People are always curious about work-from-home (WFH), remote working setups. So, I posted a Basecamp message asking our employees to share a photo of their home office, desk, table, whatever. Here’s what came in. First, the ask: And the answers, in the order they came in: Andy Didorosi, Marketing Justin White, Programmer Jonas Downey, Designer… keep reading

David and I spent nearly 2-hours giving a livestream tour of our very own Basecamp account. We wanted to show you how Basecamp uses Basecamp to run projects, communicate internally, share announcements, know what everyone’s working on, build software, keep up socially, and a whole bunch more. Our entire company runs on Basecamp, and this… keep reading

The vast majority of web applications should start life as a Majestic Monolith: A single codebase that does everything the application needs to do. This is in contrast to a constellation of services, whether micro or macro, that tries to carve up the application into little islands each doing a piece of the overall work.… keep reading

We’ve been teaching people how to do remote work well for the better part of two decades. We wrote a whole book about the topic in 2013, called REMOTE: Office Not Required. Basecamp has been a remote company since day one, and our software is sold as an all-in-one toolkit for remote work. Yeah, we’re… keep reading

Part of the Going for gold promo for the BBC UK Homepage

Part of the Going for gold promo for the BBC UK Homepage

Part of the Going for gold promo for the BBC UK Homepage

Part of the Antarctic diary promo for the BBC UK Homepage

Part of the Antarctic diary promo for the BBC UK Homepage

In a classic bit from an early Seinfeld, Jerry and Elaine are at the airport, trying to pick up the rental car that Jerry had reserved. As usual, things go poorly and get awkward fast:

Seinfeld - "Reservations"

JERRY: I don't understand...I made a reservation. Do you have my reservation?
AGENT: Yes, we do. Unfortunately, we ran out of cars.
JERRY: But, the reservation keeps the car here. That's why you have the reservation.
AGENT: I know why we have reservations.
JERRY: I don't think you do. If you did, I'd have a car. See, you know how to take the reservation--you just don't know how to hold the reservation. And, that's really the most important part of the reservation...the holding. Anybody can just TAKE them. [grabs chaotically at air]

And, how weirdly similar is that to our conflicted relationship with New Year's resolutions?

In Seinfeldspeak?

See, you know how to make the resolution, you just don't know how to keep the resolution. And, that's really the most important part of the resolution...the keeping. Anybody can just MAKE them!

Oversimplified? Probably.

But, ask yourself. Why this? And, why now? Or, why again?

Welcome to Resolvers Anonymous: I'm 'Merlin M.'

A few years ago, I shared a handful of stories on the failures that have led to my own cynicism about the usefulness of life-inverting resolutions. Because, yeah, I've historically been a big resolver.

Here's what I said when I first suggested favoring "Fresh Starts and Modest Changes" over reinventions:

Download MP3 of "Fresh Starts & Modest Changes"

Five years on, I think I probably feel even more strongly about this.

Partly because I've watched and read and heard the cyclical lamentations of folks who decided to use superficial totems (like new calendars) as an ad hoc coach and prime mover. And, partly because, in my capacity as a makebelieve productivity expert, I continue to see how self-defeating it is to pretend that past can ever be less than prologue--that we can each ignore yesterday's weather if we really wish hard enough for a sun-drenched day at the beach.

It simply doesn't work.

Companies that think they'll be Google for buying bagels. Writers who think they'll get published if they order a new pen. Obese people who think they'll become marathon runners if they pick up some new running shoes. And, regular old people with good hearts who continue to confuse new lives with new clothes.

Has this worked before? Can you look back on a proud legacy of successful New Year's resolutions that would suggest you're making serious progress by repeatedly making a list about fundamental life changes while slamming prosecco and wearing a pointy paper hat?

My bet is that most people who are seeing the kind of change and growth and improvement that sticks tend to avoid these sorts of dramatic, geometric attempts to leap blindly toward the mountain of perfection.

I'll go further and say that the repeated compulsion to resolve and resolve and resolve is actually a terrific marker that you're not really ready to change anything in a grownup and sustainable way. You probably just want another magic wand.

Otherwise you'd already be doing the things you've resolved to do. You'd already be living those changes. And, you'd already be seeing actual improvements rather than repeatedly making lists of all the ways you hope your annual hajj to the self-improvement genie will fix you.

Then, of course, we make things way worse by blaming everything on our pancakes.

Regarding "The First Pancake Problem"

Anyone who's ever made America's favorite round and flat breakfast food is familiar with the phenomenon of The First Pancake.

No matter how good a cook you are, and no matter how hard you try, the first pancake of the batch always sucks.

It comes out burnt or undercooked or weirdly shaped or just oddly inedible and aesthetically displeasing. Just ask your kids.

At least compared to your normal pancake--and definitely compared to the far superior second and subsequent pancakes that make the cut and get promoted to the pile destined for the breakfast table--the first one's always a disaster.

I'll leave it to the physicists and foodies in the gallery to develop a unified field theory on exactly why our pancake problem crops up with such unerring dependability. But I will share an orthogonal theory: you will be a way happier and more successful cook if you just accept that your first pancake is and always will be a universally flukey mess.

But, that shouldn't mean you never make another pancake.

So Loud. Then, So Quiet.

I offer all of this because today is January 7th, gang. And, for the past week, all over the web, legions of well-intentioned and seemingly strong-willed humans have been declaring their resolved intention to make this a year of more and better metaphorical pancakes.

And, like clockwork--usually around today or maybe tomorrow--a huge cohort of those cooks will begin to abandon their resolve and go back to thinking all their pancakes have to suck. Just because that first one failed.

And, as is the case every year, online and off, there won't be nearly as many breathless updates to properly bookend how poorly our annual ritual of aspirational change has fared. Which is instructive.

Not because new year's resolutions are a universally bad idea. And, not because Change is Bad. And, not because we should be embarrassed about occasionally falling short of our own (frequently unreasonable) aspirations.

I suspect we tout the resolution, but whisper the failure because we blame the cook. Or, worse, fingers point toward the pancake. Instead of just admitting that the resolution itself was simply unrealistic or fundamentally foreign.

And, that's a shame.

Remember, there's no "I" in "unreasonable"

Granted, I'm merely re-repeating a point I've struggled to make (to both others and myself) for years now. But, it will bear repeating every January in perpetuity.

Resist the urge to pin the fate of things you really care about to anything that's not truly yourself. The "yourself" who has a real life with complicated demands. The "yourself" who's going to face a hard slog trying to fold a new life out of a fresh calendar.

Calendars are just paper and staples. They can't make you care. And they can't help you spin around like Diana Prince, and instantly turn into Wonder Woman. Especially, if you're not already a hot and magical Amazon princess.

First, be reasonable. Don't set yourself up for failure by demanding things that you've never come close to achieving before. I realize this is antithetical to most self-improvement bullshit, but that's exactly the point. If you were already a viking, you wouldn't need to build a big boat. Start with where you are right now. Not with where you wish you'd been.

Also, accept that the first pancake will always suck. Hell, if you've never picked up a spatula before, be cool with the fact that your first hundred pancakes might suck. This is, as I've said, huge. Failure is the sound of beginning to suck a little less.

And, finally, also be clear about the sanity of the motivations underlying your expectations--step back to observe what's truly broken, derive a picture of incremental success that seems do-able, and really resolve to do whatever you can realistically do to actually get better. Rather than "something something I suddenly become all different."

At this point, you have logistical options for both execution and troubleshooting:

• Make a modest plan that you can envision actually doing without upending your real life;
• Build more sturdy scaffolding for sticking with whatever plan you've chosen;
• Make a practice of learning to not mind the duds--including those messed-up first pancakes;
• Or--seriously?--just accept that you never really cared that much about making breakfast in the first place. Care is not optional.

Otherwise, really, you'd never need to resolve to do anything. You'd already just be cooking a lot. Instead of being all mad and depressed about not cooking.

But, please. All I really ask of you. Don't blame the pancake. It's not really the pancake's fault.

Like me, the pancake just wants you to be happy. This and every other new year.

Merlin Mann - "Scared Shitless: How I (Mostly) Learned to Love Being Afraid of Pretty Much Everything"

Download MP4 Video of "Scared Shitless"

This is the video of a talk I did last month at Webstock in Wellington, New Zealand.

It's pretty different from a lot of stuff I've done. It's about being scared.

As I mentioned on Back to Work, Webstock is—what? Well. Webstock is unique. Truly. If you get the chance, you should go. Really.

I could not and would not have done this talk in this way had I had not been so inspired (and, frankly, so terrified) by the awesomeness of the other speakers, by the quality of their talks, and by the astounding graciousness and empathy of the audience that this particular event attracts.

Tash and Mike and their crackerjack team have made something really special here. I'm honored that they even invited me, and I'm insanely grateful for the care and hospitality that they showed to the speakers and to the attendees at every step of the way.

Seriously. Thank you.

So, yeah. I did something really weird at Webstock. Weird for me and, honestly, just plain weird for "a talk."

I'm not sure if it succeeded. But, I did the best I could to make myself (along with some really heroic friends and fellow speakers) into a legitimate guinea pig for a concept that means the world to me:

You can be scared and still do it anyway. Regardless of whatever it is.

And, you can. No. Really. You. You can do this.

You can run toward the shitstorm, let it cover you with shit, but, still never let it stop you from running.

Because, like Crazy Bob says:

"They can't eat you."

And, they can't. And, they won't. Okay?

Well, okay, then.

The DML News App offers the best in news reporting.

The post BREAKING: Founding Father of Rock Who Broke Musical Barriers, Dead at 87 appeared first on Dennis Michael Lynch.

The DML News App offers the best in news reporting.

The post REPORT: Obama rips Trump over coronavirus in private call appeared first on Dennis Michael Lynch.

Human remains located in Mulmur Township in March have been confirmed to be those of an 88-year-old Caledon man who was reported missing seven months ago.

While infection rates remain steady across the region, the Simcoe Muskoka District Health Unit is reporting more than half of all 360 cases have now recovered.

Nurses, PSWs, and front-line workers in long-term care and retirement homes have been dealing with the full reality of this pandemic every day.

With the eviction date looming for campers to leave Oppenheimer Park, B.C.'s social development minister says everyone is still expected to be out of the park by Saturday.

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.

B.C. recorded 29 more cases of COVID-19 and one more death over the last 24 hours, provincial health officials announced Friday.

T.J. Lovell had just 30 minutes to pack up his belongings from the tent city in Oppenheimer Park if he wanted access to a hotel room that he could share with his father.

The following article, Donald Trump Jr. Offers To Walk Bride-to-Be Down The Aisle After Liberal Parents Refuse To Attend Her Wedding Because Fiance Is A Conservative, was first published on 100PercentFedUp.com.

TDS or Trump Derangement Syndrome brings out the worst in a lot of people. One young Texan girl’s story of her intolerant parents who refuse to attend her wedding because she’s marrying a conservative, however, is a new low. Lawyer and conservative civil rights activist, Rogan O’Handley tweeted a Tik Tok video of a Texan […]

Continue reading: Donald Trump Jr. Offers To Walk Bride-to-Be Down The Aisle After Liberal Parents Refuse To Attend Her Wedding Because Fiance Is A Conservative ...

The following article, Breaking: Texas AG Calls for Immediate Release of Salon Owner Jailed for Opening…Texas Lt Gov Offers to Pay Salon Owner’s $7k Fine, was first published on 100PercentFedUp.com.

Salon A La Mode owner Shelley Luther opened her salon in defiance of the lockdown order for salons to stay closed during the coronavirus crisis. She boldly ripped. up the citation she was given by the police and refused to shut her doors. Luther hired a lawyer and went to court where an activist judge […]

Continue reading: Breaking: Texas AG Calls for Immediate Release of Salon Owner Jailed for Opening…Texas Lt Gov Offers to Pay Salon Owner’s $7k Fine ...

The following article, Dem Congresswoman: Biden Allegation ‘Put To Rest’ Because He ‘Went on TV’ And Denied It, was first published on 100PercentFedUp.com.

Democrat Rep. Dina Titus says that the allegations against 2020 Democrat presidential candidate Joe Biden are “put to rest” because he “went on TV” and denied them. Rep. Titus spoke with MSNBC’s Ayman Mohyeldin today about how she believes Biden because he flatly said he didn’t do it and didn’t say he doesn’t remember. It’s […]

Continue reading: Dem Congresswoman: Biden Allegation ‘Put To Rest’ Because He ‘Went on TV’ And Denied It ...

The following article, Chicago Inmate Switches Identities While Wearing Mask…Is Released from Jail, was first published on 100PercentFedUp.com.

A Cook County, Chicago inmate, was able to switch identities with another inmate allowing the wrong inmate to be released. How did they carry that off without anyone catching them? The inmate was wearing a mask when he was released, so it wasn’t as easy to identify him. Last Saturday, Quintin Henderson (pictured below), 28, […]

Continue reading: Chicago Inmate Switches Identities While Wearing Mask…Is Released from Jail ...

The following article, Breaking: Justice Department Dropping Michael Flynn Criminal Case, was first published on 100PercentFedUp.com.

Michael Flynn has finally been exonerated. The Associated Press is reporting exclusively that the Justice Department said it is dropping the criminal case against President Trump’s first national security adviser, Michael Flynn. In court documents being filed Thursday, the Justice Department said it is dropping the case “after a considered review of all the facts […]

Continue reading: Breaking: Justice Department Dropping Michael Flynn Criminal Case ...

The following article, BREAKING: Michigan’s Radical Governor Extends Lockdown AGAIN…Only One Day After Church Leaders and MI Lawmakers File Suit Against Her, was first published on 100PercentFedUp.com.

Michigan was the first state to see protests against draconian lockdown measures by an overreaching governor. On April 15, the Michigan Conservative Coalition organized an in-vehicle protest in Lansing, MI that drew an estimated 15,000 vehicles. Since that time, several protests by Michigan workers who are being devastated by the one-size-fits-all shut down of the […]

Continue reading: BREAKING: Michigan’s Radical Governor Extends Lockdown AGAIN…Only One Day After Church Leaders and MI Lawmakers File Suit Against Her ...

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” ...

In Lakeshore, it may be a little while longer before a retail cannabis store opens.

At a time when store front vacancies are growing thanks to COVID-19, a new retail cannabis store in Pillette Village is hoping to open soon.

The provincial government has announced it will support child care centres that have been closed since March with their fixed operating costs as the fight against COVID-19 continues.

One more person has died from the coronavirus in the Windsor-Essex region on Saturday and nine new cases have been reported by the health unit.

Mother's Day is Sunday, but with COVID-19, it's forcing many to change their plans.

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

10 October 2019

This paper examines the governance problems in the monetary system and global trade and regulation. It then explores whether issues have arisen because the US has given up its dominant role, and if so how these might be rectified.

Research Event

Event participants

Michael Gasiorek, Professor of Economics, University of Sussex; Director, Interanalysis; Fellow, UK Trade Policy Observatory, University of Sussex
Julia Magntorn Garrett, Research Officer, UK Trade Policy Observatory, University of Sussex
Prof Jim Rollo, Deputy Director, UK Trade Policy Observatory, University of Sussex; Associate Fellow, Global Economy and Finance Department, Chatham House
Nicolo Tamberi, Research Officer in the Economics of Brexit, University of Sussex
L. Alan Winters, Professor of Economics, Director, UK Trade Policy Observatory, University of Sussex

11 February 2020

Telomeres are specific nucleoprotein structures that are located at the ends of linear eukaryotic chromosomes and play crucial roles in genomic stability. Telomere DNA consists of simple repeats of a short G-rich sequence: TTAGGG in mammals and TTTAGGG in most plants. In recent years, the mammalian telomeric G-rich repeats have been shown to form G-quadruplex (G4) structures, which are crucial for modulating telomere functions. Surprisingly, even though plant telomeres are essential for plant growth, development, and environmental adaptions, only few reports exist on plant telomeric G4 DNA (pTG4). Here, using bulk and single-molecule assays, including CD spectroscopy, and single-molecule FRET approaches, we comprehensively characterized the structure and dynamics of a typical plant telomeric sequence, d[GGG(TTTAGGG)3]. We found that this sequence can fold into mixed G4s in potassium, including parallel and antiparallel structures. We also directly detected intermediate dynamic transitions, including G-hairpin, parallel G-triplex, and antiparallel G-triplex structures. Moreover, we observed that pTG4 is unfolded by the AtRecQ2 helicase but not by AtRecQ3. The results of our work shed light on our understanding about the existence, topological structures, stability, intermediates, unwinding, and functions of pTG4.

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

The Mycobacterium tuberculosis virulence factor EsxA and its chaperone EsxB are secreted as a heterodimer (EsxA:B) and are crucial for mycobacterial escape from phagosomes and cytosolic translocation. Current findings support the idea that for EsxA to interact with host membranes, EsxA must dissociate from EsxB at low pH. However, the molecular mechanism by which the EsxA:B heterodimer separates is not clear. In the present study, using liposome-leakage and cytotoxicity assays, LC-MS/MS–based proteomics, and CCF-4 FRET analysis, we obtained evidence that the Nα-acetylation of the Thr-2 residue on EsxA, a post-translational modification that is present in mycobacteria but absent in Escherichia coli, is required for the EsxA:B separation. Substitutions at Thr-2 that precluded Nα-acetylation inhibited the heterodimer separation and hence prevented EsxA from interacting with the host membrane, resulting in attenuated mycobacterial cytosolic translocation and virulence. Molecular dynamics simulations revealed that at low pH, the Nα-acetylated Thr-2 makes direct and frequent “bind-and-release” contacts with EsxB, which generates a force that pulls EsxB away from EsxA. In summary, our findings provide evidence that the Nα-acetylation at Thr-2 of EsxA facilitates dissociation of the EsxA:B heterodimer required for EsxA membrane permeabilization and mycobacterial cytosolic translocation and virulence.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

Bacterial type VII secretion systems secrete a wide range of extracellular proteins that play important roles in bacterial viability and in interactions of pathogenic mycobacteria with their hosts. Mycobacterial type VII secretion systems consist of five subtypes, ESX-1–5, and have four substrate classes, namely, Esx, PE, PPE, and Esp proteins. At least some of these substrates are secreted as heterodimers. Each ESX system mediates the secretion of a specific set of Esx, PE, and PPE proteins, raising the question of how these substrates are recognized in a system-specific fashion. For the PE/PPE heterodimers, it has been shown that they interact with their cognate EspG chaperone and that this chaperone determines the designated secretion pathway. However, both structural and pulldown analyses have suggested that EspG cannot interact with the Esx proteins. Therefore, the determining factor for system specificity of the Esx proteins remains unknown. Here, we investigated the secretion specificity of the ESX-1 substrate pair EsxB_1/EsxA_1 in Mycobacterium marinum. Although this substrate pair was hardly secreted when homologously expressed, it was secreted when co-expressed together with the PE35/PPE68_1 pair, indicating that this pair could stimulate secretion of the EsxB_1/EsxA_1 pair. Surprisingly, co-expression of EsxB_1/EsxA_1 with a modified PE35/PPE68_1 version that carried the EspG5 chaperone-binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in redirection and co-secretion of the Esx pair via ESX-5. Our results suggest a secretion model in which PE35/PPE68_1 determines the system-specific secretion of EsxB_1/EsxA_1.

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.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

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.

Invitation Only Research Event

Event participants

Chair: Dr Lina Khatib, Director, Middle East and North Africa Programme, Chatham House

21 January 2020

Discussions of North African integration have evoked ideas of a shared identity and a common destiny in the region. However, recent attempts to build regional blocs in North Africa have been unsuccessful. This paper examines the benefits of a ‘synergistic’ approach to North African cooperation.