Part of the Antarctic diary promo for the BBC UK Homepage

Introducing Instapaper 4.0 for iPad and iPhone

The lede here is that my pal, Marco, has just released the stellar new 4.0 version of his Instapaper suite.

This is fantastic news, and–as if you needed one more of Marco’s beta testers to say so–I do sincerely hope you’ll mark the occasion (and support his hard work) by purchasing the Instapaper iOS app(s). I promise you’ll be treating yourself to a massive update to an already excellent product.

Now, it’s fortunate and appropriate that you’ll be hearing this advice at length from a lot of people this week. Because, if it’s not already obvious, Marco’s little app (and its associated services) enjoys a rabid fanbase of sundry paragraph cultists who are as eager as I am to spread the word; and, yes, we do want you to join the Reading Nerd cult.

But, I also want to mark the occasion by adding a few thoughts on exactly what Instapaper has done, and continues to do, for me. (As you may already know, I’m a big Marco fan.)

Thing is, I want to tell you how Marco has made a magical machine for people who have decided to read.

Long-Time Fan

For years, Instapaper has been one of the best made, most used, and most beloved apps in my iOS ecosystem. It’s always lived on my iPhone’s home page, and, as you can surmise, that’s because I use Instapaper a lot. Like, a lot a lot. Specifically, I use Instapaper a lot because it helps me do four things extremely well. Four things that work together to make my life a little better.

In that typically annoying mixed order I can’t seem to stop doing, here goes.

2. Deciding WHEN to read

Second, and most obviously, I use Instapaper maybe five to ten times a day to catch up on my reading. Which is great. This is what Instapaper is actually for, right? You read stuff.

Long articles, smaller features, short books, big piles of documentation, and really just anything that I would like to read…later. More saliently, these are things that I have decided to read. This decision part’s important, but more on that in a couple minutes.

But, how does all this “stuff” I’ve decided to read get in to Instapaper?

1. Deciding WHAT to read

See, this is the really important first part. Because as much as I use Instapaper for all manner of reading, its use as an ephemeral destination for mostly ephemeral content wouldn’t be nearly so useful if I didn’t have so many ways to collect all that stuff. So, that flexibility in collecting material is where I end up using some form of Instapaper dozens of times each day.

Examples?

I have a bookmarklet for adding items to Instapaper in 4 browsers on 7 devices. I have (and use the hell out of) the “Send to Instapaper” services that are built in to everything from Google Reader to Reeder to Flipboard to Instacast to Tweetbot to Zite to you name it. I can automate in or out of Instapaper with If This Then That, I can email items directly to Instapaper–hell, I can even just copy a URL from iOS Safari, and paste it directly into the motherscratching Instapaper app.

Suffice it to say, there are many ways to get “stuff” into Instapaper. E.g.:

But, that banner dump only tells part of the story.

Yes, a big part of this is about ubiquity and ease-of-use. But, the practical result is that all those little entrees to Instapaper are available to me everywhere I might need them, and they each represent a single little click that silently adds an item of “stuff” to my Instapaper pile.

Each button is one more simple opportunity for me to decide to read.

3. Deciding WHERE to read

Now, the third part of this magic is less immediately obvious, not least because the reading experience of the Instapaper iOS apps is, for my own purposes, perfect. But, there’s more.

Because, all that support for getting stuff into Instapaper is mirrored by an endless number of ways to get stuff back out. To, in fact, read. That thing I decided to read is now everywhere.

However I ended up deciding to read something, seconds after that *click*, the real magic starts happening, and–through whatever inscrutable black art and transmogrification is happening inside the fearsome celestial engine Marco has made–that decision to read is expressed in the most elegant of results and in a startlingly broad variety of convenient places.

It’s readable on a website; it’s readable on an iPhone, and 2 iPads; it’s readable on a Kindle 3; it’s readable on the crazy number of apps and services that display Instapaper items. And, it’s even preserved for posterity in my private Pinboard archive.

So, for practical purposes, this stuff that I’ve decided to read can now go whooshing through a network of customized tubes, and gently land practically anywhere that well-formed bits may reside.

4. Just…Deciding to Read

I know most of you know these things. I know you’re familiar with the many “Features and Benefits” of Instapaper. And, I even know that most of you reading this are probably already using Instapaper–perhaps even to read this very article.

So, the point here is not simply that Instapaper is flexible, idiot-proof, and sanity-savingly redundant. Although it is all those things and many more.

The point is that my life always gets better when I decide to read things–and then actually read those things I decided to read. This is not a trivial point.

We’re all busy, and we’re all bombarded with 10,000 potential calls on our attention every day. Some days, we handle that better than others. Some days, we don’t handle it all.

All I know, is that, throughout my life, deciding to read has made that life better.

It made my life better at 7 with Henry Huggins. It made my life better at 16 with Slaughterhouse-Five. It made my life better at 20 with Absalom, Absalom!. And, it made my life way better at 25 with A Confederacy of Dunces (cf.).

And, now, for the past few years–following over a decade during which I read way more href tags than actual prose paragraphs–my life has gotten better, in part, due to Instapaper. I’ve finally gotten my hands around this “too much stuff” issue, at least insofar as it relates to words of theoretical interest. Now, I know where it goes. It goes into Instapaper.

Because, now? Yeah. Twenty-some years after a college career sucking down over 1,000 pages a week, I am finally returning to reading a lot more. Because, I am deciding to read a lot more. Instapaper means there’s no excuse for not reading a lot more. Period.

How about you?

What Are YOU Deciding?

When you’re in line at the ATM or the professional sporting event, what do you do?

If you’re like a lot of people, you hit your mobile device like a pigeon on a goddamned pellet. Then, you decide what happens.

You can decide to throw birds at pigs. You can decide to check in on which strangers are pretending to like you today. You may even decide to see what you would look like if you were really fat.

Thing is, you could also decide to read. Just for a couple minutes. Maybe more. Maybe less. Who knows. It’s your decision.

A Nudge Towards “Better”

But, if you have followed the circuitous skeins of yarn comprising this little sweater you’ve been reading, it comes down to this:

If you’ve decided that you want to read, Marco’s app will really help you. He’s removed any phony barriers you’ve built about “not having time” or “not having it with you” or “not knowing where to put it.” There are no excuses, apart from the superficial animated ones you’ve constructed out of cartoon birds.

As for me? In the last week alone, I decided to read a lot of things in Instapaper. A small sampling:

I decided to read about an American family’s educational experiment in Russia.

I decided to read about what Heidegger means by Being-in-the-World.

I decided to read about why toasters are so bad.

I decided to read about responsive web design.

I decided to read about why Charlie Kaufman wrote Being John Malkovich.

I decided to read about how Open Data could make San Francisco Public Transportation better.

I decided to read about how John Siracusa remembers Steve Jobs.

I decided, and then I read. I read, and I read.

So, thanks, Marco. You’ve made my life better by making it easier to decide to read. Then, you made it way easier to do the actual reading.

And, to you–the kind readers-of-prose-paragraphs who were inexplicably patient enough to decide to read this long article–please consider supporting Marco’s work.

Please get an account at Instapaper and, if you have an iOS dingus, please do buy the Instapaper app.

In addition to having exquisite taste in app icons and a lovely speaking voice, Marco’s just a very good human. And, good humans more than deserve our support.

Buy Instapaper 4.0 by Marco Arment.

An "appalling prank" in Innisfil sent South Simcoe Police officers on a mission to identify the culprits involved.

Labour laws haven’t changed in our province, but legal experts are already urging B.C. employers to be flexible and reasonable — while warning employees they may not be legally protected if they refuse work during the pandemic.

Vancouver resident Hugo Huynh says he's never seen the man who got out of a minivan outside his home early Monday morning and uprooted the young tree.

The following article, BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations, was first published on 100PercentFedUp.com.

Barack Obama knew. Documents released yesterday that were used to exonerate President Trump’s new NSA General Flynn, prove that President Barack Obama was aware of the details of Michael Flynn’s intercepted phone calls on December 16 with then-Russian Ambassador Sergey Kislyak. On January 5, 2017, then-Deputy Attorney General, Sally Yates attended an Oval Office meeting […]

Continue reading: BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations ...

The following article, Fed Judge Releases 21-Yr-Old Man To “Clean and Sober House” After Appearing In Court For Sexually Assaulting 12-Yr-Old Girl For One Month While Hiding In Her Bedroom, was first published on 100PercentFedUp.com.

The normalizing of pedophilia is not a far-right conspiracy theory...

Continue reading: Fed Judge Releases 21-Yr-Old Man To “Clean and Sober House” After Appearing In Court For Sexually Assaulting 12-Yr-Old Girl For One Month While Hiding In Her Bedroom ...

The following article, Busted! Late-Night Hack Comedian, Jimmy Kimmel Is Forced To Apologize For Sharing Highly Edited Video Of VP Pence To Make Him Look Bad, was first published on 100PercentFedUp.com.

Last night, Jimmy Kimmel, host of the low-rated, late-night Jimmy Kimmel Show, shared a deceptively edited video clip of Vice President Pence delivering PPE to a nursing home. Today, liberal activist Matt McDermott tweeted the videotaped segment on VP Pence that was edited to make the vice president look like he was faking a delivery […]

Continue reading: Busted! Late-Night Hack Comedian, Jimmy Kimmel Is Forced To Apologize For Sharing Highly Edited Video Of VP Pence To Make Him Look Bad ...

The following article, Rush Limbaugh Predicts Joe Biden Won’t Be The Dem Nominee: “Something’s Gonna Happen”, was first published on 100PercentFedUp.com.

Is Joe Biden going to become the Democrat nominee and run against Trump in the fall?

Continue reading: Rush Limbaugh Predicts Joe Biden Won’t Be The Dem Nominee: “Something’s Gonna Happen” ...

The following article, Obama Private Call Released: Implores Political Operatives to Help Protect Him…”We gotta make this happen”, was first published on 100PercentFedUp.com.

Michael Isikoff at Yahoo News on Friday night released audio of a call from former President Barack Obama to political operatives and the media to help protect “the rule of law” by protecting him. Obama desperately wants the Deep State and media to protect him by helping elect Joe Biden: “The fact that there is […]

Continue reading: Obama Private Call Released: Implores Political Operatives to Help Protect Him…”We gotta make this happen” ...

After being pulled over for what started as a traffic violation, two Windsor men were arrested and face multiple drug, property, and weapon related charges.

Manitoba’s unemployment rate nearly doubled between March and April, according to the monthly report from Statistics Canada released Friday morning.

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton–Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

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.

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo. Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC–treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 μm) and nitric oxide (100 μm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 μm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

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.

Chatham House is pleased to announce that Adam Ward will join the institute in a new role as deputy director.

Chatham House is pleased to announce that Tim Benton has joined the institute as a Distinguished Visiting Fellow in the Energy, Environment and Resources Department.

Chatham House is pleased to announce that Professor Tim Benton has been appointed as research director of the Energy, Environment and Resources Department.

Chatham House is pleased to announce that Rob Yates has been appointed as head of the Centre on Global Health Security.

Creon Butler has been appointed to lead the Global Economy and Finance programme at Chatham House, joining the institute at the beginning of December. He will also form part of the institute’s senior leadership team.

Invitation Only Research Event

Event participants

Diane Coyle, Professor, University of Manchester; Founder and Managing Director, Enlightenment Economics

Invitation Only Research Event

Event participants

Megan Greene, Managing Director and Chief Economist, Manulife Asset Management 

16 March 2020

Members Event Webinar

Event participants

Professor Johan Giesecke, MD, PhD, Professor Emeritus of Infectious Disease Epidemiology, Karolinska Institute Medical University, Stockholm; State Epidemiologist, Sweden (1995-05)
Professor David Heymann CBE, Distinguished Fellow, Global Health Programme, Chatham House; Executive Director, Communicable Diseases Cluster, World Health Organization (1998-03)
Chair: Emma Ross, Senior Consulting Fellow, Global Health Programme, Chatham House

Corporate Members Event Webinar

Event participants

Neil Walsh, Chief, Cybercrime and Anti-Money Laundering Department, UN Office of Drugs and Crime

Lisa Quest, Head, Public Sector, UK & Ireland, Oliver Wyman

Chair: Joyce Hakmeh, Senior Research Fellow, International Security Programme; Co-Editor, Journal of Cyber Policy, Chatham House

Further speakers to be announced.

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.

Viviane Baladi and Mark F. Demers
J. Amer. Math. Soc. 33 (2020), 381-449.
Abstract, references and article information

Alfred C. O. Vertegaal
Dec 1, 2006; 5:2298-2310
Research

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

Jonathan C. Trinidad
Aug 1, 2012; 11:215-229
Research

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

Claudio P. Albuquerque
Jul 1, 2008; 7:1389-1396
Research

Albrecht Gruhler
Mar 1, 2005; 4:310-327
Research

Ulrich Rothbauer
Feb 1, 2008; 7:282-289
Research

Jesper V. Olsen
Dec 1, 2005; 4:2010-2021
Technology

Shao-En Ong
May 1, 2002; 1:376-386
Research

The formation of translationally inactive 70S dimers (called 100S ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

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.

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.

In Staphylococcus aureus–caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1–325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1–246) as a probe for competitively defining the affinities of N-terminal SC(1–246) variants preselected by modeling. Using ProT(R155Q,R271Q,R284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ∼1 and 650 nm and activation potencies ranging from 1.8-fold that of WT SC(1–246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1–246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well-tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable with WT SC(1–246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of WT SC(1–246). The Ile16 pocket in ProTQQQ appears to favor nonpolar, nonaromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than WT Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser–His–Asp triad, proposed to serve a general acid–base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 μmol·kg−1 in brain to 1.4 μmol·kg−1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 μm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.

8 January 2020 , Volume 96, Number 1

Invitation Only Research Event