The 2012 presidential race is on. Kinda.

At the moment, it feels more like a wade through treacle - so slow is the pace of President Barack Obama's opponents. Mr Obama can be unambiguous that he is going to run because they are all showing varying degrees of hesitancy.

If the president is to get back into the White House he has to leap a number of obstacles: an economy that is so sluggish that there are constant worries it could go backwards and supporters who may be unenthusiastic about sending more troops to Afghanistan, bombing Libya and failing to close Guantanamo Bay prison. There is also huge uncertainly in the country about health care and much more we will be looking at in detail.

But the strength of opposition doesn't seem, at the moment, a particularly high hurdle.  To British eyes, the primary system is one of the most curious parts of American politics.

The elite of British political parties have only grudgingly and slowly given the power of choosing their own leader. The principle of "one member, one vote" has been slow in coming. 

Elections for leaders rarely grip in the same way as American internal elections. While any American can easily register as a Republican or Democrat and have their say about who represents them, in Britain being a party member still seems an effort of will.

Twenty-five pounds ($40) per year may not be much to play your part in conservative politics in Britain, £12 may be a bargain to have a say in the Lib Dems and it's only a penny (for those under 27) to join the Labour Party - but it still costs something.

There's a feeling that being interested in who becomes your PM or MP isn't enough. You have to be willing to sit in draughty village halls on wet Wednesdays listening.  

The biggest difference is perhaps not in just who is involved, but how late in the political cycle the choice is made. This has a real impact. Every party leader, good or bad, has an image, policy likes and dislikes and personal ticks that colour voters approach to the parties as a whole.

The British public has years to get to know Ed Miliband and decide what to think about him leading a Labour government. Here in the US, the opposition is currently either faceless or hydra-headed. There is no obvious front-runner, and any prediction about who will be the Republican candidate in 2012 is nothing more than an informed guess.

Mr Obama v Michele Bachmann would be quite a different contest to Mr Obama v John Huntsman. 

Republicans get to choose, late in the day, exactly what they want their party to stand for.

The influence of the Tea Party suggests any candidate will be economically conservative, but beyond that, it is impossible to predict very much. The candidates are so unenthusiastic about firing the starting gun, the first big debate at the Reagan library in California has been put back from next month to the autumn.

Mitt Romney, Tim Pawlenty, Newt Gingrich, Michele Bachmann and Donald Trump seem almost certain to have a go. Sarah Palin, Mike Huckabee, John Huntsman and Mitch Daniels seem less sure bets. And, of course, there are plenty of other names out there.

Mr Obama is starting the race now to make sure that whoever challenges him, his organisation will be ramped up and ready, with big bucks at its command.

IU alum and Dallas Mavericks owner Mark Cuban pitches an idea to Kentucky basketball coach John Calipari as if he was a 'Shark Tank' contestant.

The payoffs for such turnarounds can be extraordinary for the residents who live nearby and for the city as a whole.

An anonymous reader quotes CNN: The U.S. Food and Drug Administration on Friday issued an emergency use authorization for the first at-home Covid-19 test that uses saliva samples, the agency said in a news release. Rutgers University's RUCDR Infinite Biologics lab received an amended emergency authorization late Thursday. With the test, people can collect their own saliva at home and send their saliva samples to a lab for results... "Authorizing additional diagnostic tests with the option of at-home sample collection will continue to increase patient access to testing for COVID-19. This provides an additional option for the easy, safe and convenient collection of samples required for testing without traveling to a doctor's office, hospital or testing site," FDA Commissioner Dr. Stephen M. Hahn said in the FDA's press release on Friday... The test remains prescription only.

Read more of this story at Slashdot.

IndyFringe, the popular Mass Ave. theater festival, has been canceled. Social distancing requirements will not allow the artists to prepare.

St. Paul & the Broken Bones, Moon Taxi will headline Holler on the Hill, a new two-day Indianapolis music festival.

Outlaw Music Festival makes smashing Indiana debut led by national treasure Willie Nelson and rising iconoclast Sturgill Simpson.

Nearly 70 acts, including Valee and Black Moon, will perform at fourth annual Chreece hip-hop festival in Fountain Square.

Hong Kong has seen 57 new infections over the past two weeks, 50 of which were imported.

The Blyth Festival will not be staging any plays this year.

“I can’t go back,” says Philippe Lançon, who wrote a memoir about his recovery from the massacre.

The Star Wars actor faced backlash for his reaction, but he later made clear that young Theodore wasn't the target of his ire.

Maybe it’s irony. Or maybe it’s her latest attempt to pinkwash her father’s anti-family agenda.

Weak encryption technology has given rise to the phenomenon of “Zoombombing”, where uninvited trolls gain access to a video conference.

Facebook's Libra cryptocurrency could now launch in mid-November after a revamp aimed at mollifying its many critics.

Rick Bright, head of an agency tasked with developing coronavirus drugs, has said administration ignored his warnings.

Meghan Markle is reportedly set to launch her lifestyle website to rival Gwyneth Paltrow's Goop.

Ashland, Oregon Shakespeare Festival

TL;DR: A one-year subscription to Norton Secure VPN is on sale for £2.50 per month, saving you 50% on list price.

It feels like the world of VPNs is expanding, with more and more providers entering the market and increased demand from users. 

This market can be a daunting place, especially as you probably won't be familiar with most of the available providers. This doesn't mean you shouldn't consider these services, but we totally understand if you're reluctant to invest in an unknown provider.

Fortunately for those who would prefer to subscribe to an established provider, there are plenty of options from the leading names in securityNorton is one such brand, with a comprehensive VPN service on sale for just £2.50 per month. Read more...

Three months ago, Apple released a new credit card in partnership with Goldman Sachs that aimed to disrupt the highly regulated world of consumer finance. However, a well-known software developer tweeted that he was given 20x the credit line offered to his wife, despite the fact that they have been filing joint tax returns and […]

Do you know that feeling when you are hyped and ready to take on a new challenge? You are taken over by a surge of motivation and excitement and you’ve made a commitment you fully intend to stick to. It sounds familiar, right? Perhaps you declared your intention to dedicate more time to self-care, or […]

The post 7 Must-Do Hacks for When Your Motivation Dips appeared first on Dumb Little Man.

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.

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.

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” ...

20 April 2020

There is and will continue to be an edge of rivalry in Egypt’s relations with the dominant Gulf Arab powers. This paper will focus primarily on the Egypt–Gulf relationship during the Sisi era.

Research Event

Event participants

Speaker: David Butter, Associate Fellow, Middle East and North Africa Programme, Chatham House
Moderator: Mohamed El Dahshan, Associate Fellow, Middle East and North Africa Programme, Chatham House

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter PvvhBA, which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of PvvhBA, whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the PvvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of PvvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

Glial cell line–derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET–GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein–coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

Research Event

Event participants

Speaker: David Butter, Associate Fellow, Middle East and North Africa Programme, Chatham House
Moderator: Mohamed El Dahshan, Associate Fellow, Middle East and North Africa Programme, Chatham House

Jorge H. Capdevila
Feb 1, 2000; 41:163-181
Reviews

MS Brown
Jul 1, 1980; 21:505-517
Reviews

K Schoonjans
May 1, 1996; 37:907-925
Reviews

Glial cell line–derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET–GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor β superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence-divergent, having only limited similarity to other closely related members of the transforming growth factor β superfamily. Hydrogen/deuterium exchange MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen–antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

Cytochrome c oxidase (CcO) reduces O2 to water, coupled with a proton-pumping process. The structure of the O2-reduction site of CcO contains two reducing equivalents, Fea32+ and CuB1+, and suggests that a peroxide-bound state (Fea33+–O−–O−–CuB2+) rather than an O2-bound state (Fea32+–O2) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fea32+–O2, whereas Fea33+–O−–O−–CuB2+ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe–O distance of the ferryl center could best be described as Fea34+ = O2−, not as Fea34+–OH−. The distance suggests an ∼800-cm−1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fea33+–O−–O−–CuB2+ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane–associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

Prótese auditiva ancorada em osso não emite um som como o aparelho convencional. Seu diferencial é vibrar o osso da cabeça.

The post SUS vai oferecer aparelho de última geração para tipo de perda auditiva até então sem tratamento appeared first on Saúde Próspera.

The leak exposed millions of records with full names, emails, user conversations, payment logs, and IP addresses dating back to March.

Hydel, despite trailing their rivals for two straight days, easily retained their title