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.

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

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.

"I think it's always important to think about suicide as individual vulnerabilities and context," Eric Caine said.

China's National Health Commission reported 14 new confirmed coronavirus cases on May 9, the highest number since April 28, including the first for more than a month in the city of Wuhan where the outbreak was first detected late last year. While China had officially designated all areas of the country as low-risk last Thursday, the new cases according to data published on Sunday represent a jump from the single case reported for the day before. The new Wuhan case, the first reported in the epicentre of China's outbreak since April 3, was previously asymptomatic, according to the health commission.

Prime Minister Justin Trudeau called Saturday for caution and expressed concern about loosening lockdown measures in Montreal, the epicenter of Canada's coronavirus outbreak. While several Canadian provinces, including Quebec, are preparing reopening measures and a gradual revival of their economies, Trudeau stressed prudence and said that the country is not yet out of danger. Quebec is the worst-hit province in Canada, with more than half of both the country's 67,000 cases of coronavirus and 4,700 deaths.

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

"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.

The Government today welcomed the successful handling of a number of bills and subsidiary legislation by the Legislative Council House Committee (HC) at its special meeting yesterday afternoon.

In a statement, the Government said that in accordance with Article 73 of the Basic Law, one of the major functions of LegCo is to enact laws and that the HC plays the most essential role in LegCo in performing the constitutional function of making preparations for LegCo meetings.

Such work includes deciding if bills committees are required to be set up to scrutinise the bills submitted to LegCo and monitoring the progress of these bills committees.

The statement noted that starting from last October, the HC has held 17 meetings and spent more than 30 hours of discussion but still failed to elect its chairman and deputy chairman for the current term of LegCo, thereby seriously impeding the committee and jeopardising its normal operation, creating substantial backlogs of bills that affect social development, the economy and people's livelihood.

At the special meeting yesterday, the HC completed the handling of a number of bills and subsidiary legislation which had not been dealt with for seven months since last October owing to the delay in the chairman election that brought the HC to a standstill.

These include 13 bills submitted by the Government during the current legislative session, Legal Service Division reports on 31 subsidiary legislation gazetted since March 27, the motion on the endorsement of the appointment of the Chief Justice of the Court of Final Appeal and four Reports of Bills Committees.

"The Government is pleased to learn that the impasse in the HC has finally ended which enables the continual scrutiny of bills and subsidiary legislation proposed by the Government.

“The Government will continue to fully co-operate with LegCo in its work of scrutinising laws in the remaining term of office of LegCo.

“As of May 8 this year, there are 26 bills that LegCo is scrutinising. Of these, 15 were proposed during the current legislative session, while the other 11 were first read in the past two legislative sessions and respective bills committees have been formed to scrutinise them,” the statement said.

Among the 11 bills, the bills committees have completed scrutiny of seven bills which have yet to be introduced to the full council for the resumption of second reading debate.

Of the seven bills, the HC has finished scrutinising the bills committee reports of six bills, including the National Anthem Bill and the Trade Marks (Amendment) Bill 2019.

The Bills Committee on the National Anthem Bill, after 17 meetings and over 50 hours of deliberation, had reported to the HC on June 14. At the HC meeting on June 28, the HC raised no objection to the Government's plan to resume the second reading debate on the bill in the 2019-2020 legislative session.

"The national anthem is the symbol and sign of the country. The legislative principle of the National Anthem Bill is clear, that is to fully reflect the legislative purpose and intent of the Law of the People's Republic of China on National Anthem as a national law, which is to preserve the dignity of the national anthem and promote respect for the national anthem; and at the same time to give due regard to the common law system practiced in Hong Kong, as well as the actual circumstances in Hong Kong,” the statement said.

In accordance with the National Anthem Bill, a person would only commit a criminal offence if the person publicly and intentionally insults the national anthem. It would not constitute an offence to express one's opinion as long as they are not expressed in the form of public and intentional insults to the national anthem, the statement added.

“Therefore, it is completely untrue and fabricated for certain LegCo members to claim that the law is 'draconian',” the statement noted.

The Government said it hopes that LegCo members would support the Government to continue to take forward the legislative procedures of the aforementioned bills, so that the efforts by the Government, LegCo and relevant stakeholders in formulating policies would not go down the drain.

(University of Stirling) A new University of Stirling study is seeking to understand how the easing of COVID-19 pandemic restrictions on licensed premises can be effectively managed to protect emergency services.

Lethal infections by strains of the highly-pathogenic avian influenza virus (HPAIV) H5N1 pose serious threats to both the poultry industry and public health worldwide. A lack of confirmed HPAIV epitopes recognized by cytotoxic T lymphocytes (CTLs) has hindered the utilization of CD8+ T-cell–mediated immunity and has precluded the development of effectively diversified epitope-based vaccination approaches. In particular, an HPAIV H5N1 CTL-recognized epitope based on the peptide MHC-I–β2m (pMHC-I) complex has not yet been designed. Here, screening a collection of selected peptides of several HPAIV strains against a specific pathogen-free pMHC-I (pBF2*1501), we identified a highly-conserved HPAIV H5N1 CTL epitope, named HPAIV–PA123–130. We determined the structure of the BF2*1501–PA123–130 complex at 2.1 Å resolution to elucidate the molecular mechanisms of a preferential presentation of the highly-conserved PA123–130 epitope in the chicken B15 lineage. Conformational characteristics of the PA123–130 epitope with a protruding Tyr-7 residue indicated that this epitope has great potential to be recognized by specific TCRs. Moreover, significantly increased numbers of CD8+ T cells specific for the HPAIV–PA123–130 epitope in peptide-immunized chickens indicated that a repertoire of CD8+ T cells can specifically respond to this epitope. We anticipate that the identification and structural characterization of the PA123–130 epitope reported here could enable further studies of CTL immunity against HPAIV H5N1. Such studies may aid in the development of vaccine development strategies using well-conserved internal viral antigens in chickens.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8–2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168–170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

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.

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.

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

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.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

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.

Noncoercive, pay-to-go, voluntary, assisted voluntary, and nonforced returns generally can offer paid travel and/or other financial incentive to encourage unauthorized immigrants to cooperate with immigration officials and leave host countries. A look at three key rationales for governments to choose pay-to-go and other returns.

Immigration is a prominent part of the United States’ DNA, despite concerns about immigrants’ ability to integrate. An examination of recent immigrant inflows shows newcomers to the United States are integrating well, based on language proficiency, socioeconomic attainment, political participation, residential locale, and social interaction indicators.

The U.S. refugee protection system, while generous in many respects, has become less robust over the last two decades. The unique and often diverse needs of emerging refugee populations have exposed severe limitations in the standard resettlement approach.This report examines U.S. legal and policy responses to those seeking protection and addresses the barriers, gaps, and opportunities that exist.

Two competing models for selecting economic-stream immigrants are now prevalent in advanced industrialized economies: points-based and employer-led selection. Increasingly, however, hybrid selection systems are being created, implementing best practices from each selection process.

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.