per se rather than regulating splicing indirectly by altering MyD88 transcription. We conclude that alternative splicing of MyD88 may provide a sensitive mechanism that ensures robust termination of inflammation for tissue repair and restoration of normal tissue homeostasis once an infection is controlled. Full Article
se Kruppel-like factor 3 (KLF3) suppresses NF-{kappa}B-driven inflammation in mice [Immunology] By feedproxy.google.com 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
se Polarization of protease-activated receptor 2 (PAR-2) signaling is altered during airway epithelial remodeling and deciliation [Immunology] By feedproxy.google.com Published On :: 2020-05-08T03:41:14-07:00 Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases. Full Article
se Development of a novel {beta}-1,6-glucan-specific detection system using functionally-modified recombinant endo-{beta}-1,6-glucanase [Methods and Resources] By feedproxy.google.com Published On :: 2020-04-17T00:06:05-07:00 β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections. Full Article
se Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol [Glycobiology and Extracellular Matrices] By feedproxy.google.com Published On :: 2020-04-17T00:06:05-07:00 β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain. Full Article
se An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1 [Enzymology] By feedproxy.google.com 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
se Processivity of dextransucrases synthesizing very-high-molar-mass dextran is mediated by sugar-binding pockets in domain V [Glycobiology and Extracellular Matrices] By feedproxy.google.com 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
se The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase [Glycobiology and Extracellular Matrices] By feedproxy.google.com 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
se Structural basis of substrate recognition and catalysis by fucosyltransferase 8 [Protein Structure and Folding] By feedproxy.google.com Published On :: 2020-05-08T03:41:14-07:00 Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process. Full Article
se ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface [Membrane Biology] By feedproxy.google.com Published On :: 2020-05-08T03:41:14-07:00 Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation. Full Article
se Re: David Oliver: Is abuse towards doctors in government roles unfair? By feeds.bmj.com Published On :: Saturday, May 9, 2020 - 02:21 Full Article
se Re: Covid-19: how to use your time when clinical placements are postponed - Wellbeing perspective By feeds.bmj.com Published On :: Saturday, May 9, 2020 - 13:41 Full Article
se The multi-ness of fighting the short-term and long-term consequences of the COVID-19 pandemic By feeds.bmj.com Published On :: Saturday, May 9, 2020 - 16:58 Full Article
se Re: Prognosis of unrecognised myocardial infarction determined by electrocardiography or cardiac magnetic resonance imaging: systematic review and meta-analysis By feeds.bmj.com Published On :: Saturday, May 9, 2020 - 18:01 Full Article
se Call for a review of services for people with neurological disorders By feeds.bmj.com Published On :: Tuesday, August 11, 2015 - 15:35 Full Article
se A real opportunity to improve neurology services in England By feeds.bmj.com Published On :: Tuesday, August 11, 2015 - 15:56 Full Article
se Assessment and management of facial nerve palsy By feeds.bmj.com Published On :: Wednesday, September 16, 2015 - 12:46 Full Article
se US drug makers have imposed big price rises for top selling drugs, study finds By feeds.bmj.com Published On :: Tuesday, April 5, 2016 - 14:01 Full Article
se Australian neuroscientist given two year suspended sentence for falsifying Parkinson's research By feeds.bmj.com Published On :: Thursday, April 7, 2016 - 12:41 Full Article
se Seven days in medicine: 8-14 June 2016 By feeds.bmj.com Published On :: Thursday, June 16, 2016 - 23:16 Full Article
se Disease modifying therapies for relapsing multiple sclerosis By feeds.bmj.com Published On :: Monday, August 22, 2016 - 16:25 Full Article
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se The Faltering U.S. Refugee Protection System: Legal and Policy Responses to Refugees, Asylum Seekers, and Others in Need of Protection By www.migrationpolicy.org Published On :: Sun, 01 May 2011 00:00:00 -0400 The U.S. refugee protection system, while generous in many respects, has become less robust over the last two decades. The unique and often diverse needs of emerging refugee populations have exposed severe limitations in the standard resettlement approach.This report examines U.S. legal and policy responses to those seeking protection and addresses the barriers, gaps, and opportunities that exist. Full Article
se Eight Policies to Boost the Economic Contribution of Employment-Based Immigration By www.migrationpolicy.org Published On :: Wed, 01 Jun 2011 00:00:00 -0400 Drawing on experiences from Asia, Europe, North America, and the Pacific region, this report presents eight strategies that represent best practices developed by immigrant-receiving countries to increase the economic contributions of immigration. Full Article
se Rethinking Points Systems and Employer-Selected Immigration By www.migrationpolicy.org Published On :: Wed, 01 Jun 2011 00:00:00 -0400 Two competing models for selecting economic-stream immigrants are now prevalent in advanced industrialized economies: points-based and employer-led selection. Increasingly, however, hybrid selection systems are being created, implementing best practices from each selection process. Full Article