It seemed like such a great idea at the time. You wanted to put together a video for a loved one, including all their family and friends singing their praises, making their life look as epic as a Hollywood production.

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Video montages take work. But by enlisting Tribute to help you assemble your message of love, It’s all a lot more manageable.

In fact, the Tribute process is so easy that you probably won’t have to do more than a few minutes of work to produce a high-quality tribute video that brings tears of joy. No, seriously...Tribute swears 80 percent of their videos elicit actual tears of joy. Of course, there’s no way of knowing their metrics for judging the results of their 500,000 Tribute videos so far...but if they’re close, your odds for an emotional testament to your subject are pretty darn high.

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Once all your videos are in, Tribute will compile all your clips into a touching, polished montage. Read the rest

The DML News App offers the best in news reporting.

The post REPORT: German Intelligence Releases ‘Harsh’ Verdict About WHO and China appeared first on Dennis Michael Lynch.

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

School, friends and normal day to day interactions have taken a virtual shift. However, for those with limited access to the internet, devices and other technology, isolation can be challenging.

The following article, Mark Levin on Michael Flynn Bombshell Documents: This Is “Barack Obama’s Blue Dress” Without The DNA, was first published on 100PercentFedUp.com.

Mark Levin nailed the importance of the newly released documents that cleared Michael Flynn and pinned Obama to the wall. He says that the documents are “Barack Obama’s Blue Dress” without the “DNA” alluding to the blue dress from Monica Lewinsky that proved Bill Clinton’s guilt. Levin begins by praising AG Bill Barr and then […]

Continue reading: Mark Levin on Michael Flynn Bombshell Documents: This Is “Barack Obama’s Blue Dress” Without The DNA ...

The following article, Rep. Ilhan Omar Asks For Contributions To Her Campaign To Help MN Food Bank…Food Bank Director Says Omar Has Nothing To Do With Project: “I have no idea where this money is going”, was first published on 100PercentFedUp.com.

Ilhan Omar is a lot of things. First, and foremost, she’s deceitful. David Steinberg of PJ Media was one of the first investigative journalists to break the story about the anti-Semitic, freshman lawmaker’s marriage to her immigrant brother while she was still married to her first husband, who she has since divorced after having an […]

Continue reading: Rep. Ilhan Omar Asks For Contributions To Her Campaign To Help MN Food Bank…Food Bank Director Says Omar Has Nothing To Do With Project: “I have no idea where this money is going” ...

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 “ ...

The following article, OUCH! Michelle Obama’s Netflix Documentary “Becoming” Gets Panned By Critics, was first published on 100PercentFedUp.com.

Unlike Melania Trump, who’s yet to appear on the cover of a major fashion magazine since becoming America’s First Lady, Michelle Obama has always been given special treatment by the far-left media, who couldn’t put her face on enough magazine covers. When her “Becoming” documentary aired on Wednesday, it was probably expected that the media […]

Continue reading: OUCH! Michelle Obama’s Netflix Documentary “Becoming” Gets Panned By Critics ...

Invitation Only Research Event

Event participants

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

29 October 2014

29 October 2014

12 November 2014 , Volume 90, Number 6

23 February 2015

Research Event

Event participants

Charlie Parton, Counsellor, Political Section, Beijing Delegation, European External Action Service
Chair: Roderic Wye, Assciate Fellow, Asia Programme, Chatham House

17 May 2016

4 July 2016

♥..Nomyens..♥ posted a photo:

Custom faceup

Angel Philia TS.

www.nomyens.com

♥..Nomyens..♥ posted a photo:

Custom faceup

Angel Philia TS.

www.nomyens.com

ridureyu1 posted a photo:

Wearing only the best hi-tech snail shell!

ridureyu1 posted a photo:

Too bad he's named after FDR and not Teddy.

gali_nette posted a photo:

Reiterlied posted a photo:

A tribute to Pirates I made last year when winter was coming to an end.

(You can find the blog post I wrote last year at Stuck in Plastic: www.stuckinplastic.com/2019/04/week-17-ching-shih/)

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.

Shelling of Tripoli's Mitiga airport early on Saturday, part of an intensified barrage of artillery fire on the capital in recent days, hit fuel tanks and damaged passenger planes, the Transport Ministry said in a statement. Mitiga is the last functioning airport in the Libyan capital, though civilian flights stopped in March because of repeated shelling even before the country imposed a lockdown over the coronavirus pandemic. Brega Petroleum Marketing Company, part of the National Oil Corporation, said its jet fuel tanks at Mitiga caught fire after coming under attack and firemen were working to control the blaze.

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

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

In a tweet Saturday morning, Tesla's chief executive said it would file a lawsuit against county officials over not being able to run its factory.

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.

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.

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.