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!

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

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

Research, Research, Research

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

“Hi, would you like to buy some drugs?

– Nervously shakes head –

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

Kuh-Myoo-Nick-Ashion

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

Who Ya’ Gonna Call?

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

Link Up With Other Travellers And Locals!

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

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

Stay Healthy!

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

Happy tripping!

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

Part of the They've got the look promo for the BBC UK Homepage

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

Part of the Sport Relief promo for the BBC UK Homepage

Video: Merlin Mann - "Time & Attention Talk (improvised)"

Audio (mp3): "Merlin Mann - 'Rutgers Time & Attention Talk'"

This is a talk I did at Rutgers earlier this month. I kinda like it, but for a weird reason. Something something, perfect storm of technology Ragnarok, and yadda yadda, I had to start the talk 20 minutes late with no slides. Nothing.

So, I riffed.

And, I ended up talking about a lot of the new stuff you can expect to see in the Inbox Zero book—work culture, managing expectations, the 3 deadly qualities of email, and one surprising reason email's not as much fun as Project Runway.

Some people liked it. I think. I liked it. I hope you do, too.

Here's the slides I would have shown. ;-)

Many thanks, again, to my great pal, Dr. Donald Schaffner, for bringing me in for this visit. I had a great time and met some fantastic, passionate people. Much appreciated.

update 2010-04-27_13-50-00

Apologies—my friends at Rutgers (inexplicably) have placed this video under lock and key. Fortunately, I have a lock-picker called Firefox. Samizdat video available soon...

update 2010-04-27_14-42-24

Yay, fixed! Many thanks to my hero, Jesse Schibilia.

On May 16, 2010, at 10:02 AM, "Xx" wrote:

You mentioned you gave a talk at Rutgers about future proofing your passion. Is this available as a podcast? I'd love to listen!

This poor kid emailed me to ask a really simple question. And I went and saddled him with the world's most circuitously long-winded answer. Surprise, surprise.

Hey, Xx,

Thanks for the note, man. No I'm sorry its not up as audio AFAIK.

FWIW, it's a talk I'm asked to do more often lately so I wouldn't be surprised if it turns up sooner or later.

Since you were kind enough to ask, the talk—which comes out super different each time I do it— consists of a discursive mishmash of advice I wish I'd had the ears to hear in the year or five after graduating from college: primarily, that we never end up anywhere near where we'd expected, and that most of us would have been a lot happier a lot faster if we'd realized that we were often obsessing over the wrong things—starting with how much the world should care about our major. ("Liberal Arts," with a concentration in [ugh] "Cultural Studies," thanks.)

The talk started as a way to encourage students to learn enough about what they care about that any temporary derails and side roads wouldn't scare their horses too badly. But, today, I see it as something a lot bigger that's demonstrably useful to anyone who hopes to survive, evolve, and thrive in this insane world.

A handful of bits I'm (obviously) still synthesizing into something notionally cohesive:

My Kingdom for Some Context!

For myself, I wish I'd known the value of developing early expertise in interesting new skills around emerging technologies (rather than just iteratively pseudo-honing the 202-level skills I thought I "understood"). Alongside that, I wish I'd learned to embrace the non-douchier aspects of building awesome human relationships (as against "networking" in the service of landing some straight job that, as with most hungry young people, locked me into a carpeted prison of monkey work at the worst time possible).

Also how I wish I'd paid more attention to events, contexts, relationships, and change that were happening outside my immediate world —rather than becoming, say, the undisputed master of fretting about status, salary, and whether I was "a success" who had "arrived".

Hint: I was not a "success," and I had not, by any stretch, "arrived."

To my mind, "success" in the real world is much more the equivalent of achieving a new personal best; it's not about whether you won the "Springtime in Springfield SunnyD®/Q105™ 5k FunRun for Entitilitus," and got a little ribbon with a gold crest on it.

Truly, pretty much anyone who feels they've "arrived" anyplace is about to learn a) how much more they could be doing outside the narrowness of an often superficial ambition and b) the surprising number of things they had to give away through the opportunity costs and trade-offs that lead up to every theoretical milestone. It's a real goddamned thistle, and it's more than a little depressing.

Do You Still Really Want to be a Fireman?

[N.B.: I really hope you're taking bathroom breaks here, Xx]

Related, I think this is about how being an adult is not only unbelievably complicated in ways that you can't begin to imagine—that it's frequently defined by impossible decisions and non-stop layers of "hypocrisy"—but that there's an invisible but entirely real risk to doggedly chasing the theoretically laudable notion of "following your dream." Especially if it's a dream you first had while sleeping on Star Wars sheets in a racecar bed.

Not because it's a bad idea to want things or to have ambitions. Quite the opposite. More because, for a lot of us, the "dreams" of youth turn out to be half-finished blueprints for wax wings. And not particularly flattering ones at that.

By starting adult life with an autistically explicit "goal" that's never been tested against any kind of real-world experience or reality-in-context, we can paradoxically miss a thousand more useful, lucrative, or organic opportunities that just…what?…pop up. Often these are one-time chances to do amazing and even unique things—opportunities that many of us continue to reject out of hand because it's "not what we do."

It took me a full decade to learn to embrace the unfamiliar gifts that kismet loves to deliver on our busiest and most stressful days, and which gifts might (maybe/maybe not) even end up bringing the real-life, non-racecar-bed, now me a big step closer to something that's 1000 times more interesting than a hollow, ten-year-old caricature of "what I wanna be when I grow up."

Finding Your "Old Butcher"

Also related, it strikes me that the indisputable wealth of information and options that are provided by the web often comes with a harrowing hidden tradeoff. While we can certainly learn a lot on our own and become (what feels like) an instant expert on any topic in an afternoon, we usually do so in the absence of a mentor and outside the context of applying expertise to solve actual problems. In my opinion, a cadet should have to survive more than a few Kobayashi Maru scenarios before he gets to declare himself, "Captain."

Call it a guru, a wizard, an old butcher, or what have you, the mad echo chamber of a young mind often benefits from the dampening influence of an experienced grownup who can help you understand things that raw data, wikipedia entries, and lists of tips and tricks can't and wont ever do.

We benefit from a hand on the back and a gentle voice, reminding us:

• "Try not to obsess over implementation until you really understand the problem," or
• "Worry more about relationships than org charts or follower counts," or
• "Don't quit looking after you've found that first data point," or—my favorite—
• "Spend less time fantasizing about 'success' and way more time making really cool mistakes."

Conversely, though, I think this means that everything we think we know, as well as all the fancy advice that gets thrown around—absolutely including the material you're reading now—is the product of what one person knows and what another person has the ears to hear. For us. For now. For who really knows what. But it is a transaction that takes place in a very specific time and within the bounds of a set of "known" "facts." So, fair warning, doing your own due diligence never hurts.

What's Almost Not Impossible?

[N.B.: I swear to God this ends at some point, Xx]

One big pattern for "future-proofing" your passion? Keep your eyes open and your heart even "opener." And, be more than simply tolerant of the notion of change—sure, take it as read that nothing is ever fixed in place for more than a little while.

But, to the extent that your sanity can bear it, always keep an eye on the corners, the edges, and especially learn to watch for those infinitesimally tiny figures starting to shuffle around near the horizon. Because a lot of the things that seem ridiculously small and inconsequential right now will eventually cast a shadow that people will be chasing for decades. It's just that we're never sure which tiny figure that will turn out to be.

So, yeah. It really is true that no one but you cares about your major. But, trust me: everybody is interested in the person who repeatedly notices the things that are about to stop being impossible.

Be the curious one who soaks in all that "irrelevant" stuff. And, even as you stay heads-down on the "now" projects that keep the lights on, remember that the guy who invented those lights made hundreds of "failed" lightbulbs before fundamentally upending the way we think about time, family, industry, and the role of technology in how we live and work. But, yes, first he "failed" a lot a lot at something which more than a few of his contemporaries thought was pointless in the first place.

Ask: What's out there right now that's about to stop being impossible? Where will it happen first? Who will (most loudly and erroneously) declare it's total bullshit? Who will mostly get it right—but possibly too early? Who will figure out what it means to our grandkids? Who will figure out how to put it in everyone's front pocket for a quarter?

Y'know who? I'll tell you who: practically anybody BUT that guy in the racecar bed who wants to talk about his major.

Important: Merlin's Advice is Only Future-Proof to 10 Meters

A few years back, most watch manufacturers decided to come clean and stop categorically declaring that their timepieces were "waterproof." Instead, today, the more credible vendors admit their product is merely "water-resistant"—and, even then, they'll only guarantee the underwater functionality at so many meters, and for so long, and under thus and such conditions.

Truthfully, the same applies here. Nothing can actually "future-proof" anything. Anyone who claims to know the future is either a madman, a charlatan, or, often as not, both.

Thing is, regardless of the passions (or goals or values or priorities or whatever) that we hope to protect or defend, we'd all do well to remember that it is still ultimately OUR passion that's at stake.

That means we're the only one responsible for seeing that its functional components survive and adapt in a world in which each one of us has just north of zero control.

If we embrace the fact that no one can or should ever care about the health of our passions as much as we do, the practical decisions that help ensure Our Good Thing stays alive can become as "simple" as a handful of proven patterns—work hard, stay awake, fail well, hang with smart people, shed bullshit, say "maybe," focus on action, and always always commit yourself to a bracing daily mixture of all the courage, honesty, and information you need to do something awesome—discover whatever it'll take to keep your nose on the side of the ocean where the fresh air lives. This is huge.

Anything else? Yeah. Drink lots of water, play with your kid every chance you get, and quit Facebook today. No, really, do it.

Thanks again for the note, Xx, and sorry for the novella. I'll ping you if the audio ever turns up. Til then, forget your major, and break a leg!

yr internet pal,
/m

The DML News App offers the best in news reporting.

The post BREAKING: FDA issues emergency authorization for first coronavirus antigen test appeared first on Dennis Michael Lynch.

The Shelburne Retirement Residence has been devastated by an outbreak of COVID-19, with 90 per cent of its residents becoming infected.

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

Restaurants have been struggling to stay afloat since the pandemic hit, but there has been a silver lining: relaxed liquor laws mean customers can get their booze delivered along with their meals.

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 […]

Continue reading: Photos Of Kim Jong Un Spark Conspiracy Theories About A Body Double…You Be The Judge ...

The following article, MI Barber Shop Owner Defies Gov. Whitmer Shutdown Order…Opens Business…Police Officer Walks In…Says, “I love you!”…Walks Out, was first published on 100PercentFedUp.com.

Last week, we reported about how Shelley Luther, owner of the Salon Á La Mode in North Dallas, Texas, who opened up her business in defiance of lockdown orders in the city. The salon owner said that she was ignoring a citation and a cease and desist order from the city to shut down. This one […]

Continue reading: MI Barber Shop Owner Defies Gov. Whitmer Shutdown Order…Opens Business…Police Officer Walks In…Says, “I love you!”…Walks Out ...

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

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

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

The following article, BREAKING: MI Lawmakers File Lawsuit Challenging Governor’s “Improper” and “Invalid” Emergency Orders: “We’ve attempted to partner with our governor, but she’s rejected”, was first published on 100PercentFedUp.com.

In addition to crushing Michigan's economy, the governor's reckless, one-size-fits-all executive orders are harming an untold number of Michigan citizens.

Continue reading: BREAKING: MI Lawmakers File Lawsuit Challenging Governor’s “Improper” and “Invalid” Emergency Orders: “We’ve attempted to partner with our governor, but she’s rejected” ...

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

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

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

The following article, Attorney for Jailed Dallas Salon Owner Shelley Luther Slams Obama Loyalist Judge as a “Tiny Tyrant”, was first published on 100PercentFedUp.com.

Warren Norred, attorney for Shelley Luther, joined Tucker Carlson to discuss the decision by an Obama loyalist Texas judge to jail his client for reopening her salon during the COVID-19 pandemic. Norred said the judge has exposed himself for the “tiny tyrant” that he is. The judge jailed Luther and even fined her $7,000. Texas […]

Continue reading: Attorney for Jailed Dallas Salon Owner Shelley Luther Slams Obama Loyalist Judge as a “Tiny Tyrant” ...

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, Texas Gov Abbott Frees Salon Owner Shelley Luther: “Criminals shouldn’t be released to prevent COVID-19 just to put business owners in their place”, was first published on 100PercentFedUp.com.

Texas Governor Greg Abbott just changed the coronavirus order to free Salon A La Mode owner Shelley Luther from jail. Abbott tweeted out a comment about the poor treatment of the business owner: Throwing Texans in jail whose biz’s shut down through no fault of their own is wrong. I am eliminating jail for violating […]

Continue reading: Texas Gov Abbott Frees Salon Owner Shelley Luther: “Criminals shouldn’t be released to prevent COVID-19 just to put business owners in their place” ...

The following article, Video: Salon Owner Shelley Luther Released from Jail After Obama-Loyalist Judge Sentenced Her to 7 Days, was first published on 100PercentFedUp.com.

Salon A La Mode owner Shelley Luther was just released from jail today. The Texas Supreme Court and Texas Governor Greg Abbott both called for her release today (see below). When Luther left jail she was greeted by supporters who yelled “Shelley’s Free!” She thanked all of the new friends she’s made during this difficult […]

Continue reading: Video: Salon Owner Shelley Luther Released from Jail After Obama-Loyalist Judge Sentenced Her to 7 Days ...

The following article, BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations, was first published on 100PercentFedUp.com.

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 […]

Continue reading: BREAKING: New Docs Prove Obama Knew Details Of Flynn Wiretapping…Newly Surfaced Video Shows Obama Explaining How He Stays Out Of FBI Investigations ...

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, 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.

Yesterday, after 3 1/2 years of having his character and integrity called into question, President Trump's first NSA, General Michael Flynn was finally...

Continue reading: Greg Gutfeld Levels Ilhan Omar With Epic Response To Her Claim That “White Privilege” Is Reason Charges Were Dropped Against General Flynn ...

The following article, Video: Dallas Salon Owner Shelley Luther Gets Surprise Visit from Sen Ted Cruz for a Celebratory Haircut, was first published on 100PercentFedUp.com.

When Dallas, Texas salon owner Shelley Luther opened her salon in defiance of the lockdown order in Texas, she was visited numerous times by the local police and then sentenced to 7 days in jail with a fine of $7,000. After public outrage at her punishment, the Texas Supreme Court stepped in to demand her […]

Continue reading: Video: Dallas Salon Owner Shelley Luther Gets Surprise Visit from Sen Ted Cruz for a Celebratory Haircut ...

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

Health officials with the Manitoba government announced on Friday that a COVID-19 outbreak at the Health Sciences Centre has officially been declared over.

Environment Canada has issued a special weather statement for the southwestern area of Manitoba.

Instead of May flowers, Manitobans in the southwest part of the province received a blanketing of snow for Mother's Day weekend.

Research Event

Event participants

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

4 March 2020

7 May 2020

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

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

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

Heterotrimeric G proteins are the core upstream elements that transduce and amplify the cellular signals from G protein–coupled receptors (GPCRs) to intracellular effectors. GPCRs are the largest family of membrane proteins encoded in the human genome and are the targets of about one-third of prescription medicines. However, to date, no single therapeutic agent exerts its effects via perturbing heterotrimeric G protein function, despite a plethora of evidence linking G protein malfunction to human disease. Several recent studies have brought to light that the Gq family–specific inhibitor FR900359 (FR) is unexpectedly efficacious in silencing the signaling of Gq oncoproteins, mutant Gq variants that mostly exist in the active state. These data not only raise the hope that researchers working in drug discovery may be able to potentially strike Gq oncoproteins from the list of undruggable targets, but also raise questions as to how FR achieves its therapeutic effect. Here, we place emphasis on these recent studies and explain why they expand our pharmacological armamentarium for targeting Gq protein oncogenes as well as broaden our mechanistic understanding of Gq protein oncogene function. We also highlight how this novel insight impacts the significance and utility of using G(q) proteins as targets in drug discovery efforts.

The 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.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

6 January 2020

Invitation Only Research Event

Research Event

Event participants

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

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

Chatham House is pleased to announce that Adam Ward will join the institute in a new role as deputy director.

Creon Butler has been appointed to lead the Global Economy and Finance programme at Chatham House, joining the institute at the beginning of December. He will also form part of the institute’s senior leadership team.

Members Event

Event participants

Anders Fogh Rasmussen, Secretary-General, North Atlantic Treaty Organization (NATO)
Chair: Robin Niblett, Director, Chatham House