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The post REPORT: National parks visitors should plan for ‘new normal’ appeared first on Dennis Michael Lynch.

Flying in Canada during the time of COVID-19 requires a lot extra care, and CTV Senior Political Correspondent Glen McGregor gives a first-hand account on CTVNews.ca.

As Quebec begins to reopen schools and businesses, Prime Minister Justin Trudeau said he’s ‘worried’ about the province’s deconfinement plans, particularly in Montreal.

Police and City of Vancouver park rangers escorted the last person living in Oppenheimer Park out of the tent city Saturday afternoon, moments before crews with excavators moved in to clean up the mountains of trash left behind.

Many in the region had to dust off their snow shovels for at least one more dig out on Saturday morning after a polar vortex blasted parts of the province, catching many off guard.

The following article, NYC Nurse Steals Credit Card From 70-Yr-Old Dying COVID-19 Patient On Ventilator, was first published on 100PercentFedUp.com.

"She is a despicable human being"

Continue reading: NYC Nurse Steals Credit Card From 70-Yr-Old Dying COVID-19 Patient On Ventilator ...

The following article, Witness Tampering? Asst. HHS Secretary Releases Threatening Text Messages From Dem Rep. Eric Swalwell: “In clear violation of House Ethics rules “, was first published on 100PercentFedUp.com.

Now that Michael Caputo, who was previously a target of the Mueller investigation, has been cleared, he has released some pretty damning text messages from the virulently anti-Trump lawmaker from California, Rep. Eric Swalwell (D). The text messages appear to prove that Swalwell, who has spent the last 3 1/2 years calling for Trump’s impeachment, […]

Continue reading: Witness Tampering? Asst. HHS Secretary Releases Threatening Text Messages From Dem Rep. Eric Swalwell: “In clear violation of House Ethics rules “ ...

26 August 2013

6 September 2013

Invitation Only Research Event

Event participants

HE Professor Kwaku Danso-Boafo, High Commissioner for Ghana to the United Kingdom
Chair: Alex Vines OBE, Research Director, Area Studies and International Law; Head, Africa Programme, Chatham House

Research Event

Event participants

Dr Maria Ângela Bragança, Secretary of State for Cooperation, Ministry of External Relations, Angola

29 October 2014

12 March 2015 , Volume 91, Number 2

17 May 2016

Hidden in the flash. posted a photo:

With a lockdown in place it is against the rules for me to go to places I like to shoot, so I though I would try to create a series called Isolated creativity. The series is not intended to be a diary but a way of documenting thoughts and emotions via photography.

I've felt a bit like Pink Rabbit over the passed few day. I'm not fed up and depressed by the lockdown but by the people who think that it's okay to break the rules. By the tabloid media that run stories that convince people it's okay to go out and about, when it Isn't. By the political points scoring that has started to appear in all forms of media.Lastly I fed up with second home owners that have turned up during lockdown and appear to be going out and about most days.

Just like Pink Rabbit I have the blues.

Ousted vaccine expert Rick Bright said he is "frustrated at our inability to be heard as scientists."

Elizabeth Spiers wrote about an incident where Jared Kushner used the memorial of an employee to congratulate himself for success.

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.

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.

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.

The government said it's distributing the promising coronavirus drug, remdesivir, to some hard-hit states. Eventually, all 50 states should get it.

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.

"Passing along a message from Mother Nature," the National Weather Service in Binghamton, New York, tweeted alongside a photo of a car covered in light snow. "Happy Mother's Day Weekend."

Travel blogger and photographer Andy Luten drove 4,200 miles across six states to see the grounded jets, detailing the shocking state of aviation.

On Thursday, the Professional Beauty Federation of California published a press release to the “Hot Topics” section of their website. It was titled: “Time to Sue Governor Newsom.” The release came in response to Gov. Gavin Newsom’s announcement that the following morning, California would officially enter “Phase Two” of the “Safer at Home” order. Select businesses, from florists to clothing retailers to toy stores, would be able to resume operations in a limited capacity. But absent from the list of acceptable businesses: beauty salons. Newsom placed businesses like nail salons and barbershops in “Phase Three”—a stage he believes to be “months, not weeks” away. “This whole thing spread in the state of California—the first community spread—was in a nail salon,” Newsom said in a press conference last week, without providing details about the date or location of the case. “Many of the practices that you would otherwise expect of a modification were already in play in many of these salons, with people that had procedure masks on, were using gloves, and were advancing higher levels of sanitation.”The news has thrust nail salons onto the frontline of a growing coronavirus revolt in California, a battle being waged in many more American cities, like Dallas, where hairdresser Shelley Luther became a star of the anti-lockdown movement when she opted to go to jail rather than comply with an order to close her hair salon. Anti-Lockdown Protesters Are Now Facing Down Cops Outside of BarsOn Monday morning, the Professional Beauty Federation of California will file a lawsuit in federal court demanding a regulated reopening process of their salons. “We were 100 percent behind the lockdown, so that we would not overwhelm our hospitals,” the group’s legal counsel Fred Jones said in an interview with The Daily Beast. “However, after two months of the lockdown, in which, by Gov. Newsom’s own admission, we have succeeded—we have checked the mark, we have flattened the curve—we were anticipating that the governor would allow for gradual reopenings of our beauty salons under strict new guidelines.”Their argument, Jones said, hinges on the fact that, without regulated reopening, stylists will be forced underground to meet financial ends, resulting in a potentially more dangerous risk.“A lot of our stylists are on the brink of starvation in order to make their leases and make ends meet,” Jones said. “So you have a volatile combination of desperate clients and desperate stylists. We know that will lead to thousands of our stylists going underground and moving kitchen to kitchen and house to house. That’s reality. Nobody can argue that. So the real question is: how do you stop that from happening if you’re the governor? You can’t.”He suggested a gradual and controlled reopening would be safer than “stylists going house to house and spreading more than beauty.”Unmasked Protesters Storm Huntington Beach After California Governor’s ClosureSome salons statewide have already opened, defying the statewide order, like an Orange County nail spa owner who has vowed to stay open despite being handed a citation by local police, who ordered her to appear in court in July. “I have to do what I have to do. I’m fighting to provide for my children and myself and my family,” another salon owner, Breann Curtis, of The Clip Cage barbershop in Auburn, California, told Fox40 about her decision to reopen. “It’s very hard. I’m pregnant. I have children.”“Just going into debt every single day,” added Tisha Fernhoff, who owns The Beauty Bar Salon in the same Auburn shopping center. “How much longer am I supposed to just go down the rabbit hole before I just throw in the towel and go back to work?”According to Jones, the California State Board of Barbering and Cosmetology—which issues all 623,442 beauty licenses in the state—has already drafted a protocol for how salons could reopen under the current conditions. He claimed Newsom had blocked the plan from distribution, to avoid mixed messaging. (Newsom’s office didn’t respond to a request for comment and a spokesperson for the Board of Barbering and Cosmetology said their draft protocols “haven't been published because they are not finished.”)“We want him to release the plan so that our professionals can start stocking up,” Jones said. “We know we’ll need masks. Will shields be required for these services? They probably will.”If such a plan was to go into effect, Jones said, salons would use personal protective equipment widely. They would stagger appointments to avoid crowded waiting rooms, spread out work stations and shift schedules, implement a touchless pay system, and remove anything in the waiting rooms that could carry contagion. “So, sorry no more magazines and newspapers for our clientele,” Jones said. But the Centers for Disease Control and Prevention (CDC) recommends maintaining a distance of six feet from other people—a practice that would be all but impossible in salon settings. Dr. Birx Says What Trump Would Not About ProtestersThere are 53,694 licensed beauty salons in California, representing 313,734 stylists or cosmetologists, 34,093 barbers, 90,392 estheticians, 1,679 electrologists, and 129,802 manicurists, according to the State Board of Barbering and Cosmetology. All of these workers, Jones said, have to complete between 350 and 1600 hours of formal education before acquiring their license, including training in sanitization. Jones emphasized that the lawsuit stemmed from financial desperation, a sentiment shared across the country. The Labor Department announced Friday that the economy lost over 20.5 million jobs in April alone, putting the national unemployment rate at its highest since the Great Depression: 14.7 percent. But the devastation has hit the beauty sector differently than many industries. Over 80 percent of salon workers are independent contractors, meaning each stylist represents their own business. By extension, many salon owners are basically landlords, “whose income relies on those booth owners,” Jones said. As a result, most salon workers qualify for unemployment benefits under the Coronavirus Aid, Relief and Economic Security Act, signed by Trump in March—although the program is riddled with loopholes, has frequently run out of money, and may not cover their entire income, which heavily relies on tips. It is salon owners who stand to gain the most from the lawsuit. “Freelance workers do benefit on unemployment benefits,” Jones said. “But most of those Paycheck Protection Program reimbursements are based on your payments. If you’re a salon owner, you don’t have a payroll. Those stylists are their own proprietors.”On Friday, Senators Bernie Sanders (I-VT), Ed Markey (D-MA), and Kamala Harris (D-CA) introduced legislation to give a majority of Americans $2,000 a month throughout the pandemic. Asked whether the bill could provide financial relief to salon workers, while allowing them to maintain social distancing, Jones seemed doubtful that it would pass. “It’s the proverbial ‘check is in the mail’ promise,” he said. “When you’re dealing with true economic devastation, let me tell you, most of our licensees will not be banking on a divided Congress and a White House that is also divided. While Washington fiddles, our stylists are burning.” Read more at The Daily Beast.Get our top stories in your inbox every day. Sign up now!Daily Beast Membership: Beast Inside goes deeper on the stories that matter to you. Learn more.

Attorney General Bill Barr explained that the FBI did not conduct “a bonafide counterintelligence investigation” in the case that led former Trump national security adviser Michael Flynn to plead guilty to federal investigators in 2017.Barr, speaking in an exclusive interview with CBS News after the Justice Department dropped its case against Flynn on Thursday, said that his review of the case found Bureau investigators laid “a perjury trap” for Flynn in a January 2017 White House interview.“They didn’t warn him, the way that would usually be required by the Department, they bypassed the Justice Department, they bypassed the protocols at the White House, and so forth,” Barr stated. “These were things that persuaded me that there was not a legitimate counterintelligence investigation.”Former FBI director James Comey admitted in a December 2018 interview that he “sent” the agents to interview Flynn, adding that it was “something I probably wouldn’t have done or maybe gotten away with in a more organized administration.”In its Thursday court filing, the Justice Department explained that it was “not persuaded” that Flynn’s interview, which led to his guilty plea for lying to FBI agents Peter Strzok and Joe Pientka, had proper predication and was materially relevant.Comey tweeted his disappointment, following the decision, saying "the DOJ has lost its way."> The DOJ has lost its way. But, career people: please stay because America needs you. The country is hungry for honest, competent leadership.> > -- James Comey (@Comey) May 7, 2020Barr pointed to recently-released information that showed the FBI moved to close its surveillance of Flynn after finding “no derogatory information” about the retired general’s contacts with Russians, only for Strzok to keep the case open, leading to the eventual interview.“They were closing the investigation, in December [2016], they started that process and on January 4, they were closing it. When they heard about the phone call, which the FBI had the transcripts to — there was no question as to what was discussed, the FBI knew exactly what was discussed — and General Flynn, being the former director of the DIA, said to them, ‘you listen to everything, you know what was said,’” Barr explained.“So there’s no mystery about the call, but they initially tried some theories of how could open another investigation, which didn’t fly, and then they found out that they had not technically closed the earlier investigation, and they kept it open for the expressed purpose of trying to catch — lay a perjury trap — for General Flynn,” he added. A different filing released last week showed handwritten notes from an FBI official that questioned if the goal of Flynn’s White House interview was “to get him to lie, so we can prosecute him or get him fired.”Barr also did not comment on whether those that sought to entrap Flynn would face criminal charges, pointing to U.S. Attorney John Durham’s probe into the origins of the Trump-Russia investigation and saying his team was “in the middle” of “looking at the whole pattern of conduct.”“I’m going to wait until all the evidence is [in], and I get their recommendations as to what they found and how serious it is. But, if we were to find wrongdoing, in the sense of any criminal act, obviously we would follow through on that,” Barr said. “But again, just because something may even stink to high heaven, and appear to everyone to be bad, we still have to apply the right standard and be convinced that there is a violation of a criminal statute and that we can prove it beyond a reasonable doubt. The same standard applies to everybody.”

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.

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.

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.

Extracytoplasmic sugar decoration of glycopolymer components of the bacterial cell wall contributes to their structural diversity. Typically, the molecular mechanism that underpins such a decoration process involves a three-component glycosylation system (TGS) represented by an undecaprenyl-phosphate (Und-P) sugar-activating glycosyltransferase (Und-P GT), a flippase, and a polytopic glycosyltransferase (PolM GT) dedicated to attaching sugar residues to a specific glycopolymer. Here, using bioinformatic analyses, CRISPR-assisted recombineering, structural analysis of cell wall–associated polysaccharides (CWPS) through MALDI-TOF MS and methylation analysis, we report on three such systems in the bacterium Lactococcus lactis. On the basis of sequence similarities, we first identified three gene pairs, csdAB, csdCD, and csdEF, each encoding an Und-P GT and a PolM GT, as potential TGS component candidates. Our experimental results show that csdAB and csdCD are involved in Glc side-chain addition on the CWPS components rhamnan and polysaccharide pellicle (PSP), respectively, whereas csdEF plays a role in galactosylation of lipoteichoic acid (LTA). We also identified a potential flippase encoded in the L. lactis genome (llnz_02975, cflA) and confirmed that it participates in the glycosylation of the three cell wall glycopolymers rhamnan, PSP, and LTA, thus indicating that its function is shared by the three TGSs. Finally, we observed that glucosylation of both rhamnan and PSP can increase resistance to bacteriophage predation and that LTA galactosylation alters L. lactis resistance to bacteriocin.

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.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

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.

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.

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.

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.

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.

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.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

Endorepellin, the C-terminal fragment of the heparan sulfate proteoglycan perlecan, influences various signaling pathways in endothelial cells by binding to VEGFR2. In this study, we discovered that soluble endorepellin activates the canonical stress signaling pathway consisting of PERK, eIF2α, ATF4, and GADD45α. Specifically, endorepellin evoked transient activation of VEGFR2, which, in turn, phosphorylated PERK at Thr980. Subsequently, PERK phosphorylated eIF2α at Ser51, upregulating its downstream effector proteins ATF4 and GADD45α. RNAi-mediated knockdown of PERK or eIF2α abrogated the endorepellin-mediated up-regulation of GADD45α, the ultimate effector protein of this stress signaling cascade. To functionally validate these findings, we utilized an ex vivo model of angiogenesis. Exposure of the aortic rings embedded in 3D fibrillar collagen to recombinant endorepellin for 2–4 h activated PERK and induced GADD45α vis à vis vehicle-treated counterparts. Similar effects were obtained with the established cellular stress inducer tunicamycin. Notably, chronic exposure of aortic rings to endorepellin for 7–9 days markedly suppressed vessel sprouting, an angiostatic effect that was rescued by blocking PERK kinase activity. Our findings unravel a mechanism by which an extracellular matrix protein evokes stress signaling in endothelial cells, which leads to angiostasis.

The exponential growth of international travel since the 1960s has left border management systems worldwide struggling to keep up and has exposed weaknesses in states’ abilities to effectively manage their borders, especially regarding terrorist attacks, human trafficking, and illegal migration.

This report explores the migration patterns and demographics of Black African immigrants in the United States, examining their admission channels, human-capital characteristics, and labor market performance. The authors also provide an analysis of these immigrants' integration prospects.