Hear about travel to Chennai India as the Amateur Traveler talks to Amanda from millennialtraveller.com about this city in southern India. 

Hear about travel to Saint Lucia as the Amateur Traveler talks to Keiwa Simpson from discoverstluciatours.com about her beautiful island.

Hear about travel to Lombok in Indonesia as the Amateur Traveler talks to Jason Andrew Jenkins from AnEpicEducation.com about his family's recent 5 week trip to this island paradise.

Hear about travel to Namibia as the Amateur Traveler talks to Karthik Iyer about his most recent trip to this desert country.

Hear about travel to Bulgaria as the Amateur Traveler talks to Tom and Patty Burkett about their road trip from Sofia to the Black Sea.

Hear about travel to southern West Virginia as the Amateur Traveler talks to Ethan Starcher from WaywardWings.net about his home state.

Hear about travel to West Africa to Senegal and the Gambia as the Amateur Traveler talks to Brian Asher from theworldhiker.com about this under-visited region.

Hear about travel to Tbilisi and the eastern part of the Republic of Georgia as the Amateur Traveler talks to Tommo & Megsy from FoodFunTravel.com who have settled in Georgia.

The mainstream media is still a powerful entity in America, even as its influence wanes in favor of social media. The evidence of this has come again in recent polls that show Republican Gov. Rob DeSantis’ slipping poll numbers in Florida versus Democratic Gov. Andrew Cuomo’s rising numbers in New York. And the curious part…

The post Media Lies at Work: As NY Deaths Go Up, So Do Cuomo’s Polls – As FL Deaths Go Down, So Do DeSantis’ Polls appeared first on The Western Journal.

Imam Shaker Elsayed offered an explanation for the coronavirus pandemic that is not surprising considering the source: He blamed the Jews. The controversial imam didn’t stop there during his sermon at the Dar Al-Hijrah Islamic Center last month. The Falls Church, Virginia, mosque has a history of ties to terrorism, according to the New York…

The post Virginia Imam: Allah Used COVID-19 To Punish Jews appeared first on The Western Journal.

An evangelical church network in California will ignore Democratic Gov. Gavin Newsom’s executive prohibitions this month, opening sanctuary doors for communal worship despite the ongoing coronavirus pandemic. According to Fox News, California Church United, representing roughly 3,000 churches and 2.5 million congregants statewide, announced Thursday that it will return to conducting in-person services by Pentecost…

The post Huge Group of California Churches Announces Plan To Defy Governor Newsom’s Order appeared first on The Western Journal.

The health expert who helped shaped Britain’s lockdown policies in response to the coronavirus has resigned after admitting he broke his own rules by having his married lover visit him at his home. Professor Neil Ferguson, 51, of Imperial College in London was a prominent member of the Scientific Advisory Group for Emergencies. The Telegraph…

The post Scientist Behind Social Distancing Breaks Own Rules To Cheat with Married Woman, Resigns appeared first on The Western Journal.

What one expert called “something out a monster cartoon” has now arrived in the United States. The Asian giant hornet, which can decimate bee colonies and is responsible for 50 deaths a year in Japan, is now in Washington state, according to The New York Times. A new threat reaches the United States: A massive…

The post Giant Asian ‘Murder Hornets’ Spotted in US for First Time appeared first on The Western Journal.

CTVNews.ca asked Canadians to share how they were using their CERB payments and got a flood of responses. Most said they were covering the basics -- housing, groceries, transportation and medicine.

The Assad government sought to promote regime-sponsored cultural events through a marketing campaign called "Summer in Syria," but the effort didn't exactly go as planned.

By the time Martin Smith reached Syria this past summer, the war was already in its fifth year, but life in regime-controlled areas was still largely a mystery.

For Russian President Vladimir Putin, Syria is not just about supporting the Assad regime in Syria. It's about Russia's place in the world.

At least 65,116 individuals have been "forcibly disappeared" by the Syrian government, according to a new report by Amnesty International.

Just days after the attack in Paris, America’s top intelligence official suggested that recent leaks about classified surveillance programs were partially responsible.

The coronavirus pandemic has many of us nostalgic for a simpler time — maybe when we were teenagers and/or watching our favorite early oughts sitcoms. 

Actress Cobie Smulders seems to be feeling that itch, and sensed we are too. The former How I Met Your Mother actress decided to recreate her character Robin Scherbatsky's hit song from the series, "Let's Go to The Mall."

For those who need a refresher, "Let's Go to The Mall" was released during Robin's short career as a pop star (under the name Robin Sparkles) in her homeland of Canada. While it has all the flair of an '80s banger — jelly bracelets, worshipping the mall — it was released in the '90s because apparently that's when '80s culture reached Canada.  Read more...

An array of scientific evidence suggest that in some cases, the bacteria in your gut–your microbiome–could be tied to neurological and psychological disorders and differences, from anxiety and autism to Parkinson's and schizophrenia. The journal Science published a survey of the field and the Cambridge, Massachusetts start-up Holobiome that hopes to use insight into this "psychobiome" to develop treatments for depression, insomnia, and other conditions with a neurological side to them. From Science:

For example, many people with irritable bowel syndrome are also depressed, people on the autism spectrum tend to have digestive problems, and people with Parkinson’s are prone to constipation.

Researchers have also noticed an increase in depression in people taking antibiotics—but not antiviral or antifungal medications that leave gut bacteria unharmed. Last year, Jeroen Raes, a microbiologist at the Catholic University of Leuven, and colleagues analyzed the health records of two groups—one Belgian, one Dutch—of more then 1000 people participating in surveys of their types of gut bacteria. People with depression had deficits of the same two bacterial species, the authors reported in April 2019 in Nature Microbiology.

Researchers see ways in which gut microbes could influence the brain. Some may secrete messenger molecules that travel though the blood to the brain. Other bacteria may stimulate the vagus nerve, which runs from the base of the brain to the organs in the abdomen. Bacterial molecules might relay signals to the vagus through recently discovered “neuropod” cells that sit in the lining of the gut, sensing its biochemical milieu, including microbial compounds.

I have a love/hate relationship with musical reaction videos. There are kids reacting to heavy metal (or The Beatles), vocal teachers and opera singers reacting to rock vocalists, millennials reacting to classic rock, and on and on. Some of these are quite moving, for instance, a 20-something hearing Zep's "Since I've Been Loving You" for the first time or a vocal teacher reacting to a Black Metal vocalist's cookie monster growl.

In these two videos, Heline, a classical flutist and music teacher, listens to Jethro Tull's Ian Anderson performing "Bouree" at a 1969 concert and then she listens and reacts to a performance from 1976.

I didn't realize that Anderson had only been playing the flute for a year and had no formal training at the time of the 69 video (their first tour). Heline can appreciate his chutzpah in the first performance and the fact that he's only been playing for a year(!), but is perhaps predictably critical of his playing. She is more impressed with what she sees and hears in the 76 performance.

Personally, I always thought his playing was inspired and his playing, vocalizing, singing and playing, and his other stage antics (the goofy one-legged stance) were a perfect example of "the street finding its own uses for things," using technology in ways in which it was never intended. As with all things artistic, your mileage may vary.

Image: YouTube Read the rest

In one way or another, social learning has been part of our lives for a long time. We spend a large amount of time at work. During this time, we are bound to learn from our colleagues and get a chance to talk on mutual topics of interest. Moreover, social media has given an outlet […]

The post Social Learning as a Way to Foster Productivity in the Workplace appeared first on Dumb Little Man.

Radoslaw Dobrolecki, US business development director at RTB House, discusses how artificial intelligence can help predict customer behavior at scale.

Omer Golan, founder and CEO of Outernets, discusses how artificial intelligence can improve the digital out-of-home industry.

TV time among adult viewers up 10 minutes per day in 2020 April 23, 2020 (London, UK) – The UK’s social distancing measures due to coronavirus are having a measurable […]

Our Estimate for Time Spent with TV in 2020 Is Revised Upward by 5 Minutes April 24, 2020 (New York, NY) – Over the course of just a few months, […]

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.

When someone mentions “Peru,” images of bucolic mountain pastures, ancient Incan ruins, and verdant Amazonian jungle are probably the first thing that pop into your mind. But this beautiful South American nation boasts miles and miles of gorgeous Pacific coastline, and it is also home to some amazing beaches. Let’s take a look at some of best beaches Peru has to offer.

Cabo Blanco: Arguably the most famous beach along Peru’s 2,500 kilometers of coastline, Cabo Blanco is routinely hailed as one the best surf spots in the world. When Peruvian surfer Gordo Barreda was scouting the area for great waves, he randomly paid a visit to the village, where he stumbled upon the now famous Peruvian Pipeline. A hollow, powerful left-hanging wave, it is arguably one of the best places to catch a wave on the continent’s entire Pacific coastline. Even before Barreda’s famous discovery, this fishing hamlet long enchanted visitors with its small-town charm and thriving fishing industry. In the 1950s and 1960s, fishermen routinely made the trek to Cabo Blanco in search of the area’s legendary large marlin. The famous writer Ernest Hemmingway spent several months here during the filming of the movie adaptation of his novel “The Old Man and the Sea.” During his stay, he reportedly caught a 700-pound marlin.

Mancora: Once a quaint fishing village, Mancora has exploded onto Peru’s backpacker scene as a major hub in the last decade or so. Located right in the middle of Peru’s sunniest and warmest region, you can relax on beautiful beaches during the day and then party the night away. Mancora is great for travelers on a budget, and cheap hostels abound.

Vichayito: If you want great beaches without a slew of rowdy partiers, Vichayito is an excellent option. Situated about 7 kilometers to the south of Mancora, this is an ideal spot for families. The water is ideal for swimming and kite surfing, and the beaches are clean and quiet.

Punta Hermosa: Just an hour drive from Peru’s capital, Punta Hermosa is popular with Limeños looking for a summer retreat from the city. While not as spectacular as the beaches of Paracas or Mancora, Punta Hermosa’s proximity to Lima makes it a great option for looking for a quick weekend escape from the city.

Asia: Peru’s most opulent beach, Asia is all about glitz and glamour. The upper echelons of Peruvian society have luxurious summer homes at this beach resort town, making it a hub of wealth. The beaches are great, but what really make Asia stand out are its high-end restaurants, luxury shopping center, and dazzling nightclubs.

Paracas: The Paracas National Reserve boasts some of Peru’s most dramatic desert landscape. Here, enormous sand dunes and dramatic sandstone rock formations meet the azure waters of the Pacific. The reserve, which consists of a total of 335,000 hectares of tropical desert on the Paracas Peninsula, is intended to preserve the area’s rich marine ecosystem as well as protect its unique cultural heritage (the site was of great significance to the Paracas, a pre-Colombian indigenous group that inhabited the area between roughly 800 BC and 1000 BC). There are no formal hotels within the reserve, though there are many sites popular with beach campers.

If you are looking for more luxurious accommodations, they can certainly also be found. Though beach destinations to the north of the country often attract the majority of tourist attention, it is Paracas where you will find the most luxurious beach vacations Peru has to offer. Just a few miles from the park’s entrance, you will find Hotel Paracas, a Luxury Collection Resort complete with 5-star accommodations, three pools, and a luxury spa.

Marcona: For those really looking to get off of Peru’s beaten tourist trail, we recommend a visit to the rugged, windswept beaches of Marcona, located roughly 8 hours south of Lima. The waters here can be quite cold year-round thanks to the Humboldt current, which brings water up from Antarctica along the Pacific coast of Chile and southern Peru. But the cold temperatures mean that waters are teeming with marine life. With any bit of luck, visitors might catch a glimpse of one of the zone’s enormous seal colonies. The beaches are known for their dramatic rock formations and rough surf, but they are a great place for those looking for options totally free of tourists.

So they you have it, our pick of the best Peruvian beaches. Happy adventuring in Peru!

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

Part of the Antarctic diary promo for the BBC UK Homepage

To mark International Polar Year, BBC science producer Martin Redfern is spending a month on board a research ship in the Antarctic. Follow his exploits online.

Part of the Sport Relief promo for the BBC UK Homepage

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, Legal Immigrant in Michigan Sends a Message to MI Gov Whitmer and Media Criticizing Freedom-Loving Protesters, was first published on 100PercentFedUp.com.

The media and even some governors are trying to paint the Americans who are protesting to open businesses up as racists and even Nazis. The Governor of Michigan recently made a derogatory statement about the protesters implying they are racists. Painting a negative picture of the people who want their freedom and businesses demonizes our […]

Continue reading: Legal Immigrant in Michigan Sends a Message to MI Gov Whitmer and Media Criticizing Freedom-Loving Protesters ...

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, ‘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, BREAKING: President Trump’s Fiery Interview On Fox & Friends…”These are dirty politicians and dirty cops…They put our nation in danger with other nations, including Russia” [VIDEO], was first published on 100PercentFedUp.com.

This morning during a nearly one hour interview with Fox & Friends, President Trump addressed the decision by the DOJ to drop the case against the innocent General Michael Flynn. Trump ripped into the “dirty politicians and dirty cops” who went after General Michael Flynn. President Trump called the players involved in the horrible plot […]

Continue reading: BREAKING: President Trump’s Fiery Interview On Fox & Friends…”These are dirty politicians and dirty cops…They put our nation in danger with other nations, including Russia” [VIDEO] ...

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 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.

Ontario’s provincial parks and conservation areas will reopen this week but campgrounds and beaches will continue to be off-limits for now.

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.

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 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.

The protein-tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors. Mutations in the SHP2 gene have been linked to many different types of human diseases, including developmental disorders, leukemia, and solid tumors. Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography, and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the WT enzyme, the interactions between its N-SH2 and protein-tyrosine phosphatase domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and a potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enable the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr-53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

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.

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.