NEXT JAMES BOND odds favourite Tom Hardy has overtaken Richard Madden in the race to replace Daniel Craig after No Time To Die.

Clubs in League One and League Two fear their seasons may be ended prematurely as early as next week.

Liverpool and Newcastle are leading the way for Kalidou Koulibaly.

Chelsea and Manchester United have joined Tottenham in the pursuit of Paris Saint-Germain star Thomas Meunier.

The Legislature should declare a voting holiday to make it easier for all Hoosiers to vote on election day, a letter to the editor says.

The IndyCar schedule has undergone another massive change, but this time, the series has managed to add a race back onto the slate.

John and Tucker Hartung of Lamar Advertising removed an Indianapolis 500 billboard along Lafayette Road, April 7, 2020. The race is delayed to August.

Watch Gary Varvel's time lapse video of how he draws the Indiana Senate race.

Braun and Donnelly tout their support for Trump's policies

For 15 years, Lucas Oil Raceway's Wild Wednesday has offered residents around Indianapolis the opportunity to drive fast, drag race — legally.

SONOMA, Calif. – The men and women who pull racing helmets over their heads are a different breed, defying speed and danger mortals cannot imagine.

Right now, there's a 1 in 8 chance the Hoosiers land the consensus top-10 talent out of Minnesota. But they're competing with NCAA royalty.

"I could miss 15 shots, and I always have the mindset that the next one's going in," Grace Berger says. "I'm not worried about those other shots."

There are two main candidates for the Indiana governor's race in 2020: Republican incumbent Eric Holcomb and Democratic challenger Woody Myers. Here's what we know.

"I could miss 15 shots, and I always have the mindset that the next one's going in," Grace Berger says. "I'm not worried about those other shots."

The cousin of Grace Millane, who was murdered in New Zealand, says she would have been "so proud".

Watch highlights as Pascal Wehrlein takes a lights-to-flag win in the third Formula E Race At Home Challenge at Monaco which saw a "shunt that never ends" on the opening lap.

"You can see the fear in their faces," says medic as health workers in Yemen brace for Covid surge.

Watch Lewis Hamilton and Sir Stirling Moss race vintage Mercedes Silver Arrows at Monza in 2015.

Watch highlights from the crash-filled second race of the Formula E Race At Home Challenge as Maximillian Gunther wins on the Electric Docks circuit.

Formula 1 chairman Chase Carey has admitted that the sport's owners are preparing for "the remote possibility of no racing in 2020".

Renault's Daniel Ricciardo expects the first race of the season to be chaotic after the long break enforced by the coronavirus crisis.

With more and more F1 drivers making the move to esports during the lockdown and doing well, could an expert sim racer easily make the leap the other way?

South Korea is testing more people per capita than anywhere else - and could be a role model for others.

India does not have the PPE kits it needs to protect its doctors and police from Covid-19 infection.

Pulitzer-winning cartoonist Mike Luckovich had a middle-of-the-night inspiration after Super Tuesday.

Quarterbacks have won 10 of the past 11 MVP awards, and the six rushers who have won over the past 30 years have been historically good, either establishing a record for touchdowns or rushing for more than 2,000 yards in a season.

Favorites have won 99 of 318 races (31 percent) in the Breeders’ Cup.

The Braves' Acuna appears to be a runaway winner right now, but a closer look shows Soto had a tremendous impact on the Nationals.

Here’s what we know for sure about the NFC playoff picture: the Saints, Rams and Bears can make postseason plans. The rest is up for grabs.

• Dockworkers in Belgium are wearing bracelets to enforce social distancing.
• The bracelets were already used to detect if someone fell into the water, but now they will sound an alarm if workers get to close to each other.
• Manufacturers say there is no privacy issue and the bracelets don't track workers' locations, despite concerns.
• Visit Business Insider's homepage for more stories.

Quarantine and social distancing are going high-tech as countries and companies embrace wearables. In Antwerp, Belgium, dockworkers are instructed to wear bracelets that enforce social distancing rules while they work.

Europe, where more than 100,000 people have died from COVID-19, is slowly starting to reopen in some places. Stay at home orders are expiring in many countries, while nonessential travel has stopped across the EU, and countries look towards the summer to anticipate what kind of travel might be possible. 

People are beginning to go back to work, which in some sectors means inevitable close contact, especially in many essential jobs. Social distancing bracelets in Belgium are one idea bing tested to see what the future of work might look like after coronavirus.

Here's how it works. 

SEE ALSO: People arriving in Hong Kong must wear tracking bracelets for 2 weeks or face jail time. Here's how they work.

The black, plastic bracelets are worn on the wrist like a watch.

They're made by Belgian company Rombit, which says that they are "a fully integrated personal safety and security device, specifically designed for highly industrial environments."

Source: Romware

Rombit already made bracelets useful in the port setting, which could be used to call for help if a worker fell into the water or another accident occurred.

Europe is slowly starting to go back to work, but fears of a second wave are making officials cautious.

Contact tracing is one solution being explored around the world, and the manufacturers of the bracelet believe it could also be used for contact tracing.

Source: The Associated Press

European health guidances say to wash hands, wear masks, and keep at least 1.5 meters, or about five feet, apart.

When two workers are less than five feet apart, the bracelets will sound warnings.

Rombit CEO John Baekelmans told Reuters that the bracelets won't allow companies to track employees' locations, because the devices are only connected to each other. He says there is no central server.

Source: Reuters

Workers in the control tower will be the first to test the bracelets early this month.

Then, the Port of Antwerp will likely expand the devices to tug boat workers.

Baekelmans told Reuters that Rombit already had hundreds of requests in 99 countries, and is hoping to ramp up production to 25,000 in a few weeks.

Some states still haven’t recovered from the last recession. They could face worse now.

Part of the Going for gold promo for the BBC UK Homepage

28 February 2020

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.

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.

CJ Fielding
Aug 1, 1997; 38:1503-1521
Reviews

EJ Blanchette-Mackie
Jun 1, 1995; 36:1211-1226
Articles

Corporate Members Event Nominees Breakfast Briefing Partners and Major Corporates

Event participants

Dr Lindsay Newman, Senior Research Fellow, US and the Americas Programme, Chatham House
Chair: Dr Leslie Vinjamuri, Dean, Queen Elizabeth II Academy for Leadership in International Affairs; Director, US and the Americas Programme, Chatham House

Invitation Only Research Event

Event participants

John Zogby, Founder and Senior Partner, John Zogby Strategies
Chair: Dr Lindsay Newman, Senior Research Fellow, US and Americas Programme, Chatham House

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

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.

Membrane-bound proteins have been proposed to mediate the transport of long-chain FA (LCFA) transport through the plasma membrane (PM). These proposals are based largely on reports that PM transport of LCFAs can be blocked by a number of enzymes and purported inhibitors of LCFA transport. Here, using the ratiometric pH indicator (2',7'-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein and acrylodated intestinal FA-binding protein-based dual fluorescence assays, we investigated the effects of nine inhibitors of the putative FA transporter protein CD36 on the binding and transmembrane movement of LCFAs. We particularly focused on sulfosuccinimidyl oleate (SSO), reported to be a competitive inhibitor of CD36-mediated LCFA transport. Using these assays in adipocytes and inhibitor-treated protein-free lipid vesicles, we demonstrate that rapid LCFA transport across model and biological membranes remains unchanged in the presence of these purported inhibitors. We have previously shown in live cells that CD36 does not accelerate the transport of unesterified LCFAs across the PM. Our present experiments indicated disruption of LCFA metabolism inside the cell within minutes upon treatment with many of the "inhibitors" previously assumed to inhibit LCFA transport across the PM. Furthermore, using confocal microscopy and a specific anti-SSO antibody, we found that numerous intracellular and PM-bound proteins are SSO-modified in addition to CD36. Our results support the hypothesis that LCFAs diffuse rapidly across biological membranes and do not require an active protein transporter for their transmembrane movement.

Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.

A. Knightly and C. Li
Memoirs of the AMS 224 (2012).
Abstract, references and article information

Researcher: Christine Darden, NASA (retired) Description: Christine Darden on working at NASA.

Greg Martin and Nathan Ng
Trans. Amer. Math. Soc. 373 (2020), 3561-3607.
Abstract, references and article information

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.