SAN JUAN, PUERTO RICO – Recently, U.S.

Die einen befürworten Schutzmaßnahmen, die anderen protestieren vehement: Rhetoriker Olaf Kramer erklärt, warum die Coronakrise für heftige gesellschaftliche Spannungen sorgt - und wie wir miteinander reden sollten.

Andre Harrell, a record executive who shepherded the careers of Mary J. Blige and Sean "Diddy" Combs and combined the sounds of R&B and hip-hop with his label, Uptown Records, has died.

Unilever is recalling Wall’s Mini Calippo (Orange and Lemon-Lime) multi-packs because they may contain small pieces of metal. The presence of metal makes this product unsafe to eat.

A Saskatoon family physician wanted to find a way to help send supplies to the northern community of La Loche and set an initial goal for $15,000 - but within days the donations total over $26,000.

Purdue Boilermakers play seven games in West Lafayette during the 2018 season, including their first four

Purdue coach Matt Painter and staff will explore the July evaluation period over the next three weeks. But the recruiting foundation for 2019 is set.

Robert Marve, who quarterbacked Purdue to the 2012 Heart of Dallas Bowl, has been accused of battery and sexual assault in Florida.

The road to Indianapolis: Why Colts fell in love with Michael Pittman Jr.

Charlotte Web writes: Since 2001 the TIOBE Index has been ranking top results for the search query +"<language> programming" on the top 25 search engines. "This month, C moved up past Java and entered the number one position," reports JAXenter. "There's a new number one. (Or, should we say an old number one?)" "Java and C were already very close in April, but this month C surpasses Java again," explains Paul Jansen CEO TIOBE Software. He also points out that the last time C was number one was back in 2015, suggesting that today embedded software languages like C and C++ "are gaining popularity because these are used in software for medical devices." "On another note, it is also worth mentioning that Rust is really getting close to the top 20 now (from #27 to #21 within one month)." "Perl, on the other hand, might be on its way off of the charts," argues JAXenter, "if it continues its downward trend. This month it saw a rate of change of -0.51%. It is currently number 18 on the list, but in May 2019 it was number 13." Python also passed C++ to take the #3 spot, while C# overtook Visual Basic for the #5 spot. ("Classic Visual Basic" also lost the #16 spot to PL/SQL). Even PHP rose a notch, pushing past SQL to take the #8 spot, and Scratch also moved up one, overtaking Objective C for the #19 position.

Read more of this story at Slashdot.

Emacs "predates Linux, and was once far more popular," writes LWN.net, while adding that the text editor "has fallen into relative obscurity over the years." Then it reports on a "mega-thread" on the emacs-devel mailing list about how to make Emacs more appealing and attractive to users: The discussion started with a post from "ndame" asking why Emacs is "so square"; the appearance of things like buttons could be improved with rounded corners, they said. Richard Stallman, one of the original authors of Emacs, seemed somewhat dismissive in his reply: "Perhaps we should implement a mode that puts cosmetics on Emacs so it will appeal to those who judge by the surface of things." But Stefan Kangas thought there was more to it than that: I think it's unfortunate if we assume that this is all bells and whistles. Graphical design elements can also improve usability. I also don't know that it's helpful to assume that the rest of the world will take the enlightened stance.... He wondered if there was "any reason not to improve the default look". Stallman said that there are some technical barriers in finding someone interested in and capable of doing the work needed, but there is an overarching problem that needs to be addressed first: The code to interface Emacs to X-based GUIs needs rewriting by an expert, and has needed it for decades. Until it gets that rewrite, changes in it are likely to break something. Stallman did agree that the graphical design could improve usability, "but I have a feeling that the changes that would help are deeper issues than the shape of corners". It was a long and interesting discussion, touching on the popularity of both Vim and Visual Studio Code, while another post questioned whether Emacs should even be prioritizing its menu bar and tool bar. One post suggested "starter kits" to make the text editor more friendly to newcomers, another suggested making Cntrl-C cut-and-paste the default key binding, and one asked whether it was Emacs' terminology and keyboard shortcuts that might be confusing to users coming from Microsoft Word. "You are basically making a commitment to being or becoming a power user..." argued another post. "If you just want to do 'casual' text editing emacs is a very weird choice in 2020."

Read more of this story at Slashdot.

"Is Meritocracy an Idea Worth Saving?" asks The Chronicle of Higher Education, reporting on a special forum held recently at the University of North Carolina's Program for Public Discourse. "This discussion took place before Covid-19 changed everything. But the topics — the definition of meritocracy, the role of universities in a just society, the composition of socioeconomic class, and the real purpose of education — are as relevant as ever." Moral philosopher Anastasia Berg, a junior research fellow at the University of Cambridge: Obviously certain roles in society and certain honors should be going to someone who is most competent for them: the Nobel Prize, or a teaching award, or who should perform eye surgery on us. The question is whether this is the right measure for determining who should be entering universities. There are objections from the left and from the right. I find the left ones persuasive, which is to say, in effect, that the pretensions to meritocracy are not borne out, if we actually look at who gets into colleges. We find out that there's huge correlation between the kind of material support that people have, and their ability to perform on the kind of exams that allow people to get into colleges. But what I also find problematic has to do with what has formerly been thought of as a conservative critique, although I think that leftists and liberals and progressives should be as concerned about it as anyone else: The current way of running college admissions concentrates talent, ambition, and competence in very few areas — on the coasts, in a very few universities — and draws potential leaders from communities elsewhere. Moreover, the current system leaves people blind to all the ways in which they owe gratitude to a community, for all the help that allowed them to achieve. New York Times opinion columnist Ross Douthat: It's useful to remember that the term "meritocracy" was coined as a description of a dystopia, in a book by a British civil servant written in the late '50s called The Rise of the Meritocracy. It was a tongue-in-cheek evocation of some pompous civil servant from somewhere around our own era, looking back on what he saw as the self-selection of the cognitive elite to rule over a society that was drained of talent, drained of ambition, and had all power centers outside the elite deprived of leadership and talent from within. It's reasonable to look at class divisions in the United States and much of the West and say that at least a partial version of that dystopia has come to pass. College-educated and more-than-college-educated Americans cluster together in geographic hubs in ways that they did not 50 or 60 years ago. It's a fascinating discussion, in which writer Thomas Chatterton Williams argues "it takes a kind of privilege to sneer at meritocratic measures that allow people to advance." But Berg also makes the observation that at least half of Americans won't ever have a college degree. "If that's the way to make citizens, what do we do with the rest? We have to make room for the dignity of other paths."

Read more of this story at Slashdot.

The road to Indianapolis: Why Colts fell in love with Michael Pittman Jr.

Reggie Brooks bullish on Notre Dame RB Chris Tyree and the Holtz's Heroes Foundation

Volunteers from six Santa Clarita Valley high schools step forward to help those shut in by the COVID-19 pandemic.

When someone mentions “Peru,” images of bucolic mountain pastures, ancient Incan ruins, and verdant Amazonian jungle are probably the first thing that pop into your mind. But this beautiful South American nation boasts miles and miles of gorgeous Pacific coastline, and it is also home to some amazing beaches. Let’s take a look at some of best beaches Peru has to offer.

Cabo Blanco: Arguably the most famous beach along Peru’s 2,500 kilometers of coastline, Cabo Blanco is routinely hailed as one the best surf spots in the world. When Peruvian surfer Gordo Barreda was scouting the area for great waves, he randomly paid a visit to the village, where he stumbled upon the now famous Peruvian Pipeline. A hollow, powerful left-hanging wave, it is arguably one of the best places to catch a wave on the continent’s entire Pacific coastline. Even before Barreda’s famous discovery, this fishing hamlet long enchanted visitors with its small-town charm and thriving fishing industry. In the 1950s and 1960s, fishermen routinely made the trek to Cabo Blanco in search of the area’s legendary large marlin. The famous writer Ernest Hemmingway spent several months here during the filming of the movie adaptation of his novel “The Old Man and the Sea.” During his stay, he reportedly caught a 700-pound marlin.

Mancora: Once a quaint fishing village, Mancora has exploded onto Peru’s backpacker scene as a major hub in the last decade or so. Located right in the middle of Peru’s sunniest and warmest region, you can relax on beautiful beaches during the day and then party the night away. Mancora is great for travelers on a budget, and cheap hostels abound.

Vichayito: If you want great beaches without a slew of rowdy partiers, Vichayito is an excellent option. Situated about 7 kilometers to the south of Mancora, this is an ideal spot for families. The water is ideal for swimming and kite surfing, and the beaches are clean and quiet.

Punta Hermosa: Just an hour drive from Peru’s capital, Punta Hermosa is popular with Limeños looking for a summer retreat from the city. While not as spectacular as the beaches of Paracas or Mancora, Punta Hermosa’s proximity to Lima makes it a great option for looking for a quick weekend escape from the city.

Asia: Peru’s most opulent beach, Asia is all about glitz and glamour. The upper echelons of Peruvian society have luxurious summer homes at this beach resort town, making it a hub of wealth. The beaches are great, but what really make Asia stand out are its high-end restaurants, luxury shopping center, and dazzling nightclubs.

Paracas: The Paracas National Reserve boasts some of Peru’s most dramatic desert landscape. Here, enormous sand dunes and dramatic sandstone rock formations meet the azure waters of the Pacific. The reserve, which consists of a total of 335,000 hectares of tropical desert on the Paracas Peninsula, is intended to preserve the area’s rich marine ecosystem as well as protect its unique cultural heritage (the site was of great significance to the Paracas, a pre-Colombian indigenous group that inhabited the area between roughly 800 BC and 1000 BC). There are no formal hotels within the reserve, though there are many sites popular with beach campers.

If you are looking for more luxurious accommodations, they can certainly also be found. Though beach destinations to the north of the country often attract the majority of tourist attention, it is Paracas where you will find the most luxurious beach vacations Peru has to offer. Just a few miles from the park’s entrance, you will find Hotel Paracas, a Luxury Collection Resort complete with 5-star accommodations, three pools, and a luxury spa.

Marcona: For those really looking to get off of Peru’s beaten tourist trail, we recommend a visit to the rugged, windswept beaches of Marcona, located roughly 8 hours south of Lima. The waters here can be quite cold year-round thanks to the Humboldt current, which brings water up from Antarctica along the Pacific coast of Chile and southern Peru. But the cold temperatures mean that waters are teeming with marine life. With any bit of luck, visitors might catch a glimpse of one of the zone’s enormous seal colonies. The beaches are known for their dramatic rock formations and rough surf, but they are a great place for those looking for options totally free of tourists.

So they you have it, our pick of the best Peruvian beaches. Happy adventuring in Peru!

One of the most popular hikes in South America, the Inca Trail is definitely the experience of a lifetime. A centuries-old trail, it begins in the ancient Incan capital of Cusco and winds some 88 kilometers to some of the world’s most iconic ruins: Machu Picchu. Of course, the entirety of Inca Trail is absolutely stunning, from stunning snowcapped Andean peaks to centuries-old ruins situated in bucolic river valleys. However, these 5 highlights are truly bound to make your jaw drop.

Wayllabamba. The endpoint of the first day’s trek along the Inca Trail, Wayllabamba, which means “grassy plain” in Quechua, is the perfect spot to watch the sunset behind the dramatic Andean peaks. This grassy plain overlooks a stunning spot of Andean scenery, with centuries-old Incan terraces winding through the surrounding mountainsides. There is even a village nearby where travelers can mingle with local villagers.

The Valley of Llulluchapampa. Trekkers will start off the first portion of day two of the journey hiking through the picturesque Valley of Llulluchapampa. As you gradually ascend in altitude, you will even be afforded perfect views of stunning snowcapped cliffs.

Runkuracay: This unique oval structure, sometimes colloquially know as the “Egg Hut,” is believed to have been a kind of rest stop for Incan travelers, called a tambo, providing them with a place to spend the night and rest their animals. It is the perfect place to enjoy a mid-hike break and marvel at the beauty of Incan architecture.

Sayacmarca: First discovered by the famous Hiram Bingham when he wandered along a road extending from Machu Picchu, the dramatic Sayacmarca is situated at a fork in an old Incan road in a dense subtropical forest full of butterflies and hummingbirds. Quechua for “Dominant Town,” these unique ruins have an almost mystical air about them and are arguably the most impressive along the Inca Trail (except for Machu Picchu itself, of course!). It is believed that Sayacmarca was actually built by the Colla, a major enemy of the Incas, and that the Incas took over the site following their conquest of the group.

Phuyupatamarca: Dubbed “La Ciudad entre la Niebla” (“The City above the Clouds”), this major archeological site is situated a staggering 3,200 meters above sea level. Apropos to the nickname, Phuyupatamarca is very often surrounded by dense, white clouds. The ruins, dramatically constructed into a steep cliffside, contain five stone baths that fill up with freshwater during the rainy season. It is believed that these baths were used for religious ceremonies. Visitors can also check out the site’s elaborate hydraulic system, a true testament to impressive capabilities of Incan engineering. Of all of the Incan ruins in the region, Phuyupatamarca is arguably the most intact and therefore a truly spectacular site for trekkers passing through.

Huiñay Huayna (Wiñay Wayna): Huiñay Huayna (traditionally spelled Wiñay Wayna in Quechua, the language of the Incas) was constructed into a steep hillside overlooking the Urubamba River. In addition to the site’s ancient houses and temples, it also boasts an incredibly complex system of Incan terraces, formerly used for agriculture. The name of the site roughly translates to “Forever Young,” and many trekkers report that these ruins are the most beautiful found along the trail.

These are just some of what you will see along the Inca Trail: Learn more about popular Inca Trail itineraries here

Residents of Quebec’s regions want Montrealers to stay away, considering the city has the highest concentration of COVID-19 infections in Canada.

The fiancee of murdered journalist Jamal Khashoggi says Newcastle United and the Premier League must put moral values ahead of financial gains.

The Most Useful Websites - self published using Amazon KDP.

The fiancee of murdered journalist Jamal Khashoggi says Newcastle United and the Premier League must put moral values ahead of financial gains.

Here are the winning Lotto and Lotto Plus numbers.

South Africans must insist on innovative solutions and new policies to ensure that when the country emerges from this governance and economic crisis, it does so not only aware of the country’s weaknesses but also primed to effect the necessary changes, writes Pieter du Toit.

French tennis chief Bernard Guidicelli admitted that Roland Garros could be staged behind closed doors.

My friend Steve Davee posted this fun project to Instructables. It's a perfect project for shut-in parents and kids to do together.

The main body parts of the bots are keyboard keycaps and Q-tips/cotton swabs. An eccentric weight (pager) motor provides the bouncy movement that makes your vibrobots go.

Dip the swabs in watercolor paints, place the little critter on some paper, and watch your little tabletop Jackson Pollockbot go to town.

Image: YouTube Read the rest

I photographed this Buloke Mistletoe (Amyema linophylla) flower near Stanhope in Victoria.

The DML News App offers the best in news reporting.

The post REPORT: Actor says ‘rule of law is dead’ until Barr is ‘impeached’ appeared first on Dennis Michael Lynch.

The DML News App offers the best in news reporting.

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

The manager of a Vancouver barber shop warns that once many B.C. salons are back open, they may have to implement a price increase for their services.

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

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

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

17 May 2016

Invitation Only Research Event

Event participants

Professor Philippe Sands QC, Barrister, Matrix Chambers
Chris Whomersley, Deputy Legal Adviser, Foreign and Commonwealth Office (2002-14)
Professor Julia Xue, Academy Senior Fellow, International Law Programme, Chatham House
Chair: Elizabeth Wilmshurst, Distinguished Fellow, International Law Programme, Chatham House

The White House has halted talks with Congress over any further coronavirus stimulus package as it waits for more information about how U.S. state reopenings affect the economy, White House top economic adviser Larry Kudlow told reporters on Friday. The Senate and House of Representatives have already passed four major bills to address the novel coronavirus outbreak, including three aimed at stabilizing the economy as most Americans have sheltered in place and unemployment has soared.

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

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

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.

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

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

β-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form β-cholesterylglucoside (β-GlcChol) in vitro. β-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate β-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (β-GalChol), in addition to β-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for β-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for β-GalChol formation. Liquid chromatography–tandem MS revealed that β-GlcChol and β-GalChol are present throughout development from embryo to adult in the mouse brain. We found that β-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of β-GalChol biosynthesis appeared to be during myelination. We also found that β-GlcChol and β-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form β-GalChol. This is the first report of the existence of β-GalChol in vertebrates and how β-GlcChol and β-GalChol are formed in the brain.

Actinobacillus pleuropneumoniae (App) is the etiological agent of acute porcine pneumonia and responsible for severe economic losses worldwide. The capsule polymer of App serotype 1 (App1) consists of [4)-GlcNAc-β(1,6)-Gal-α-1-(PO4-] repeating units that are O-acetylated at O-6 of the GlcNAc. It is a major virulence factor and was used in previous studies in the successful generation of an experimental glycoconjugate vaccine. However, the application of glycoconjugate vaccines in the animal health sector is limited, presumably because of the high costs associated with harvesting the polymer from pathogen culture. Consequently, here we exploited the capsule polymerase Cps1B of App1 as an in vitro synthesis tool and an alternative for capsule polymer provision. Cps1B consists of two catalytic domains, as well as a domain rich in tetratricopeptide repeats (TPRs). We compared the elongation mechanism of Cps1B with that of a ΔTPR truncation (Cps1B-ΔTPR). Interestingly, the product profiles displayed by Cps1B suggested processive elongation of the nascent polymer, whereas Cps1B-ΔTPR appeared to work in a more distributive manner. The dispersity of the synthesized products could be reduced by generating single-action transferases and immobilizing them on individual columns, separating the two catalytic activities. Furthermore, we identified the O-acetyltransferase Cps1D of App1 and used it to modify the polymers produced by Cps1B. Two-dimensional NMR analyses of the products revealed O-acetylation levels identical to those of polymer harvested from App1 culture supernatants. In conclusion, we have established a protocol for the pathogen-free in vitro synthesis of tailored, nature-identical App1 capsule polymers.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein–cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin–γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens–epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone–client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone–client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin–client complexes could contribute to lens aging and presbyopia.

The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (∼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10–13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane–localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

Extracellular matrix-evoked angiostasis and autophagy within the tumor microenvironment represent two critical, but unconnected, functions of the small leucine-rich proteoglycan, decorin. Acting as a partial agonist of vascular endothelial growth factor 2 (VEGFR2), soluble decorin signals via the energy sensing protein, AMP-activated protein kinase (AMPK), in the autophagic degradation of intracellular vascular endothelial growth factor A (VEGFA). Here, we discovered that soluble decorin evokes intracellular catabolism of endothelial VEGFA that is mechanistically independent of mTOR, but requires an autophagic regulator, paternally expressed gene 3 (PEG3). We found that administration of autophagic inhibitors such as chloroquine or bafilomycin A1, or depletion of autophagy-related 5 (ATG5), results in accumulation of intracellular VEGFA, indicating that VEGFA is a basal autophagic substrate. Mechanistically, decorin increased the VEGFA clearance rate by augmenting autophagic flux, a process that required RAB24 member RAS oncogene family (RAB24), a small GTPase that facilitates the disposal of autophagic compartments. We validated these findings by demonstrating the physiological relevance of this process in vivo. Mice starved for 48 h exhibited a sharp decrease in overall cardiac and aortic VEGFA that could be blocked by systemic chloroquine treatment. Thus, our findings reveal a unified mechanism for the metabolic control of endothelial VEGFA for autophagic clearance in response to decorin and canonical pro-autophagic stimuli. We posit that the VEGFR2/AMPK/PEG3 axis integrates the anti-angiogenic and pro-autophagic bioactivities of decorin as the molecular basis for tumorigenic suppression. These results support future therapeutic use of decorin as a next-generation protein therapy to combat cancer.

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.