Many suburban churches plan to keep their doors shut this weekend despite an easing of restrictions on public gatherings by Gov. Eric Holcomb.

A film that has never previously been broadcast shows crowds gathered at City Hall and Royal Avenue.

Saudi plans to build a new tech city are coming up against two hurdles: funding and a local tribe.

Hundreds of thousands of families live in overcrowded homes. What is lockdown life like for them?

A chronology of key events

Key facts, figures and dates

Key facts, figures and dates

A chronology of key events

Key facts, figures and dates

Mediterranean sediments are shown to have up to 1.9 million tiny plastic pieces per square metre.

Scientists say the microbe - found in the wild near Lake Victoria - has enormous potential.

1. Coronavirus LIVE Updates: Maharashtra's Tally of Infections Crosses 20,000 with 1,165 New Cases, 48 Deaths in Last 24 Hours  News18
2. Coronavirus India News LIVE | Tamil Nadu COVID-19 tally zooms to 6,535 with 526 new cases; 44 die  Moneycontrol
3. Retirement age hiked for all Tamil Nadu government staff  Deccan Chronicle
4. Andra Prasdesh to Reduce Retail Liquor Outlets to 2,934 by May End  News18
5. In Pictures | Day-45 of coronavirus lockdown leaves people in limbo  The Hindu
6. View Full coverage on Google News

Premier Doug Ford is asking Ottawans dreaming of a visit to their Quebec cottage or loved ones to “stay within our own province until this gets over with.”

The SeaBubble is a new form of urban transportation that could offer Parisians a watery alternative to hailing a taxi, driving a car or hopping on an electric scooter.

Here’s what we know for sure about the NFC playoff picture: the Saints, Rams and Bears can make postseason plans. The rest is up for grabs.

• Facebook is taking a harder line on misinformation related to coronavirus than it has on other health topics in the past.
• This decision may increase the pressure on the company to act more decisively against other forms of harmful falsehoods that spread on its social networks.
• Facebook is banning events that promote flouting lockdown protests, and is removing the conspiracy theory video "Plandemic."
• But false claims that vaccines are dangerous still proliferate on Facebook — even though they contribute to the deaths of children.

Amid the pandemic, Facebook is taking a harder line on misinformation than it has in the past. That decision may come back to haunt it.

As coronavirus has wreaked havoc across the globe, forcing lockdowns and disrupting economies, false information and hoaxes have spread like wildfire on social media. Miracle cures, intentional disinformation about government policies, and wild claims that Bill Gates orchestrated the entire health crisis abound.

In the past, Facebook has been heavily criticised for failing to take action to stop its platform being used to facilitate the spread of misinformation. To be sure, coronavirus falsehoods are still easily found on Facebook — but the company has taken more decisive action than in previous years:

• For starters, Facebook is now displaying warning messages to people who have shared false information about COVID-19. They're imperfect — Stat reported that they may be too vague in their wording to have a major impact — but it's a step further than Facebook has taken on misinformation in the past.
• The company is also taking down event pages for events that reject mainstream science on coronavirus by calling on people to flout lockdown rules.
• And it is banning "Plandemic," a conspiratorial video about coronavirus that has been going viral on social media and contains numerous falsehoods.

But Facebook's actions to combat COVID-19 misinformation may backfire — in the sense that it has the potential to dramatically increase pressure on the company to take stronger action against other forms of misinformation.

The company has long struggled with how to handle fake news and hoaxes; historically, its approach is not to delete them, but to try to artificially stifle their reach via algorithmic tweaks. Despite this, pseudoscience, anti-government conspiracy theories, and other falsehoods still abound on the social network.

Facebook has now demonstrated that it is willing to take more decisive action on misinformation, when the stakes are high enough. Its critics may subsequently ask why it is so reticent to combat the issue when it causes harm in other areas — particularly around other medical misinformation.

One expected defence for Facebook? That it is focused on taking down content that causes "imminent harm," and while COVID-19 misinformation falls into that category, lots of other sorts of falsehoods don't.

However, using "imminence" as the barometer of acceptability is dubious: Vaccine denialism directly results in the deaths of babies and children. That this harm isn't "imminent" doesn't make it any less dangerous — but, for now, such material is freely posted on Facebook.

Far-right conspiracy theories like Pizzagate, and more recent, Qanon, have also spread on Facebook — stoking baseless fears of shadowy cabals secretly controlling the government. These theories don't intrinsically incite harm, but have been linked to multiple acts of violence, from a Pizzagate believer firing his weapon in a pizza parlour to the Qanon-linked killing of a Gambino crime boss. (Earlier this week, Facebook did take down some popular QAnon pages — but for breaking its rules on fake profiles, rather than disinformation.)

And Facebook is still full of groups rallying against 5G technology, making evidence-free claims about its health effects (and now, sometimes linking it to coronavirus in a messy web). These posts exist on a continuum, with believers at the extreme end attempting to burn down radio towers and assault technicians; Facebook does take down such incitements to violence, but the more general fearmongering that can act as a gateway to more extreme action remains.

This week, Facebook announced the first 20 members of its Oversight Board — a "Supreme Court"-style entity that will review reports from users make rulings as to what objectionable content is and isn't allowed on Facebook and Instagram, with — in theory — the power to overrule the company. It remains to be seen whether its decisions may affect the company's approach for misinformation, and it still needs to appoint the rest of its members and get up and running.

For now, limits remain in place as to what Facebook will countenance in its fight against coronavirus-specific misinformation.

CEO Mark Zuckerberg said the company would immediately take down posts advertising dangerous false cures to COVID-19, like drinking bleach. It is "obviously going to create imminent harm," he said in March. "That is just in a completely different class of content than the back-and-forth accusations a candidate might make in an election."

But in April, President Donald Trump suggested that people might try injecting a "disinfectant" as a cure, which both has the potential to be extremely harmful, and will not cure coronavirus.

Facebook is not taking down video of his comments.

Do you work at Facebook? Contact Business Insider reporter Rob Price via encrypted messaging app Signal (+1 650-636-6268), encrypted email (robaeprice@protonmail.com), standard email (rprice@businessinsider.com), Telegram/Wickr/WeChat (robaeprice), or Twitter DM (@robaeprice). We can keep sources anonymous. Use a non-work device to reach out. PR pitches by standard email only, please.

SEE ALSO: Facebook announced the first 20 members of its oversight board that will decide what controversial content is allowed on Facebook and Instagram

Join the conversation about this story »

NOW WATCH: A cleaning expert reveals her 3-step method for cleaning your entire home quickly

• Oracle's bid to become a bigger player in the cloud has become more aggressive in the COVID-19 crisis, highlighted by a new partnership with Zoom.
• The tech giant is up against stronger rivals led by Amazon, Microsoft and Google, but the need for more cloud capacity sparked by the sudden pivot to remote work has created opportunities for the Silicon Valley behemoth.
• Here are the 10 Oracle executives who are playing key roles in CEO Safra Catz and founder Larry Ellison bold cloud offensive.
• Click here for more BI Prime stories.

Oracle has been through some jarring changes in the last seven months. 

The tech giant lost a well-regarded and experienced co-CEO when Mark Hurd died in October after taking leave for health reasons, leaving Safra Catz as the solo CEO. Now, like other major tech companies, Oracle is grappling with the impact of the coronavirus crisis.

But Oracle has been through tough times in its 43-year history. In fact, the Silicon Valley giant has been known to seize opportunities during rough spots. It's already seen some success during this crisis, too: Oracle just scored a big win when videoconferencing company Zoom — suddenly facing a surge in demand — chose to expand on Oracle Cloud, instead of other platforms like top cloud provider Amazon. Oracle is generally considered a smaller player in the cloud wars, behind giants Amazon, Microsoft, Google, and Alibaba.

Yes, Oracle still has a long way to go to match its rivals' reach, but its strategy of expanding its capacity by building more data centers seems to be paying off, IDC President Crawford Del Prete told Business Insider.

That increased capacity and Oracle's "world class" applications are key in the cloud words, Del Prete said: "Oracle is one of the few companies able to deliver both at scale in order to compete."

While Catz and founder, executive chairman, and chief technology officer Larry Ellison the lead company, they're also relying on key top executives, including cloud veterans from rival Amazon, to advance Oracle's cloud strategy. 

Nearly all are white men, something Oracle has criticized for in the past: Over 30 members of Congress slammed the company late last year about the lack of diversity in its leadership team and on its board.

Meet the 10 top executives playing important roles in Oracle's cloud offensive:

SEE ALSO: Oracle is known for making bold M&A moves in a recession and it's sitting on a fresh $20 billion. Here are the 7 companies experts think it could acquire as the coronavirus crisis drives down valuations

SEE ALSO: Experts lay out five moves that Oracle founder Larry Ellison, one of tech's best tacticians, might take in a coronavirus-driven downturn

Don Johnson left Amazon to focus on Oracle's cloud infrastructure.

Title: Executive vice president, cloud infrastructure

Reports to: Larry Ellison

Johnson  played a key role in Amazon's dramatic expansion in the cloud before joining Oracle in 2014.

He was instrumental in setting up Oracle's cloud engineering development center in Seattle and in the tech giant's expanding data center footprint.  Johnson has also led another major Oracle initiative: forming a cloud partnership with Microsoft.

Oracle's chief corporate architect Edward Screven has been with the company since 1986.

Title: Chief corporate architect

Reports to: Larry Ellison

Screven is an Oracle veteran who helped lead the company through all of the major industry changes of the past 30 years.

He admits that cloud market-leader Amazon had a head start, but says that there are benefits to following it. 

"We definitely started after Amazon: The bad news is they have market share, the good news is we get to learn a lot," he told Business Insider in an interview in May 2019. "Mindshare, that may be their biggest asset. But there is no technology they have that is concerning to me at all."

As one of Oracle's top technologists, he's focused on making Oracle's cloud infrastructure more secure, with more sophisticated and efficient ways to manage data. 

"We have hundreds of thousands of customers that store their most important data in Oracle databases," Screven said. "We could do a far better job for them than any other cloud provider. We are doing a far better job for them."

Clay Magouyrk leads cloud infrastructure engineering and played a key role in forging Oracle's new alliance with Zoom.

Title: Executive vice president, cloud infrastructure engineering

Reports to: Don Johnson

Magouyrk is another veteran of Amazon Web Services who joined the Oracle team in Seattle in 2014. 

He was Oracle's point-man in forging its new partnership with Zoom, which was seen as a major victory for Oracle.

"They needed capacity," Magouyrk told Business Insider last month "They reached out to us and we were like, 'Awesome, we can work with you.' Within a day, we had their application up and running."

Magouyrk was a founding team member of Oracle's cloud engineering development center in Seattle, which is spearheading the company's cloud infrastructure efforts.

Ariel Kelman left Amazon Web Services to become Oracle's chief marketing officer.

Title: Chief Marketing Officer

Reports to: Safra Catz

One of the biggest hurdles for Oracle is the public perception that it's a minor player in the cloud. In other words, it's a marketing problem.

This is where Kelman comes in. Before Oracle brought him on board in January 2020, Kelman led rival Amazon's cloud marketing efforts, and served as a marketing executive at Salesforce for six years before that.

"Ariel is a super smart hire for Oracle," analyst Ray Wang of Constellation Research told Business Insider. "He brings the cred in the market and understands how to counter all of Amazon's tactics and long-term strategy. He has the ear of Larry and Safra and is making progress with some great hires on his team."

Juergen Lindner left SAP to lead Oracle's software-as-a-service marketing strategy.

Title: Senior vice president, software-as-a-service marketing

Reports to: Ariel Kelman, chief marketing officer

Lindner spent most of his career helping SAP outsell Oracle in the traditional business software market: both dominated teh market for software installed in private data centers. 

He switched sides and roles four years ago to support Oracle's bid to become a stronger player in cloud software, also referred to as software-as-a-service, where businesses access applications through cloud platforms and pay via a subscription, usually based on the number of users granted access. 

Lindner has said it became clear to him that Oracle had a better strategy for the cloud-software era.

"Oracle has architected a very sustainable cloud infrastructure and applications strategy," he told Business Insider last year.

Steve Daheb left Citrix to lead Oracle's cloud marketing strategy.

Title: Senior vice president, cloud go-to-market

Reports to: Ashley Hart, senior vice president, global marketing cloud platform and database

Daheb joined Oracle in 2015 after serving as the chief marketing officer of Citrix, a cloud pioneer that first let businesses set up computing networks on web-based platforms instead of on-premise data centers, leading to dramatic IT cost savings.

Daheb witnessed the unexpected rise of Amazon in cloud computing, which began in the early : 2000s when the online retail giant realized it could make some extra money by giving businesses access to its massive but underutilized computing infrastructure, hosted from its data centers.

"Amazon had spare computing resources to rent out," he told Business Insider last year. "It's like, 'Hey, man, I got an extra room in the house during the summer when it's not spike retail time. There's nobody in there, so why don't I put this thing on Airbnb and see if anybody wants it?'"

Amazon Web Services has led the industry ever since. 

Like others on the Oracle team, Daheb thinks the software giant's technology and track record of working with major players across industries will eventually propel it to the front of the cloud pack.

"There's a level of understanding we have and a level of empathy we have for enterprise users: We serve the major banks, we serve transportation, we serve healthcare," he said. "We brought this enterprise mentality to it."

Juan Loaiza, who has been with Oracle since 1988, is in charge of mission-critical database technologies.

Title: Executive vice president, mission-critical database technologies

Reports to: Larry Ellison

Loaiza is another Oracle veteran who has been with the company for more than 30 years and is currently focused on its bid to expand the reach of its flagship database product.

The tech giant's cloud-based automated data-management platform Autonomous Database uses machine learning to quickly repair and update itself.Loaiza has compared the status of this fairly new initiative to the development of the self-driving car:

"It took a long time to get to a point where we are now and say, 'The next step is a self-driving car,'" he told Business Insider last year. "It's got to be safe. It has to have seatbelts and airbags and a navigation system. All that stuff was necessary before you take it to the next stage." 

The database is ready for that next stage. 

Jason Williamson left Amazon to lead Oracle's outreach to startups.

Title: Vice president, Oracle for Startups

Reports to: Mamei Sun, Ellison's chief of staff

Startups have played an important role in the growth of cloud computing and Oracle has launched a big push to establish closer ties with these smaller companies, given that they could eventually become the biggest power players. 

Williamson has been the company's point-man in this effort, as he develops ways to make Oracle's products and services more accessible to startups.

Williamson is another veteran of Amazon Web Services where he led the cloud giant's private-equity team before joining Oracle in 2017.

Evan Goldberg cofounded NetSuite, which is now part of Oracle.

Title: Executive vice president, NetSuite

Reports to: Safra Catz

Goldberg is part of the elite club of Oracle alums who went on to launch successful enterprise-software companies. (Salesforce CEO Marc Benioff is perhaps the best-known.)

Goldberg left a long career at Oracle in the late 1990s to launch NetSuite, a cloud-based provider of financial- and accounting-management services. He was the chief technology officer alongside CEO Zach Nelson, another Oracle alum, and Ellison was actually one of their early backers.

Oracle acquired the company in 2016 and it now has more than 18,000 customers. 

Steve Miranda has been with Oracle since 1992 and leads cloud-applications development.

Title: Executive vice president, applications product development

Reports to: Ellison

Miranda is an Oracle veteran in charge of different aspects of the company's cloud-software business, including product development and strategy.

This covers applications used for major business operations, like supply-chain management, human resources, and enterprise performance management.

What is a Cron Job? Cron Jobs are basically scheduled tasks that are created automatically by WordPress or a WordPress Plugin that you have activated on your website. They are similar to an alarm clock that goes off regularly to trigger an automative response. Before we delve into explaining how you can view and control […]

The post How to View and Control WordPress Cron Jobs appeared first on Tips and Tricks HQ.

As cruise ships get larger and cruising becoming more popular, cruise ports can get very crowded with cruise passengers. There can be between 15,000 and 20,000 passengers arriving at a port one any one day. What can you do to avoid the crowds and have a great time in a cruise port away from the mass of other cruising people there too? Over the many cruises I have done, over 73 at time of making this, I have found 8 great and proven ways to avoid the crowds in a cruise port. Hope these help you have a great time in every port you call on on your cruise vacation.

SUPPORT THE CHANNEL BY:
Buying my Cruise T-shirts: http://bit.ly/TFTStore
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Gary Bembridge's Tips For Travellers aims to help you make more of your precious travel time and money on land and when cruising the oceans or rivers of the world. To help you, in every video I draw on my first-hand tips and advice from travelling every month for over 20 years and 60+ cruises.

Follow Tips For Travellers on:
- Instagram: http://www.instagram.com/garybembridge
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Octoshape announced a partnership with INVISO to deliver Internet TV contribution services throughout Latin America.

"At INVISO, we seek the most innovative and high quality products to serve our customers through the brands we represent,” said Jose Luis Reyes, Vice President for Sales and Operations, INVISO. “In the case of Octoshape, we found a company and a product that bring these qualities to our supply chain, sales and service.”

Octoshape offers an innovative cloud-based solution that provides instant infrastructure for the distribution of both linear and video on demand content. The Octoshape Infinite Uplink service provides point-to-point distribution of TV signals over the Internet for source signal acquisition to traditional IPTV and cable headends as an alternative to traditional methods like satellite and video fiber.

The administration is celebrating the brain drain and helping the real swamp.

velocityconf: Help your dev + ops teams be cross-functional and more successful. http://t.co/1mqGK3zh0U Free webcast 5/22 w/ @lnxchk

Micronesia

Croatia Backroads

The Amateur Traveler talks to to Gary Arndt about the 4 micro-states in Europe: Andorra, Liechtenstein, Monaco, San Marino.  "They all have very unique histories and the one thing that they are all very small"

Hear about travel to Croatia as the Amateur Traveler talks to Jay Ternavan of JaywayTravel.com about this country on the coast of the Adriatic.

Jay says, "for me Croatia has everything you could look for in a travel experience"

Hear about travel to the countries of Kiribati, Tuvalu and Nauru in Micronesia as the Amateur Traveler talks to Stefan from Rapid Travel Chai about these small difficult to reach island nations. 

Church bells rang out across the city of Barrie on Friday morning to commemorate the anniversary of the Victory in Europe Day on Friday.

17 June 2019

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

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.

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Nucleoside analogues are a valuable experimental tool. Incorporation of these molecules into newly synthesized DNA (i.e. pulse-labeling) is used to monitor cell proliferation or to isolate nascent DNA. Some of the most common nucleoside analogues used for pulse-labeling of DNA in cells are the deoxypyrimidine analogues 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC). Click chemistry enables conjugation of an azide molecule tagged with a fluorescent dye or biotin to the alkyne of the analog, which can then be used to detect incorporation of EdU and EdC into DNA. The use of EdC is often recommended because of the potential cytotoxicity associated with EdU during longer incubations. Here, by comparing the relative incorporation efficiencies of EdU and EdC during short 30-min pulses, we demonstrate significantly lower incorporation of EdC than of EdU in noninfected human fibroblast cells or in cells infected with either human cytomegalovirus or Kaposi's sarcoma-associated herpesvirus. Interestingly, cells infected with herpes simplex virus type-1 (HSV-1) incorporated EdC and EdU at similar levels during short pulses. Of note, exogenous expression of HSV-1 thymidine kinase increased the incorporation efficiency of EdC. These results highlight the limitations when using substituted pyrimidine analogues in pulse-labeling and suggest that EdU is the preferable nucleoside analogue for short pulse-labeling experiments, resulting in increased recovery and sensitivity for downstream applications. This is an important discovery that may help to better characterize the biochemical properties of different nucleoside analogues with a given kinase, ultimately leading to significant differences in labeling efficiency of nascent DNA.

The cell surfaces of many bacteria carry filamentous polypeptides termed adhesins that enable binding to both biotic and abiotic surfaces. Surface adherence is facilitated by the exquisite selectivity of the adhesins for their cognate ligands or receptors and is a key step in niche or host colonization and pathogenicity. Streptococcus gordonii is a primary colonizer of the human oral cavity and an opportunistic pathogen, as well as a leading cause of infective endocarditis in humans. The fibrillar adhesin CshA is an important determinant of S. gordonii adherence, forming peritrichous fibrils on its surface that bind host cells and other microorganisms. CshA possesses a distinctive multidomain architecture comprising an N-terminal target-binding region fused to 17 repeat domains (RDs) that are each ∼100 amino acids long. Here, using structural and biophysical methods, we demonstrate that the intact CshA repeat region (CshA_RD1–17, domains 1–17) forms an extended polymeric monomer in solution. We recombinantly produced a subset of CshA RDs and found that they differ in stability and unfolding behavior. The NMR structure of CshA_RD13 revealed a hitherto unreported all β-fold, flanked by disordered interdomain linkers. These findings, in tandem with complementary hydrodynamic studies of CshA_RD1–17, indicate that this polypeptide possesses a highly unusual dynamic transitory structure characterized by alternating regions of order and disorder. This architecture provides flexibility for the adhesive tip of the CshA fibril to maintain bacterial attachment that withstands shear forces within the human host. It may also help mitigate deleterious folding events between neighboring RDs that share significant structural identity without compromising mechanical stability.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

27 September 2017

US policymakers should give special consideration to a more open immigration policy for highly skilled professionals from the EU. This would ultimately benefit the US economy.

Research Event

Event participants

Tim Jinks, Head of Drug-Resistant Infections Programme, Wellcome
Jim O’Neill, Chair, Review on Antimicrobial Resistance; Chair, Chatham House
Haileyesus Getahun, Director of Global Coordination and Partnership on Antimicrobial Resistance, World Health Organization 
Juan Lubroth, Chief Veterinary Officer, Food and Agriculture Organization (Videolink)
Jyoti Joshi, Head, South Asia, Center for Disease Dynamics, Economics & Policy
Estelle Mbadiwe, Coordinator-Nigeria, Global Antibiotic Resistance Partnership
Charles Clift, Senior Consulting Fellow, Chatham House; Report Author

Roland Bruderer
May 1, 2015; 14:1400-1410
Research

M. Wang
Aug 1, 2012; 11:492-500
Technological Innovation and Resources

Christian Tagwerker
Apr 1, 2006; 5:737-748
Research

Martin R. Larsen
Jul 1, 2005; 4:873-886
Technology

The linear ubiquitin assembly complex (LUBAC) is an essential component of the innate and adaptive immune system. Modification of cellular substrates with linear polyubiquitin chains is a key regulatory step in signal transduction that impacts cell death and inflammatory signaling downstream of various innate immunity receptors. Loss-of-function mutations in the LUBAC components HOIP and HOIL-1 yield a systemic autoinflammatory disease in humans, whereas their genetic ablation is embryonically lethal in mice. Deficiency of the LUBAC adaptor protein Sharpin results in a multi-organ inflammatory disease in mice characterized by chronic proliferative dermatitis (cpdm), which is propagated by TNFR1-induced and RIPK1-mediated keratinocyte cell death. We have previously shown that caspase-1 and -11 promoted the dermatitis pathology of cpdm mice and mediated cell death in the skin. Here, we describe a reciprocal regulation of caspase-1 and LUBAC activities in keratinocytes. We show that LUBAC interacted with caspase-1 via HOIP and modified its CARD domain with linear polyubiquitin and that depletion of HOIP or Sharpin resulted in heightened caspase-1 activation and cell death in response to inflammasome activation, unlike what is observed in macrophages. Reciprocally, caspase-1, as well as caspase-8, regulated LUBAC activity by proteolytically processing HOIP at Asp-348 and Asp-387 during the execution of cell death. HOIP processing impeded substrate ubiquitination in the NF-κB pathway and resulted in enhanced apoptosis. These results highlight a regulatory mechanism underlying efficient apoptosis in keratinocytes and provide further evidence of a cross-talk between inflammatory and cell death pathways.

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.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Gijs den Besten
Sep 1, 2013; 54:2325-2340
Reviews

W Gamble
Nov 1, 1978; 19:1068-1070
Articles

M. Mahmood Hussain
Jan 1, 2003; 44:22-32
Reviews

Saverio Cinti
Nov 1, 2005; 46:2347-2355
Research Articles