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Joshua Summers, CEO of linear television supply-side platform clypd, discusses the major advanced targeting trends expected to infiltrate traditional TV ad buying within the next year.

From better campsite cooking to car maintenance to smartphone-free communication, here are 12 gadgets to up your car camping game.

The post Up Your Car Camping Game with These 12 Clever Gadgets appeared first on Vagabondish.

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So you've decided to hike Machu Picchu? Well great! There's no doubt that you're in for the experience of a lifetime. However, the truth of the matter is that now you've got some additional decisions to make. You see, there's more than one route to arrive to the world-famous Inca site, and even once you've arrived there are a few choices to make as well.

But don't fret! If you're looking to hike Machu Picchu, we've got the information you need to start planning your trip.

Hike Machu Picchu: Arriving to the Site

1. Classic Inca Trail

The Classic Inca Trail is a four-day trek stretching some 42 kilometers along an ancient road built by the Incas themselves. This is undoubtedly the most famous route to Machu Picchu, and it's the one way that offers the opportunity to enter the city through its storied Sun Gate. Frankly, we've got tons of information on the Inca Trail on other pages of our site, so we won't waste too much space being redundant here. Check out these pages for more information on the trail's itinerary as well as for access to an Inca Trail map.

2. One-Day Inca Trail

Whether you're crunched for time or simply don't feel physically up to four full days of trekking, don't fret--a small taste of the world-famous Inca Trail is still available to you! A number of tour companies offer an abridged version of the trek, either one full day of hiking or a shortened day followed by a night of camping and a dawn arrival at Machu Picchu. Besides the Inca Trail's famed destination, the one-day version of the trek also takes you past Wiñay Wayna, another favorite ruins along the trail. The bad news here is that you'll still need to reserve one of only a few hundred daily Inca Trail passes, meaning that, just like the full Inca Trail, you'll need to book this trek months in advance.

3. Lares Trail

Just north of the world-famous Sacred Valley sits the comparatively lesser-known Lares Valley, and whereas the former can be swarmed with tourists especially during the high season, life in the Lares Valley continues humming along relatively unchanged. So whether you're looking for a simpler, more culturally-immersive experience or if you just didn't book an Inca Trail pass in time, the Lares Trail is an excellent option! Lucky for you, we've already gathered plenty of information on the Lares Trail including the trek's daily itinerary and a side-by-side comparison of the Lares and Inca Trails if you're struggling to decide how to hike Machu Picchu.

4. Vilcabamba Traverse Route

Warning--this option is neither for the faint of heart nor the out of shape! The Vilcabamba Traverse Route is one of the newest on the Peru trekking circuit, clocking in at nearly 100 kilometers over the course of a week of trekking. Though very difficult, the route is especially rewarding--think a variety of diverse biomes and the ruins of Choquequirao, often compared to Machu Picchu although less than 40% of the site has been excavated (pictured above). Though visited by only 5,000 or so brave trekkers last year, be warned--the local government is pushing a plan to build a cable car to the site! If you want to see the ruins in their current, undisturbed state, it would be best to go sooner than later.

Hike Machu Picchu: Once You're There

5. Huayna Picchu

Just because you've finally arrived at Machu Picchu doesn't mean your hiking experience has to end! The famous mountain soaring in the background of most iconic Machu Picchu photos is possible to hike, and the experience is certainly recommended. The views from the top are great, and on the way down you can even take a back route to the storied Temple of the Moon. For more information on Huayna Picchu, you can check out this past post on our site.

So that's it for today! If you're planning to hike Machu Picchu and have any questions you'd like to ask us, feel free to reach out in the comments section below. And, if you've already done one of these hikes, feel free to share your experience as well. We appreciate all of your comments!

Machu Picchu, Peru

Although it's one of the most well-known archaeological wonders of the world, Machu Picchu still holds plenty of secrets and is on our shortlist of must-see destinations on a Peru trip.

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Machu Picchu is an enigma, some would say a paradox because it is known as both the best known yet least known about of the Inca sites. Since its discovery on July 24, 1911, by North American Hiram Bingham, Machu Picchu has been considered one of the world's greatest architectural and archaeological monuments, due to its extraordinary magnificence and harmonious structure. Machu Picchu is definitely one of the most fascinating sites in Peru.

At 2,400 meters above sea level, in the province of Urubamba, department of Cusco, Machu Picchu surprises us because of the way its stone constructions are spread over a narrow and uneven mountain top, bordering a sheer 400 meter cliff side of the Urubamba River canyon.

Why and how was Machu Picchu built?

Machu Picchu is a citadel shrouded in mystery, and to this day archaeologists have not definitively uncovered the purpose of this city of stone. The site covers an area of about one square mile, and stands in a region that the Incas considered to be magical, due to the meeting of the Andes mountains with the mighty Amazon river. When 135 bodies were discovered while exploring the site, 109 of which were female, some believed that Machu Picchu could have been a monastery where acllas (young girls) were trained to serve the Inca and the Willac Uno (High Priest). Others said it may simply have been an advance settlement for further expansions planned by the Incas. Perhaps the mystery may never be fully explained.

The surprising perfection and beauty of Machu Picchu's walls, built by joining stone to stone without using any cement or adhesive whatsoever, has led to many theories developing around how the city was constructed as well. It is said that a bird by the name of Kak'aqllu knew the formula for softening rock but by command, perhaps by the ancient Inca gods, had its tongue torn out. Others say there was a magic plant that could dissolve and compress stone. Nonetheless, mysteries and myths aside, the obvious wisdom and skill of the city's ancient builders -- evidenced by Machu Picchu's many squares, aqueducts, watchtowers, observatories and its sun clock -- is quite clear.

Many people may be drawn to Peru by Machu Picchu, yet it is considered by many of our guests, to be just one of many of the ruins featuring on the "highlights reel"of their trip. See reviews to read more

How to get to Machu Picchu - One day or multi day trails

You can take a one day trip to Peru from Cuzco or Lima, and walk up to this citadel in the clouds high in the Andes, or you can take some time to get acclimatised and trek via several trails that lead to Machu Picchu, most taking around 4 to 5 days to complete. Alot of people begin their Peru trip with the intent of visiting Machu Picchu, but don't know how much more there is to see and do in and around Machu Picchu.

After all, if you are going to Peru to experience a South American trip of a lifetime, why not learn about all the activities and other ruins there are to discover.

Popular activities on our Peru trips (including Machu Picchu):

1. Hiking the Lares or Classic Inca Trail
2. Exploring Machu Picchu - facts about Machu Picchu
3. Hiking in the Amazon jungle
4. Sea kayaking on Lake Titicaca
5. Staying with a local family on Amantani Island
6. Hiking Sacsayhuamán fortress
7. Hiking and cycle in the Sacred Valley of the Incas
8. Exploring Cuzco
9. Cycling through Andean villages and La Raya Pass
10. Hiking Amantani and Taquile Islands

[link to above pages Will]

You may be surprised at the number of activities you can do in Peru, in fact it's a surprise to alot of people that it is possible to enjoy these "non Machu Picchu focused" activities at all. Our philosophy is a little different to many tour companies, we believe that if you are going to travel all the way to a new country to experience a whole new culture, why not experience as many perspectives, local cultures and ruins as you can while you are there.

Obviously the most popular trail chosen by visitors wanting to visit Machu Picchu is the Inca Trail. Some people prefer the Lares trail because it offers a much more immersive experience in Peruvian village culture. If you wants to experience some of the traditions and village life the early Inca's enjoyed, you can stay with their descendants in one of the many villages along the Lares Trail.

If you want to hike the traditional route,  take a sneak peak below at some of what the Inca Trail has to offer.

Hike to Machu Picchu on the ancient Inca Trail

The Inca Trail between the Sacred Valley of the Urubamba River and the mysterious abandoned citadel of Machu Picchu is one of the world's classic treks. Climbing out of the river valley, crossing rugged mountain passes over 13,000 ft high, the trail winds through the Andes, passing numerous significant Inca ruins en route before descending through the Sun Gate to the silent stone city of Machu Picchu. To hike the Inca Trail is a thrilling experience and a great privilege. You need a permit from the Peruvian government to set foot on it, and there are strict limits on the number of permits issued each year.

But the Inca Trail is much more than a great hike. It is one small portion of an incredible network of such trails crossing high mountain ranges, bleak deserts, and raging Andean rivers, tying the Inca Empire together. At its peak expansion, Tahuantinsuyo (or The Four Corners as the empire was known) extended from what is now southern Colombia in the north, to central Chile in the south, covering a distance of about 5500 km (3400 mi). To rule such a vast domain, the emperor, or Inca, forged a remarkable communications system of approximately 18,600 miles of trails, paved through much of its length, stepped where need be, through tunnels where necessary, and using gossamer suspension bridges built of straw ropes to cross rivers unfordable in the wet season.

The roads served to move the conquering Inca armies, and were generally wide enough for a minimum of two warriors to travel abreast. A system of runners stationed at rest houses known as tambos sped messages along the roadways, much like the Pony Express mail of the old American West. The Inca, at his empire's capital in Cuzco, could receive news from far away Quito as rapidly as a letter crosses between the two cities in today's mail.

As remarkable as this highway system was in the days when it was built, used and maintained, it is an astounding testimony to its construction that so many segments remain serviceable today, after half a millennium of neglect. Clearly the Inca highway system ranks as one of the greatest engineering achievements of pre-industrial man.

The full Inca Trail is approximately 40km long. Spread over 4 days, this amounts to about five hours walking per day, although you can walk at your own pace - you are not forced to walk with your group the whole time. It is not a difficult walk, although there are a couple of high passes, and a steep climb on the second day, so a basic level of fitness is required.

Alternative Routes To Get To Machu Picchu other than the Inca Trail (link to another page how to get to machu picchu)

Lares Trail (link)

Inca Trail (link)

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History of Machu Picchu

Archaeological evidence uncovered around the site suggests that the area was first used for agricultural purposes back in 760 B.C.

The war of Vilcambamba Pachacutec in 1440 established the first settlement at the site. It was called the Tahuantinsuyo Empire which was later followed by the formation of the government of Manco Capac.

It is thought that Machu Picchu was first inhabited by 300-1000 inhabitants, who were of the highest Class or "llactas".

The valleys around these areas were important for their agricultural contribution, however after death of the Emperor Pachacutec, it lost it's importance, with the establishment of new sites like Ollaytantambo and Vilcambamba. The building of these new sites by his successors, in more accessible terrain made Machu Picchu less appealing.

From 1527 to 1532, two brothers Huáscar and Atahualpa fought against each other in a civil war over the Inca Empire. Their father, Inca Huayna Capac had given each brother a section of the empire to manage, one in Huáscar in Cuzco and Atahualpa in Quito. When Huayna Capac and his heir, Ninan Cuyuchi, died somewhere between 1525 and 1527, the two brothers Atahualpa and Huáscar went to war over who should rule.  The population who had come to live in the Machu Picchu area from rural or remote locations left after the war ended to return to where they came from. Later another brother, Manco Inca was sent into exile in Vilcambamba, and Machu Picchu was deserted.

Antonio Raimondi was an Italian geographer and scientist from Milan who visited Machu Picchu in 1851. In 1867 Augusto Berns arrived to mine the site.

Hiram Bingham re-discovered the ruins in 1911. He documented and publicised his "discovery".

Hiram Bingham

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

[jump to video]

Long story (not very) short? One night in 2003--after killing it in front of audience of about 30 lucky people in Oakland--The Long Winters needed a place to crash, and my wife and I were happy to oblige. 

So, they drove their Big Stinky Blue Van over the bridge, slept on our floor, and by breakfast the next morning, it'd become clear to me that I'd provided lodging to a man who was not only very likely a member of my karass--he was also one of the smartest bullshit artists I'd ever met. 

Almost eight years later, although I don't see him nearly as much as I'd like, I still count the guy as one of my best pals ever.

That's John Roderick. And, I think you need to know about him.

John doesn't read this site--he's more of a Twitter person--so I don't risk feeding his astounding excess of dignity by saying he's one of the most gifted writers and bon vivants of our generation. He's just the best. In large part because he's congenitally incapable of suffering bullshit.

This was never more apparent than the Saturday morning in 2007 when we sat in my back yard and talked about a lot of stuff. Playing guitar, advertising on the web, the evil work of promoters, and why everyone is always trying to shortchange everyone on copper pipe. 

That talking became a four-part interview I ran on the late and occasionally lamented The Merlin Show, and, to this day, it's one of my favorite things I've been lucky enough to post to the web.

So, y'know how I'm definitely "not for everyone?" Well, John is really "not for everyone."

He's opinionated and arrogant and undiplomatic and unironically loves Judas Priest--meaning everyone will find at least one thing not to like about him. Despite being hairy and enjoying laying on your bed, John is not exactly a teddy bear. 

But, John's also right a lot. And, he never sands off the edges of his personality or opinions to make you theoretically "like" him. Which, it will come as no surprise to you, is a big reason I love the guy more than a free prime rib dinner. 

So, why the jizzfest about that awful jerk, John Roderick?

Because, as I noted the other day on the Twitter, in our first episode of Back to Work I misattributed a line that should have been credited to John. Which in itself is unimportant, except inasmuch as finding that link to correct the error got me watching our 50-some minutes of chatting again. I also received some at-responses and emails that reminded me how much people enjoyed our chat. 

But, really it made me realize how much that rambling morning in my back yard still resonates so much with stuff I care a lot about. Independence. Agency. Directness. And, never apologizing for wanting to get paid. Also, guitars and talkative hippies.

So, anyway. John. 

I edited all four parts of the video into one big (streamable/downloadable) movie that should make it way easier to watch at a sitting. Should that interest you. Which it may not. Which, as ever, is totally fine, and kind of the point.

But. If you like Dan and my new show (and, seriously—God bless you magnificent bastards who helped briefly make B2W the most popular podcast in the world [gulp]), I think you'll really like this interview a lot too. I hope so, anyway.

Thus, submitted for your disapproval, permit me to present my four-year-old visit with the acerbic, opinionated, and reportedly unlikeable bullshit artist whom I respect and adore more than just about anybody. 

Meet Hotrod.

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The following article, Photos Of Kim Jong Un Spark Conspiracy Theories About A Body Double…You Be The Judge, was first published on 100PercentFedUp.com.

For several weeks rumors of the North Korean dictator’s sickness and ultimately, his death, have been making their rounds in the media. North Korea’s state-run media released photos of  Kim Jong Un that were allegedly taken on May 1, at the opening of a fertilizer factory in Sunchon, N. Korea. Twitter users who’ve studied the images are […]

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The following article, Univ of Pittsburg Researcher, China Native, Dr. Bing Liu, “On the verge” of COVID19 Breakthrough Is Murdered…Alleged Gunman, Hao Gu, Kills Himself, was first published on 100PercentFedUp.com.

A 37-year-old China native and "outstanding researcher" at the University of Pittsburgh...

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

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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 […]

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

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The following article, Greg Gutfeld Levels Ilhan Omar With Epic Response To Her Claim That “White Privilege” Is Reason Charges Were Dropped Against General Flynn, was first published on 100PercentFedUp.com.

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

Little Richard, the self-proclaimed "architect of rock 'n' roll" whose piercing wail, pounding piano and towering pompadour irrevocably altered popular music while introducing black R&B to white America, has died Saturday. He was 87.

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

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.

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

10 June 2014

23 April 2020

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11–7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Charles Collot, Frank Merle and Pierre Raphaël
J. Amer. Math. Soc. 33 (2020), 527-607.
Abstract, references and article information

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

Yue Chen
May 1, 2007; 6:812-819
Research

Yi Zhang
Sep 1, 2005; 4:1240-1250
Research

SUMOylation is a posttranslational modification (PTM) at a lysine residue and is crucial for the proper functions of many proteins, particularly of transcription factors, in various biological processes. Zinc finger homeobox 3 (ZFHX3), also known as AT motif-binding factor 1 (ATBF1), is a large transcription factor that is active in multiple pathological processes, including atrial fibrillation and carcinogenesis, and in circadian regulation and development. We have previously demonstrated that ZFHX3 is SUMOylated at three or more lysine residues. Here, we investigated which enzymes regulate ZFHX3 SUMOylation and whether SUMOylation modulates ZFHX3 stability and function. We found that SUMO1, SUMO2, and SUMO3 each are conjugated to ZFHX3. Multiple lysine residues in ZFHX3 were SUMOylated, but Lys-2806 was the major SUMOylation site, and we also found that it is highly conserved among ZFHX3 orthologs from different animal species. Using molecular analyses, we identified the enzymes that mediate ZFHX3 SUMOylation; these included SUMO1-activating enzyme subunit 1 (SAE1), an E1-activating enzyme; SUMO-conjugating enzyme UBC9 (UBC9), an E2-conjugating enzyme; and protein inhibitor of activated STAT2 (PIAS2), an E3 ligase. Multiple analyses established that both SUMO-specific peptidase 1 (SENP1) and SENP2 deSUMOylate ZFHX3. SUMOylation at Lys-2806 enhanced ZFHX3 stability by interfering with its ubiquitination and proteasomal degradation. Functionally, Lys-2806 SUMOylation enabled ZFHX3-mediated cell proliferation and xenograft tumor growth of the MDA-MB-231 breast cancer cell line. These findings reveal the enzymes involved in, and the functional consequences of, ZFHX3 SUMOylation, insights that may help shed light on ZFHX3's roles in various cellular and pathophysiological processes.

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

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.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11–7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

31 January 2020

NAD+ is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD+ biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD+. Some members of the NadE family use l-glutamine as a nitrogen donor and are named NadEGln. Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity, and biolayer interferometry assays, we show that PII and NadEGln physically interact in vitro, that this complex relieves NadEGln negative feedback inhibition by NAD+. This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadEGln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD+, representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadEGln, thereby enabling the control of NAD+ biosynthesis according to the nutritional status of the bacterial cell.

8 January 2020 , Volume 96, Number 1

4 March 2020 , Volume 96, Number 2

Invitation Only Research Event

Event participants

Paul D’anieri, Professor of Public Policy and Political Science, University of California, Riverside
Vlad Mykhnenko, Associate Professor of Sustainable Urban Development, St Peter’s College, University of Oxford
Chair: Orysia Lutsevych, Research Fellow and Manager, Ukraine Forum, Chatham House

Webinar Research Event

Clete Willems, Partner, Akin Gump; Deputy Director, US National Economic Council, 2018 - 19
Chair: Marianne Schneider-Petsinger, Senior Research Fellow, US and Americas Programme, Chatham House

Research Event

Lord Mark Malloch-Brown, Chairman, SGO; Former Deputy Secretary-General and Chief of Staff, United Nations
Dr Elizabeth Cousens, President and CEO, United Nations Foundation
Ambassador Nicholas Burns, Roy and Barbara Goodman Family Professor of the Practice of Diplomacy and International Relations at the Harvard Kennedy School; US Under Secretary of State for Political Affairs, 2005 – 2008
Chair: Dr Leslie Vinjamuri, Director, US and the Americas Programme, Chatham House

6 February 2014 , Volume 70, Number 1

From the point of view of a Saudi policy-maker, the country, the region and the world look to be increasingly complex places

5 June 2014 , Volume 70, Number 3

The Golan Heights is home to thousands of Druze who cling on tenaciously while looking over their shoulder at the chaos in their homeland

You’ve got a short story that goes on too long? A chapter that reads at a dying snail’s pace? A challenge entry that trails beyond the maximum word count? You’re also relatively new to writing and haven’t yet dragged your way through all the millions of writing articles? If yes to the last and anything else to the rest, here’s your article.

1. Remove all redundancies. I repeat: Remove all redundancies. I repeat: ….

“Unknown strangers” or “asked a question” can be removed right away. While it may sound silly, there’s a good chance you have a few of those rummaging around in your work for you to weed out.

More significantly you will want to root out the various strings of wordy words writers conjure up. “At that point in time,” can be replaced with “back then,” or even “then,” if the placement in the sentence allows it. “Despite the fact” can be “although.” “Less than great,” is just a polite man’s “mediocre.”

As a lone appreciator of purple prose I won’t tell you to always cull all of these combos, for there’s a time and place for everything and it’s called colle… a long-form story, but if you’re trying to shorten things up these are the first words to go.

Continue reading Keeping It Short at Mythic Scribes.