Kingston’s top health officials say that there are no current cases of COVID-19 active in the city.

Ontario's provincial parks and conservation areas will reopen this week but campgrounds and beaches will continue to be off-limits for now.

Dr John Wright is intrigued by some singers who became ill long before the UK's first known Covid-19 case.

Filed under: North America, United States

Continue reading Sorry Indianapolis, You're No Longer the Sole Location Offering TSA PreCheck Enrollment Background Checks

Sorry Indianapolis, You're No Longer the Sole Location Offering TSA PreCheck Enrollment Background Checks originally appeared on Gadling on Mon, 16 Dec 2013 15:07:00 EST. Please see our terms for use of feeds.

We reassessed our hierarchy of needs, and survival always outweighs the rest. To be blunt, we would rather have load shedding than risk dying, writes Adriaan Basson.

Some kids took advantage of the opportunity to show their appreciation for their moms on Saturday afternoon at the Love You Mama event.

Frank Soo, the first and only player of Asian heritage to represent England's national football team, has been honoured by Google.

SuperSport United winger Aubrey Modiba says a conversation with club CEO Stanley Matthews convinced him to stay despite interest from Mamelodi Sundowns.

With the UK government set to tighten its lockdown restrictions, fresh doubts have been cast over the viability of a British Grand Prix.

Fear is ripe soil for the unscrupulous. With so much uncertainty and concern over our health and the broader world economy, cybercriminals have been playing on that fear to steal a few extra dollars out of the most scared and vulnerable. The U.S. Secret Service warned that phishing attacks were up significantly and scams over fake COVID-19 treatments have led to seizures and arrests.

From companies and organizations to individuals, it’s never been more important for everyone to have their cybersecurity measures on high alert. And whether you’re looking to protect your own assets or you’ve been tasked with safeguarding a company and all its workers, the vital work of white hat hackers is absolutely essential these days.

The training in The Ultimate 2020 White Hat Hacker Certification Bundle can put you in a position to understand all aspects of maintaining cybersecurity for a communication system of virtually any size, a lucrative career that can earn you a six-figure income.

The four-part Complete Cyber Security Course (taught by cybersecurity expert and noted consultant Nathan House) is a 360-degree starting point for any cybersecurity career. Starting at the beginning, each part of this multi-pronged introduction will help guide you through vital knowledge, from network hacking techniques and vulnerability scanning to all the defense methods that assure every laptop, desktop, smartphone and tablet in your network remain secure.

The hacker training continues with the rest of the courses in the collection, including building security analysis tools using Python and learning how to analyze web app security vulnerabilities and solutions using frameworks like Ruby on Rails and PHP. Read the rest

The DML News App offers the best in news reporting.

The post REPORT: Three Times Joe Biden Was Reportedly Involved with the Russia Collusion Hoax appeared first on Dennis Michael Lynch.

Canadians working aboard two cruise ships who weren't allowed to come to shore because of concerns about COVID-19 are finally able to return home.

The following article, California Sheriff Refuses to Arrest People Defying Stay-at-Home Order: “There cannot be a new normal”, was first published on 100PercentFedUp.com.

Riverside, California Sheriff Chad Bianco spoke to the Riverside Board of Supervisors on May 5th to say that he will not enforce the stay-at-home order in California. He tells people who are afraid of contracting the coronavirus that they should stay home if they want to. Bianco continues with the suggestion that any business owner […]

Continue reading: California Sheriff Refuses to Arrest People Defying Stay-at-Home Order: “There cannot be a new normal” ...

26 August 2013

18 June 2014

Invitation Only Research Event

Event participants

Dr Leena Koni Hoffmann, Centre for Population, Poverty and Public Policy Studies; Author, Who Speaks for the North? Politics and Influence in Northern Nigeria; ERANDA Junior Research Fellow, Africa Programme, Chatham House (2013)
Chair: Elizabeth Donnelly, Assistant Head, Africa Programme, Chatham House

Research Event

Event participants

Dr Doyin Okupe, Senior Special Assistant on Public Affairs to the President of Nigeria 

Invitation Only Research Event

12 November 2014 , Volume 90, Number 6

27 January 2015

Members Event

Event participants

Parag Khanna, Author, Connectography: Mapping the Global Network Revolution

Members Event

Event participants

Dr Mukulika Banerjee, Director, South Asia Centre, London School of Economics
Nandan Nilekani, Co-founder and Chairman, EkStep; Chairman, Unique Identification Authority of India (UIAI) (2009-14)
Dr Gareth Price, Senior Research Fellow, Asia Programme, Chatham House
Mihir Swarup Sharma, India Columnist, Bloomberg View; Senior Fellow, Observer Research Foundation, New Delhi
Chair: James Crabtree, Contributing Editor, Financial Times; Senior Visiting Fellow, Lee Kuan Yew School of Public Policy

6 January 2017 , Volume 93, Number 1

♥..Nomyens..♥ posted a photo:

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Angel Philia TS.

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

GAZIANTEP, Turkey—After five years fighting to preserve Bashar al-Assad’s regime in Syria, Russia now appears inclined to dispose of its infamous client. Assad’s persistent brutality and corruption, and his inability to establish even the semblance of a functioning state, has grown to be a burden Moscow would prefer not to bear.And then there’s the problem of Iran. Assad, members of his family, and his Alawite clansmen enjoy close, perhaps unbreakable, bonds to the regime in Tehran and to Iranian-backed militias in Syria. All of which undermines Moscow’s primary mission there: to rehabilitate the Assad regime as a symbol of stability capable of attracting hundreds of billions of dollars of foreign investment for reconstruction, which Russian firms would then be poised to receive. As long as Assad’s relatives continue to function as a mafia and give free rein to Iranian troops using Syria as base of operations to threaten Israel and plan attacks against U.S. troops in Iraq, those countries likely to foot the bill for Syrian reconstruction—the nations of Europe and the Gulf—are unlikely to come up with the cash. Amid Escalating Syrian Carnage, Turkey Shoots Down Assad’s PlanesThis has not gone unnoticed by the United States.“Assad has done nothing to help the Russians sell this regime,”James Jeffrey, the U.S. special envoy for the Coalition to Defeat ISIS, told reporters in a State Department briefing on Thursday. “You find Assad has nothing but thugs around him, and they don't sell well either in the Arab world or in Europe. We have heard repeatedly from Russians we take as credible that they understand how bad Assad is.” The Syrian president’s “refusal to make any compromises” in order to secure diplomatic recognition and acceptance for his regime has jeopardized “hundreds of billions of dollars in reconstruction assistance” for Syria, according to Jeffrey. Yet the Trump administration is unlikely to exploit this growing rift. “Getting Russia out of Syria,” Jeffrey said, “has never been our goal. Russia has been there for 30 years. It has a long-term relationship with Syria. We don’t think it has been healthy for the region. We don’t think it really is even healthy for Russia. But that’s not our policy.”  MEDIA FRENZYJeffrey’s statements come just one week after Russian state media unleashed a slew of reports and editorials targeting Assad, portraying the beleaguered president as hopelessly corrupt and unfit to govern, and suggesting the time had come to replace him with a new leader.The first batch of articles was published by the Russia’s Federal News Agency (FNA), an outlet owned by Yevgeny Prigozhin, a Russian oligarch and chairman of several companies implicated in the 2016 U.S. elections scandal. Appearing over the course of a mere three hours on April 17, they would shake Syria to its core. The first of the three articles in question highlighted a corruption scheme carried out by the regime in summer 2019 in which the Syrian prime minister purportedly lied to citizens about oil and gas scarcities in order to justify the occurrence of long power outages while selling Syrian electricity to businessmen in Lebanon. The second piece cited an opinion poll claiming only 32 percent of Syrians would vote for Assad in the country’s upcoming 2021 presidential election. The third and final article, entitled, “Corruption is Worse than Terrorism,” chastized President Assad for personally failing to combat corruption, prevalent at all levels of the state.  That these were published by Prigozhin’s news agency was the kind of signal it would be hard for Assad to miss. Prigozhin, who first built his fortune as a caterer, is sometimes known as “Putin’s chef.” But of particular relevance to Syria is his role as chairman of the Wagner Group, whose mercenaries have fought alongside Assad regime forces since October 2015 and helped the latter take back control of key revenue generating infrastructure such as the al-Sha’ir gas field in Homs province.Deputy Assistant Secretary Christopher Robin told the same State Department briefing Thursday, “Wagner is often misleadingly referred to as a Russian private military company, but in fact it’s an instrument of the Russian government which the Kremlin uses as a low-cost and low-risk instrument to advance its goals.”The article on corruption would also point out, suggestively, that the Assads are not the only powerful family in Syria, “there are also the Makhloufs.”Rami Makhlouf, who is in fact Bashar al-Assad’s first cousin, is Syria’s wealthiest man, and also, it would seem, Russia’s man. Certainly he has strong ties to the Kremlin and for years has been one of the most vocal critics of Iran’s presence in Syria. In July 2018, the al-Watan newspaper, one Syria’s most prominent pro-regime mouthpieces and owned by Rami Makhlouf since 2006, published a then unprecedented public rebuke to Iran, accusing it of sponsoring Islamist fanaticism throughout the Middle East alongside Turkey and Qatar, the main backers of Syria’s opposition. (Rami Makhlouf’s father Muhammad and brother Hafiz meanwhile are alleged by some to be living in Russia.) The April 17 articles published by Prigozhin’s FNA preceded the release of a wave of other articles and items in the media over the next 12 days that would further drive home the point that Moscow was considering options other than Assad to rule Syria. TASS, Russia’s largest state-run news agency, wrote in one editorial that, “Russia suspects that Assad is not only unable to lead the country anymore, but also that the head of the Syrian regime is dragging Moscow towards the Afghani scenario.” This is like evoking the Vietnam War for an American audience, a reference to the Kremlin’s botched campaign through the 1980s that helped bankrupt the Soviet Union and finally break it apart.Amid this coverage, TASS would also take swipes at Iran, claiming that the Islamic Republic has “no interest in achieving stability in the region, because it considers it a battlefield with Washington”.On April 30, the Russian International Affairs Council (RIAC), a think tank established by Moscow’s Ministry of Foreign Affairs, released a scathing report saying Russia was in talks with other parties to the Syrian conflict to draw up plans for a political resolution that did not include Bashar al-Assad as president. The report highlighted purported Russian efforts to compel the Syrian regime to commit to ceasefires with both American-backed and Kurdish-led Syrian Democratic Forces (SDF), and the Turkish-backed Free Syrian Army (FSA) opposition, while beginning steps to form a new unity government that would include representatives from both. That day, Rami Makhlouf, whose assets were frozen five months earlier as part of a tax dispute, uploaded a video onto his personal Facebook page accusing the Assad regime of corruption. In a state known for carrying out the full-scale slaughter of those who test its authority, Makhlouf’s videos, coming on the heels of the unprecedented Russian attacks in the media, sent shockwaves throughout the country.  THE ROYAL FAMILYWhile the Makhlouf clan clearly has thrown its lot in with Russia, key members of Bashar al-Assad’s immediate family and others with ties to Qardaha in Syria’s largely Alawite Latakia province, are among the most prominent Iranian-backed militia leaders in Syria. It’s an alliance that traces back to his father Hafez al-Assad, who was born in Qardaha, and who forged ties with the Iranian revolution almost from its beginning more than 40 years ago. The Iranians responded by offering religious legitimacy to the Alawite sect, which is regarded as heretical by Sunnis and indeed by many Shi’a.These Qardaha militia leaders have regularly engaged in armed clashes against Russian backed units. They are among the most egregious violators and abusers of power, overseeing wide networks of corruption similar to those lamented in the Russian media. And foremost among them is Bashar’s younger brother, Maher al-Assad. Since April 2018, Maher al-Assad has commanded the Syrian Army’s 4th Armored Division, one of country’s oldest, best equipped and overwhelmingly Alawite brigades. After the 2011 outbreak of the Syrian revolution, when the loyalty of much of the army was in doubt, it became a refuge for numerous Alawite-Shi’a dominated pro-regime militias.Currently, the 4th Armored Division’s members control many smuggling operations throughout the country, in cities from Albu Kamel on Syria’s eastern border with Iraq to Latakia on the Syrian coast, where the port was leased to Iran on October 1 last year. It has since become one of the biggest export hubs for drugs headed to markets in Europe, the Middle East and North Africa. Examples abound: On July 5, 2019, Greek coast guard and drug enforcement officials announced the biggest drug bust in history, seizing 5.25 tons (33 million pills) of Captagon amphetamines worth $660m hidden in shipping containers loaded at the Latakia port in Syria. That followed a long string of such seizures made by Greek authorities. More recently, in late April, customs officials in both Saudi Arabia and Egypt also announced the seizure of similar quantities of drugs in containers traced back to Latakia. Local reports have accused a range of actors including Maher al-Assad’s 4th Division, Hizbollah, Rami Makhlouf, and others of profiting from the massive drug exports emanating from the port. In January 2019 the 4th Armored Division launched attacks on the Russian-backed Tiger Forces unit in an attempt to wrest control of smuggling routes between regime- and opposition-held territory in Idlib province. The clashes led to the death of 70 fighters. These and other skirmishes prompted Russia to back a major campaign to arrest 4th Division and other Iranian-backed units throughout the country beginning in April 2019, which succeeded in rounding up numerous mid-ranking Iranian-backed officers. Among those targeted in the campaign was Bashar Talal al-Assad, a cousin to the president (similar name, different people) who was wanted on drug and weapons trafficking charges. Unlike others who were detained in the roundup, Bashar Talal al-Assad and his ‘Areen Brigade managed to fight off Russian-backed forces that sought to arrest him in Qardaha. He then pledged to attack Russia’s Hmeimim military base, located 17 miles east of Latakia city, in the event the regime sought to arrest him again.For Russia, the threat of such attacks on its military infrastructure is a real concern. The Hmeimim base—from which Moscow has directed its entire military campaign in Syria—had already been subject to a series of attacks from January to October 2018 by other Iranian-backed militias in the area. The threat posed by both Iran’s acquisition of the Latakia port and its support for local Assad family proxies in Syria’s coastal region is exacerbated by the fact that Tehran has also begun making progress toward completing construction of its Shalamcha railroad, which, via stops in Basra, Baghdad, Albu Kamel and Damascus, will give Tehran direct access to the Syrian and Lebanese coasts. If Iran succeeds in integrating the Latakia port with the Shalamcha rail line, this will cut off Hmeimim from Russian forces in central and southern Syria and enable Tehran to quickly deliver weapons to proxy forces in Latakia that are already engaged in clashes against Russian-backed groups. WORLDWIDE CONSENSUSMoscow’s inability to control Iranian backed Syrian militiamen engaged in widespread crime, corruption, and assaults on Russian forces has infuriated the Kremlin. But Russia is not the only major player on the ground with scores to settle against Iran, and the Russian military leadership in Syria has ignored if not largely encouraged Israeli strikes on Iranian troops throughout the country.It may not be coincidental that the Israeli attacks have increased in pace and scope since April, following the flurry of Russian media articles attacking Assad and his regime. “We have moved from blocking Iran’s entrenchment in Syria to forcing it out of there, and we will not stop,” Israel’s new defense minister, Naftali Bennett, declared on April 28. Without Russia, Iran has found itself the odd man out in Syria, the single party still seeking to push for war at a time when most other international players have been struck with fatigue and simply seek to put Syria’s pieces back together. President Recep Tayyip Erdoğan of Turkey, the last patron of Syria’s battered FSA opposition, has himself made peace with Moscow, effectively agreeing last March to cede control of wide swaths of rebel held territory after a particularly bloody Russian led campaign against the last FSA holdout in Idlib province that ended in victory for regime forces. Ironically, Erdoğan’s long-held desire to overthrow Syria’s president may still come to fruition, albeit not as he expected, as Assad’s ouster may come at the hands of Russia itself, and not the revolution. 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.

South Dakota Governor Kristi Noem sent letters to two Sioux tribes demanding they remove COVID-19 checkpoints because they interfere with traffic.

Nepal protested India's inauguration of a new road to China that passes through territory claimed by Kathmandu on Saturday, with police arresting dozens demonstrating close to India's embassy. Indian defence minister Rajnath Singh on Friday inaugurated via video link the 80-kilometre (50 miles) long road from Ghatiabagarh in northern Uttarakhand state to the Lipu Lekh pass high in the Himalaya. The pass is claimed by Nepal based on an 1816 treaty that defines its western border with India.

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

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.

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.

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.

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.

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.

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.