With a bright, 8.2-inch HD display and the flexibility to work in almost any vehicle, the Lanmodo Automotive Vast Night Vision System is a near-perfect alternative to factory-installed systems.

The post Brighten Up Your After-Dark Road-Tripping with Lanmodo’s Vast Automotive Night Vision System appeared first on Vagabondish.

Perhaps you've read somewhere or other that there are over 3,000 traditional Peruvian festivals held throughout the year? It's a popular claim that pops up time and again in guidebooks and on websites, and though we ourselves haven't verified the number, it certainly wouldn't surprise us! Peru is a country that holds onto its traditions while increasingly embracing innovation as well, a perfect storm for festivals and fiestas of all types to flourish and to grow.

If you're planning to visit Peru, you might find yourself intrigued by the prospect of observing, or even participating in, one of these many different Peruvian festivals. If that's the case, you're in luck--we've taken the liberty to compile a list of some of our favorite Peruvian festivals, including one for each month of the year in hopes that you'll find something that corresponds with the dates of your trip. Keep in mind that this is very much just the tip of the iceberg--all of these Peruvian festivals are fantastic, but there are many more that we've had to leave out simply due to the nature of our list. If we haven't included any options that work out with your travel itinerary, don't fret--get to researching and we're sure you'll find the festival for you.

So without further ado, let's get to it! Here is our list of twelve awesome Peruvian festivals, one for each month of the year:

1. January--Trujillo Marinera Festival

Trujillo, a coastal city in northern Peru, is the traditional home of the marinera, an elegant couple's dance making use of handkerchiefs as props. Since 1986 it's been the official national capital of this dance, a status celebrated annually in a month-long marinera festival held throughout the city! The Festival de Marinera, as it's known in Spanish, features dance competitions, parades, and even exhibitions of the Peruvian Paso, a breed of horse also recognized as part of the cultural patrimony of the Trujillo region. One of our favorite Peruvian festivals, this one is a can't miss if you find yourself in the area during January.

2. February--Fiesta de la Candelaria

The Fiesta de la Candelaria is not only the largest and most famous festival in Peru, but also one of the largest in South America as a whole--in fact, throughout the continent, it's only dwarfed by the world-famous Carnival in Rio de Janeiro and the Carnaval de Ouro in Bolivia. The first of many religious Peruvian festivals on our list, the Fiesta de la Candelaria celebrates the Virgin of Candelaria, the patron saint of the town of Puno where the festivities are held. Music and dance are at the core of this festival, all of it performed by elaborately-costumed participants numbering well over 40,000. An incredible two week-long synthesis of indigenous and Catholic traditions, this is one of the most iconic Peruvian festivals.

3. March--Festival Internacional de la Vendimia

Depending upon where Easter falls during a given year, the month of March can either be very busy or very quiet as far as major Peruvian festivals are concerned. That said, we figured we may as well take a break from the religious festivals to focus on something else with a different sort of appeal--wine! The Ica Region of Peru is famous for a number of things, the Nazca Lines and the desert oasis of La Huacachina among them, but those in-the-know also recognize it as the finest wine-producing region in the country. This role has been celebrated annually in March since the 1950s during the Festival Internacional de la Vendimia, or the International Harvest Festival, in which a queen is famously chosen to ceremoniously stomp the first grapes of the season.

 4. April--Festividad del Señor del los Temblores del Cusco

El Señor de los Temblores, or the Lord of the Earthquakes, is the patron saint of Cusco and the celebratory focus of this major Holy Week celebration. The story goes that during an earthquake here in 1650, a canvas image of Jesus Christ was held to the sky as prayers were offered, causing the tremors to subside and saving the town from ruin. Since then, this statue commemorating the original image has become the focal point of an important annual celebration. Each Easter Monday at 7 PM, the statue is removed from Cusco's cathedral and paraded throughout the city. Amazingly, it's said that the statue was not originally black, but rather that its color is due to years of exposure to smoke from incense during religious ceremonies.

5. May--Fiesta de las Cruces

First of all, let's be clear: the Fiesta de las Cruces, or the Festival of the Crosses, is not a uniquely Peruvian celebration. However, the way the Peruvians celebrate it does make it one of the most important Peruvian festivals. You see, the Festival of the Crosses is not held in just one spot in the country. Rather, the festival is celebrated in towns and cities throughout Peru, each putting their own unique spin on things. Though the name may conjure up solemn images, don't worry--festivals throughout the Peruvian highlands include music, dancing, and even events such as bullfighting as part of the celebrations.

6. June--Inti Raymi

Inti Raymi, the ancient Inca Festival of the Sun, is one of the most traditional Peruvian festivals on our list, indigenous in design and untouched by Catholic tradition. Held annually on June 24th, Inti Raymi is meant to celebrate the Winter Solstice--remember that we're in the Southern Hemisphere here! Banned by the Spanish and the Catholic Church after 1535, the modern incarnation of the celebration began in 1944 based on the few historical records of the festivities that managed to survive. Since then it has grown in size and scope, and though sometimes derided as tourist pageantry by detractors, its historical and cultural significance definitely make it worth checking out among the many Peruvian festivals.

7. July--Fiesta de la Virgen del Carmen

Another fine example of traditional Andean culture blended with Catholicism, the Fiesta de la Virgen del Carmen is held annually around the 16th of July in the relatively small town of Paucartambo, some four hours outside of Cusco. This three or four day festival is nominally religious but also one of the biggest and wildest parties in Peru, drawing visitors from all over the country, many of whom pass the nights sleeping under the stars as there's simply not enough space in town! If you know what you're looking for, you'll actually notice some serious Peruvian history depicted in the festival's songs, costumes, and dance--for example, black-masked dancers represent African slaves imported to work the silver mines here during the colonial era.

8. August--Día de Santa Rosa de Lima

Santa Rosa de Lima was the first native-born American saint canonized by the Catholic Church, and her legacy is celebrated throughout the world. However, it should come as no surprise that the grandest festivities take place in her home country of Peru. On August 30th, the anniversary of her death, celebrations and memorials take place throughout the country, the most famous being in Santa Rosa de Quives just outside of the capital city itself.

9. September--Mistura Culinary Festival

As you should know by now, Peruvian cuisine is some of the world's best and Lima in particular features what is probably South America's most impressive culinary scene. And as if the food wasn't enticing enough year round, the prospect of eating your way through Peru becomes even more appealing each September when the Mistura Culinary Festival rolls around. Featuring over 200 restaurants and bars not to mention plenty of outdoor vendors and food carts, well over half a million visitors stop by to enjoy the festival's offerings over the course of some ten days in mid-September. Though certainly not one of the most traditional Peruvian festivals on our list, this is without a doubt the most delicious.

10. October--Procesión del Señor de los Milagros

The story might sound a bit familiar--an 18th Century earthquake destroyed a good part of town leaving only this iconic image of Jesus Christ standing. This was considered a miracle, the image became even more venerated, and with time the celebration of its feast day became the largest religious celebration in Peru featuring one of the longest processions of any religious gathering in the world. If you're just looking to have fun, this one probably isn't for you--however, this considerably more solemn celebration is one of the most important Peruvian festivals and we couldn't leave it off our list.

11. November--Puno Week

Perhaps the name Puno rings a bell? If so, you've got a good memory--this is the same city where our February festival choice of the Fiesta de la Candelaria took place! Puno Week takes place during the beginning of November and centers around November 5th, also known as Puno Day. The purpose of Puno Day, and by extension Puno Week, is to celebrate the legendary birth of one Manco Cápac, said to be the first Inca. This involves a very interesting reenactment of his arrival on the shores of Lake Titicaca bordering the city--and it's also just a great excuse to party the whole week long.

12. December--Santuranticuy

As we wind down our list, a few things should by now be obvious: the first is that Peruvians love their festivals, and the second is that many of these Peruvian festivals feature religious--and here that means Catholic--themes. The final entry on our list is of course no exception--Santuranticuy is held exclusively in the city of Cusco each year on Christmas Eve, December 24th, and is essentially a massive outdoor marketplace set up to celebrate the holiday. The festival's name, which means "sale of saints," is quite appropriate, as the primary draw here are dolls, sculptures, and figurines used to decorate the nacimientos, or Catholic nativity scenes, found in many Peruvian homes during the Christmas season. However, even if you aren't religious, you're sure to find something of interest at this most shopper-friendly of Peruvian festivals.

Planning a little solo jaunt across the land? If this is your first time planning your virgin solo adventure, I understand completely how excited and nervous you are. I was once in your shoes.

Before you slap on your backpack and march out that door, here’s a small checklist of things you might want to do to ensure a better trip.

Research, Research, Research

It doesn’t matter if you’re headed into uncharted territory or a bustling metropolis, it only helps if you already know what to expect when you set foot in your destination. A quick google search should give you enough information on things like the local culture, geography, weather and political climate to start with. Oh, and do not forget to check out the local laws, either-written and unwritten. People have in the past gotten into trouble for using the wrong gesture or for looking the wrong person in the eye. Did you also know that shaking your head can be interpreted as ‘yes’ in India?

“Hi, would you like to buy some drugs?

– Nervously shakes head –

“Great! Here’s two kilograms of cocaine!”

Kuh-Myoo-Nick-Ashion

Learn a few words and phrases from the local language and the appropriate situations to use them in. You would be surprised at how much a ‘Yallah’ or a ‘Habibi’ can get people to warm up to you in the Middle East. Also, knowing the language makes ordering food that much easier. Just saying.

Who Ya’ Gonna Call?

It’s very useful to have in hand a bunch of contacts that you can get in touch with if-knock on wood- you should ever find yourself in trouble, eg: Your home country’s Embassy/Consulate(stolen passports are a thing in some countries you know?), local emergency numbers etc. If you have friends or relatives in the country/area, make sure you have their numbers as well. There’s no telling what kind of emergency you might encounter on the road.

Link Up With Other Travellers And Locals!

This is for many people, the single greatest reward of solo travel-meeting new people. There’s so much you discover from engaging with people from different cultures and backgrounds. Yes, I’m being captain obvious here, but it’s a point that just had to be reiterated.  Being alive in the internet age has its perks.

A slew of apps and websites have made it that much easier for travelers to get in touch with people at their destinations. It doesn’t matter if you’re just hanging out with your host from ‘AirBnB’ and ‘Couchsurfing’ or finding fellow travelers to share in your adventure from ‘Penroads’, you’re guaranteed to meet some cool people on your journey. As a matter of fact, Penroads is the best way for you to connect with international travellers coming in from all over the world, so it’s definitely a useful tool to have (It’s a shameless plug. I am so sorry. But it really works!).

Stay Healthy!

Last but never the least, take care of your body. Stay hydrated. It’s no good if your mind yells “Onward!” and your body groans “Hell no!”. Find out if you need to get vaccinated before setting off. Carry some basic medicines and bandages if you can, especially if you’re venturing into the wilderness. On occasion you might have to be careful about where and what you eat as well. Talk to someone who has travelled to India and you just might hear the story about how the delicious chicken Tikka from a street vendor gave them a bad case of ‘Delhi Belly’.

Happy tripping!

Parag Murali is the Marketing & Happiness Manager at penroads.com. He combines his love for travelling and people into a daily passion for bringing travellers together. You can contact him at parag@penroads.com for just about anything, so feel free!

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

In this livesteam, David and I answer audience questions about how to work remotely. At Basecamp we’ve been working remotely for nearly 20 years, so we have a lot of experience to share. This nearly 2-hour video goes into great detail on a wide variety of topics. Highly recommended if you’re trying to figure out… keep reading

For many, moving from everyone’s-working-from-the-office to everyone’s-working-at-home isn’t so much a transition as it is a scramble. A very how the fuck? moment. That’s natural. And people need time to figure it out. So if you’re in a leadership position, bake in time. You can’t expect people to hit the ground running when everything’s different.… 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

Part of the Food for thought promo for the BBC UK Homepage

Part of the Sport Relief promo for the BBC UK Homepage

The DML News App offers the best in news reporting.

The post JUST IN: Actress and leader of #MeToo movement accuses Bill Maher of sexually harassing her appeared first on Dennis Michael Lynch.

The province is investing $1.5 million to support a housing project in Muskoka to help people with no place to call home.

The calendar may say May, but that didn't stop Mother Nature from blasting some wintry weather in Muskoka on Friday.

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

The following article, BREAKING: CBS News Allegedly Staged Fake COVID19 Testing In MI…Makes Dem Gov Whitmer Look Like She’s Doing More To Help Citizens Than Fed Government [VIDEO], was first published on 100PercentFedUp.com.

Project Veritas founder James O’Keefe has done it again. He’s uncovered yet another deceitful piece of coverage on the Wuhan coronavirus pandemic in America by CBS News. O’Keefe begins his interview with a man whose identity is being hidden, “You’re telling me—you’re 100% certain, that CBS News, CBS News Corporation national—staged a fake event. They […]

Continue reading: BREAKING: CBS News Allegedly Staged Fake COVID19 Testing In MI…Makes Dem Gov Whitmer Look Like She’s Doing More To Help Citizens Than Fed Government [VIDEO] ...

The following article, 77-Yr-Old MI Barber Defies Gov Whitmer’s Shutdown Order: ”I don’t need another mother” Shop Will Stay Open Until “Jesus walks in or until they arrest me” [VIDEO], was first published on 100PercentFedUp.com.

Emergency orders that “non-essential businesses” remain closed during the Covid-19 crisis have devastated small business owners across America. A wave of business owners across the nation are defying executive orders by their governors and opening their businesses anyhow. Yesterday, we reported about a small business owner in the state of Michigan, where Governor Gretchen Whitmer […]

Continue reading: 77-Yr-Old MI Barber Defies Gov Whitmer’s Shutdown Order: ”I don’t need another mother” Shop Will Stay Open Until “Jesus walks in or until they arrest me” [VIDEO] ...

The following article, ‘Schiff in Panic Mode’: Sources Say Russia Probe Transcripts Affirm Officials Found Nothing on Collusion, was first published on 100PercentFedUp.com.

Transcripts of House Intelligence Committee interviews have been cleared for release, and Adam Schiff is reportedly in panic mode. Unfortunately for Schiff, top law enforcement and intel officials said they saw no evidence of collusion between the Trump campaign and Russia in the transcripts. Republicans sought and got approval for the release of the transcripts […]

Continue reading: ‘Schiff in Panic Mode’: Sources Say Russia Probe Transcripts Affirm Officials Found Nothing on Collusion ...

The following article, MSNBC’s Brian Williams Chuckles With Dem Strategist as He Gloats, Mocks Trump About Tragic Downturn in Economy: “They were going to lose before this hit. They’re just going to lose worse now”, was first published on 100PercentFedUp.com.

James Carville spoke out before the coronavirus crisis to say that there is no way  Joe Biden has a chance at beating President Trump in the 2020 election. Well, He’s singing a different tune now at the expense of Americans suffering through this horrible pandemic and economic crisis. James Carvill is a Democratic strategist who […]

Continue reading: MSNBC’s Brian Williams Chuckles With Dem Strategist as He Gloats, Mocks Trump About Tragic Downturn in Economy: “They were going to lose before this hit. They’re just going to lose worse now” ...

The following article, Mother of the Year, Michelle Obama Explains How Having Kids Was A “Concession”… Had To Give Up Her “Aspirations and Dreams” [VIDEO], was first published on 100PercentFedUp.com.

While campaigning for her community organizer turned presidential candidate husband, Barack, Michelle Obama told a crowd of his supporters in Milwaukee, Wisconsin that for the first time in her life, she was proud to be an American. Four years later, Michelle Obama was a keynote speaker at the DNC convention, where she told Democrats how […]

Continue reading: Mother of the Year, Michelle Obama Explains How Having Kids Was A “Concession”… Had To Give Up Her “Aspirations and Dreams” [VIDEO] ...

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 ...

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

A beverage company in the U.S. is inviting moms to let out a ‘quaranscream’ this Mother’s Day on a designated ‘zero-judgement venting hotline’ for a chance to win its wine spritzers.

Previous studies have shown that sphingosine kinase interacting protein (SKIP) inhibits sphingosine kinase (SK) function in fibroblasts. SK phosphorylates sphingosine producing the potent signaling molecule sphingosine-1-phosphate (S1P). SKIP gene (SPHKAP) expression is silenced by hypermethylation of its promoter in acute myeloid leukemia (AML). However, why SKIP activity is silenced in primary AML cells is unclear. Here, we investigated the consequences of SKIP down-regulation in AML primary cells and the effects of SKIP re-expression in leukemic cell lines. Using targeted ultra-HPLC-tandem MS (UPLC-MS/MS), we measured sphingolipids (including S1P and ceramides) in AML and control cells. Primary AML cells had significantly lower SK activity and intracellular S1P concentrations than control cells, and SKIP-transfected leukemia cell lines exhibited increased SK activity. These findings show that SKIP re-expression enhances SK activity in leukemia cells. Furthermore, other bioactive sphingolipids such as ceramide were also down-regulated in primary AML cells. Of note, SKIP re-expression in leukemia cells increased ceramide levels 2-fold, inactivated the key signaling protein extracellular signal-regulated kinase, and increased apoptosis following serum deprivation or chemotherapy. These results indicate that SKIP down-regulation in AML reduces SK activity and ceramide levels, an effect that ultimately inhibits apoptosis in leukemia cells. The findings of our study contrast with previous results indicating that SKIP inhibits SK function in fibroblasts and therefore challenge the notion that SKIP always inhibits SK activity.

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.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

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 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.

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.

Tau is a microtubule-associated protein that plays a major role in Alzheimer's disease (AD) and other tauopathies. Recent reports indicate that, in the presence of crowding agents, tau can undergo liquid–liquid phase separation (LLPS), forming highly dynamic liquid droplets. Here, using recombinantly expressed proteins, turbidimetry, fluorescence microscopy imaging, and fluorescence recovery after photobleaching (FRAP) assays, we show that the divalent transition metal zinc strongly promotes this process, shifting the equilibrium phase boundary to lower protein or crowding agent concentrations. We observed no tau LLPS-promoting effect for any other divalent transition metal ions tested, including Mn2+, Fe2+, Co2+, Ni2+, and Cu2+. We also demonstrate that multiple zinc-binding sites on tau are involved in the LLPS-promoting effect and provide insights into the mechanism of this process. Zinc concentration is highly elevated in AD brains, and this metal ion is believed to be an important player in the pathogenesis of this disease. Thus, the present findings bring a new dimension to understanding the relationship between zinc homeostasis and the pathogenic process in AD and related neurodegenerative disorders.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Cone photoreceptors in the retina enable vision over a wide range of light intensities. However, the processes enabling cone vision in bright light (i.e. photopic vision) are not adequately understood. Chromophore regeneration of cone photopigments may require the retinal pigment epithelium (RPE) and/or retinal Müller glia. In the RPE, isomerization of all-trans-retinyl esters to 11-cis-retinol is mediated by the retinoid isomerohydrolase Rpe65. A putative alternative retinoid isomerase, dihydroceramide desaturase-1 (DES1), is expressed in RPE and Müller cells. The retinol-isomerase activities of Rpe65 and Des1 are inhibited by emixustat and fenretinide, respectively. Here, we tested the effects of these visual cycle inhibitors on immediate, early, and late phases of cone photopic vision. In zebrafish larvae raised under cyclic light conditions, fenretinide impaired late cone photopic vision, while the emixustat-treated zebrafish unexpectedly had normal vision. In contrast, emixustat-treated larvae raised under extensive dark-adaptation displayed significantly attenuated immediate photopic vision concomitant with significantly reduced 11-cis-retinaldehyde (11cRAL). Following 30 min of light, early photopic vision was recovered, despite 11cRAL levels remaining significantly reduced. Defects in immediate cone photopic vision were rescued in emixustat- or fenretinide-treated larvae following exogenous 9-cis-retinaldehyde supplementation. Genetic knockout of Des1 (degs1) or retinaldehyde-binding protein 1b (rlbp1b) did not eliminate photopic vision in zebrafish. Our findings define molecular and temporal requirements of the nonphotopic or photopic visual cycles for mediating vision in bright light.

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.

Manganese (Mn) is an essential micronutrient required for the normal development of many organs, including the brain. Although its roles as a cofactor in several enzymes and in maintaining optimal physiology are well-known, the overall biological functions of Mn are rather poorly understood. Alterations in body Mn status are associated with altered neuronal physiology and cognition in humans, and either overexposure or (more rarely) insufficiency can cause neurological dysfunction. The resultant balancing act can be viewed as a hormetic U-shaped relationship for biological Mn status and optimal brain health, with changes in the brain leading to physiological effects throughout the body and vice versa. This review discusses Mn homeostasis, biomarkers, molecular mechanisms of cellular transport, and neuropathological changes associated with disruptions of Mn homeostasis, especially in its excess, and identifies gaps in our understanding of the molecular and biochemical mechanisms underlying Mn homeostasis and neurotoxicity.

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.

26 February 2020

Over the past two decades, China has increased its presence in North Africa in terms of trade and investment. This paper looks at China’s policy within the context of its Africa and Middle East policies to better understand its approach to Morocco and Tunisia.

Webinar Research Event

Event participants

Mohammed Masbah, Director, Moroccan Institute for Policy Analysis; Associate Fellow, MENA Programme, Chatham House
Moderator: Lina Khatib, Director, MENA Programme, Chatham House

6 April 2020

29 April 2020

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

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.

US Secretary of State John Kerry and Iranian Minister of Foreign Affairs Dr Mohammad Javad Zarif have been voted as the winners of this year’s Chatham House Prize.

The Moving Energy Initiative starts four new clean energy projects for refugees.

Our project on Democracy and Technology in Europe is now entering its final phase. Now we want your help in shaping the final report.