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28 to 30 cm.. tall and around 210 weight g.
The Bronze winged Parrot (Pionus chalcopterus) has the general Brown, darker in head, neck, chest and flanks, This plumage with blue-violet edge.
The throat whitish and pink; wings Bronze Green.
Flight is evident violet blue at the top of the wing the Green bottom, that contrasts with the Red of the lower abdomen. Dorsally Violet Blue is notorious in the rump, shoulder and tail. The feathers of the tail are dark blue with a shade of red at the base.
The bill is of color beige, area of naked skin surrounding the eyes of pink. The legs They are pink.
- Sound of the Bronze winged Parrot.
It inhabits in rainforest, semi-húmeda, crops and forest edge between 120 and 2800 m.
They are birds gregarious and noisy. Every tomorrow is mobilized in flocks in search of food. Within the flocks are can observe to the couples, that form of life, and to interact with a variety of behavior of contact that reaffirm the conjugal relationship.
Prefer for their nests broken rods, hollow of logs and palm trees. They nest in the months of October-may. The female often put in two to four eggs they are incubated during three weeks approximately, after which are born them chicks naked or just covered with down. The chicks remain with their parents until the next reproductive shekel.
Will congregate in cornfields in groups up to of 40 individuals, feeding is of pulp and seeds many fruits and other material ingredients as nectar and flower petals, complementing occasionally its diet with insects.
Its population is distributed by the mountain range of the Andes, from Colombia, Northwest of Venezuela; to the South, by the mountains to the Northwest of Peru.
• Red list category of the UICN current: Least concern
• Population trend: Decreasing
The size of the population World of the Bronze winged Parrot It has not been quantified, but this species is described as 'rare' (Stotz et to the., 1996).
The population was suspected of being in decline due to ongoing habitat destruction.
This species is characterized be decreasing in Colombia and West Ecuador, due to the logging of forest subtropical, which has been severe and quick on the slopes of the Andes (Juniper and Parr 1998).
The evident anecdotal, based on the frequency of sightings, suggests that the species has declined in Pineapples, South of Ecuador, c.15-20 years (M. By D Sanchez. Diaz in litt. 2011). Also is says that has been extirpated from areas previously occupied as the slopes of them Andes and valleys of the Cauca and the Magdalena river in Colombia, again due to loss of habitat (Juniper and Parr 1998).
In Venezuela the species is scarce and local, limited, to a large extent, to the slope West of them Andes in Mérida and Sierra de Perija, with occasional records in Táchira (CJ Sharpe in litt. 2011). These areas have experienced rapid deforestation during at least the last 24 years for the crop and the livestock.
Surveys conducted in the Northwest of Peru in the Decade of 1990 they appeared to detect a sharp decrease in population from 1993 (Rosales et to the., 2007), Although this species is known as Nomad and their local numbers may fluctuate.
Alibronceado parrot in captivity:
Rare in captivity, It is not a main target of poachers (CJ Sharpe in litt. 2011, R. Clay in litt. 2011), Although it is still present in illegal trade of Peru (F. Angle in litt. 2011)
Intelligent, playful and curious. You can be nervous, loving with your caregiver and aggressive with other people. It has a tendency to obesity.
It requires a spacious cage unless the bird pass periods of time outside of it. Not must keep is to open air where the temperatures fall below them 5 ° C.
With feed a wide variety of seeds, fruits and berries.
It is a relatively healthy bird, While it may be more susceptible to infection aspergillosis that other species.
In captivity they lay two eggs the incubation takes 26 days.
Alternative names:
– Bronze winged Parrot, Bronze-winged Parrot (English).
– Pione noire, Pionus noir (French).
– Glanzflügelpapagei, Glanzflügel-Papagei (German).
– Curica-asa-de-bronze (Portuguese).
– Cotorra Oscura (Colombia).
– Cotorra Negra (Venezuela).
– Cotorra Oscura, Loro Alibronceado, Loro de Alas Bronceadas, Cheja alibronceada (Spanish).
Scientific classification:
– Order: Psittaciformes
– Family: Psittacidae
– Genus: Pionus
– Scientific name: Pionus chalcopterus
– Citation: (Fraser, 1841)
– Protonimo: Psittacus chalcopterus
Bronze winged Parrot (Pionus chalcopterus)
- Avibase
- Parrots of the World – Forshaw Joseph M
- Parrots A Guide to the Parrots of the World – Tony Juniper & Mike Parr
- Birdlife
(1) – IBC.lynxeds.com
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- Sounds: Sergio Chaparro-Herrera


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This is not about Canada
It must be clear that Canada is not the one in need of support from the international community. It is the Saudi feminists and activists who have made this call for solidarity who need this support.
The effects of Saudi authorities' actions towards Canada are minimal. The same cannot be said of Saudi civil society.
The reason why this call was made in the first place is because it was becoming clear that Saudi authorities were suffocating all breathing room for activists.
When this is referred to as a "diplomatic fallout" that must be resolved between "two sides", Saudi civil society —and its demands—are erased from the narrative. This is not an infringement on but a support of national sovereignty of a people over the whims of one man.
Neutrality, here, is nothing more than an expression of indifference towards state repression when committed by an ally. It does nothing but embolden the Crown Prince to further escalate his all-out war against civil society.
The Crown Prince is creating a dystopian reality where dozens of opinion writers have to become overnight experts on his arbitrary enemy of the week. Remaining silent and not expressing support for each and every decision he makes could land a public figure in jail.
Saudi newspapers have finally come to care about human rights violations, but only in Canada, e.g. Human Rights Watch reports about discrimination First Nations women experience. Needless to say, the same attention is never given to reports on systemic discrimination that Saudi women face.
Never will you find in Saudi media any mention of HRW 'Boxed In' report—among many other reports about Saudi Arabia's plethora of human rights violations—, an extensive report about sexist discrimination in the country, the jailed activists were working to abolish.
This is the reality that we and our communities are dealing with at the moment. This is why we have made a call for international solidarity. People continue to live in fear, of state repression, and of where Mohammad bin Salman's rash, self-destructive decisions will take our country.
This is not a time to back down. We must maintain our solidarity with Saudi activists and feminists, and continue to condemn the state's ongoing campaign to crush civil society.
This is not about backing Canada, it is about supporting Saudi communities working towards a better future.

subset: commoncrawl


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Thomson Confinement Food Home Delivery
Meal Activation
Here are the points you may like to know:
- We require two working days notice to deliver your first meal.
- Please submit the form in this page and we shall call you back to confirm the exact delivery date and meal.
- Or, you may call us at 63508848 during office hours for meal activation.
- We deliver daily including weekends and public holidays, EXCEPT Christmas Day, New Year, first and second days of Chinese New Year. Meals for these four days will be deferred. Meal rotation will not be affected.
- Delivery Time: Lunch 10am-1pm; Dinner 4:00pm-7:00pm


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We're less than two weeks away from the release of the LHSAA Football playoff brackets. Kevin Foote and James Bewers look at the current power rankings, and talk local teams' chances of getting in. Lafayette Daily Advertiser
There are have been many great offenses in the Acadiana area over the years.
A special few, though, just have this glamorous appeal to them.
These are units opposing defenses just fear. There almost doesn't appear to be a way to stop attacks of their caliber.
St. Thomas More's offense in 2018 is certainly one that's achieved that reputation.
It indeed may be the most complete offense I've seen in 38 years of watching high school football in this area.
And when pondering Friday's District 4-4A matchup between STM and the Carencro Golden Bears at Cougar Stadium, the thought hit me that another one of those highlight-film offenses for the ages was the Kevin Faulk-ignited rushing attack of the 1990s at Carencro.
"They're probably on their second set of lights on their scoreboard this year with the way they've been lighting that thing up," Courville joked about STM's offense this season.
Coach Shane Savoie's certainly has the ability to score from anywhere on any given play with the explosive running of William Cryer or with the precise passing game of Caleb Holstein.
"They do present you with a lot of dilemmas," Courville said. "You kind of get baffled when you start talking about it. Every skill position — and it doesn't make a difference which personnel grouping they're in — every skill position has guys who can hurt you.
"If you try and double up on one, somebody else can hurt you. They just keep you back on your heels."
Courville was an assistant coach on those Faulk-era Bears teams, including the 1992 state championship squad. In fact, Courville did quite a bit of reliving those special memories this past summer as his offense shifted to a veer concept under new offensive coordinator Gavin Peters this season.
"Absolutely, I did a lot of that this summer," Courville said. "Going to the veer this year, we ran a lot of inside- and outside-veer stuff in those days. It was more of a ground attack back then. It was a three-headed monster. It was just which two were going to hurt you."
Savoie was playing for St. Thomas More in those days thankfully not in the same district as those Golden Bears.
"We got to play them in a jamboree game one year, and it was pretty humbling to say the least," Savoie laughed.
Much like this year's Cougars, Carencro's offensive weapons were the talk of the town in those days.
"They were something special for sure," Savoie remembered. "A lot of it had to do with the scheme they were running, but the scheme really fit the talent they had at the time.
"The biggest difference was you had Derrick Beavers, you had Kevin Faulk, you had Ernest Lazard and the big tight ends. They were just so physically dominant and athletic. I'd say some of Acadiana's years with Ted (Davidson) were pretty close to them."
Much like STM has been this season, the Golden Bears were must-watch TV in the 1990s.
"I can remember watching the highlights," Savoie said. "We would get home from games just to try to get the TV on so we could see what Kevin Faulk did that night and what he shaved into his head that week. He used to shave designs into his head each week."
The star power was everywhere.
Those Bears teams even had stars on defense and at placekicker.
While the Cougars utilized the new-school spread offense, things weren't nearly as complex for coach Mac Barousse's offense in the 1990s. Barousse, who was quite familiar with arguably the parish's first Hollywood offense with the Mighty Lions of the 1970s as a Lafayette High graduate, knew thoroughbreds he had in his arsenal and he simply gave them the reins.
More: Can anyone stop STM's juggernaut offense?
"Especially on the high school level, you always have to tailor what you're doing to your personnel," Savoie said. "We sit down each year and identify who our playmakers are, and then we try to tailor what we do to try to get them the ball in space and give them the best chance possible to make plays."
In other words, sure Savoie enjoys the new-age passing game. But if he had a Kevin Faulk, he wouldn't be chunking it on a regular basis.
"If I had a Kevin Faulk, or even if I was doing what I'm doing now with a Mikie Mahtook, we would be doing a whole lot of different things with quarterback runs and different things with him because of his ability as an athlete," Savoie explained.
This year, Savoie possesses a big-armed quarterback, a host of talented receivers, a powerful offensive line and a running back who does it all. The result is the Cougars are averaging an incredible 60 points a game.
More: Cougars, sports help Howard family deal with tragic loss
"But one year, we had two Division I tight ends (Jordan Murray and Evan Tatford), so we ran a lot of two tight ends that year," Savoie said. "It's the same kind of base package, but how we got to different things is different based on the talent we had. We can manipulate it based on our guys."
Naturally any coach would take either offense in a second. This STM offense will be talked about for decades and here I am still bringing up the Bears of the 1990s.
But as much as he's agonizing over trying to devise a plan for these Cougars, Courville would vote for the dominant rushing attack if he had to vote.
More: Allen, defense ignite Carencro win
"I'll be totally honest with you, I'm still more — I don't call it old-school; I call it real-school. The ground attack gives you TOP — time of possession," Courville said. "Our defense continues to get better, but our ground attack with a timely passing game is what's helping our TOP and helping our defense.
"It's absolutely easier to play defense with a good ground attack. I'm hoping that's what's going to happen this weekend. It's going to need to happen to hopefully keep that scoring machine on the sidelines."
The undefeated LCA Knights were looking to shut out their opponent for the eighth week in a row against the undefeated Vermilion Catholic Eagles. The Eagles ended the streak, but the Knights came away with the victory.


text: Baked potatoes (yes — white potatoes!) are an excellent source of potassium, which can help beat bloat and counterbalance sodium. Since they've got filling fiber, spuds also help you stay fuller, longer. Avoid the deep fryer, though. Potatoes are a nutrient-dense food as long as they're not served the french-fry way.


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Update for your love machine
Version: M3s V1.7
File size: 45.5MB
Release time:Updated August 27, 2018
Developer: ShanLingDownload
1. The text on the left side of the status bar is replaced by the "play" and "pause" icons, and corresponds to the playback status;
2. Add: "Lyrics mode". After opening, the lyrics interface screen is always on;
3. Add the "PO/LO" port identifier to the status bar;
1. Go to the product website of Shanling official website to download the latest firmware update.upt.
2, to ensure that the player is fully charged, unzip the compressed package, put the update.upt upgrade file in the compressed package into TF In the card.
3. In the system settings, select the system upgrade. After the upgrade is completed, the system automatically restarts and the upgrade is completed.
4. If the card is pulled out during the upgrade process, the machine will display the words insert TF Pls. At this time, only the card needs to be reinserted.
You can continue to upgrade.
5. If you choose not to find the upgrade file after upgrading the system, please check whether the compressed package is extracted and the upgrade file is placed.
The root directory or the upgrade file is damaged during the transfer process. The upgrade file update.upt needs to be placed in the root directory of the memory card.
If the upgrade file is damaged during transmission, please re-import the upgrade file into the memory card.
Driver: M3s USB driver
File size: 1.60MB
Release time: 2018-10-15
Developer: ShanLingDownload
Please visit the official website http://www.shanling.com to enter the product page to download and install the latest firmware.
Open the machine to enter the "System Settings" interface, select the "USB Mode" item and set it to the "DAC" state and return to the main interface.
After connecting to the computer, please extract the file "M3s USB driver.rar" and install the "setup.exe" file to the computer.
Right click on the "Sound" option on the computer and select "Playback Device" and check "Speaker SHANLING AUDIO DEVICE" as the default device, as shown above.
Please log in to the official website http://www.shanling.com to enter the M3s product page for related downloads.


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We all know the gut-wrenching, stomach-dropping feeling associated with hydraulic equipment breaking down on the job. It can mean lost time and money, neither or which are good. So, where do you turn when your hydraulic machines breakdown? Who do you trust to get the job done right in a timely, efficient, and affordable manner? Truthfully, you should rely on Bernell Hydraulics to repair your hydraulic equipment.
When your hydraulic equipment breaks, you need to act fast. No one wants to experience downtime, especially when you have quotas and deadlines to meet. Thankfully, Bernell Hydraulics offers repair services around the clock. That's right, 24/7 emergency hydraulic repairs in Riverside, CA and the surrounding areas. It doesn't matter what time or day it is, Bernell Hydraulics will send an experienced mobile tech out to diagnose the problem and get you back up and running fast.
You might be wondering what makes Bernell Hydraulics so special. We get it, you want to make sure that you are trusting your hydraulic equipment with someone worthy that has the expertise necessary to get the job done right. Well, at Bernell Hydraulics, we have over 43 years of experience in the hydraulics industry. Our technicians and engineers are some of the best in the business and you can rest assured that we will be able to get your equipment back up and running. Since 1977 we've been providing hydraulic repair services in the Inland Empire area of southern California and our resume speaks for itself!
At Bernell Hydraulics, we don't just excel at hydraulic repairs. In addition to our 24/7 repair services, we also have four conveniently located retail stores offering full lines of quality hydraulic components. Our online store also provides world-wide access to hydraulic parts and components. Plus, we also provide hydraulic maintenance services for those who don't have time to regularly inspect and maintain their equipment. At Bernell Hydraulics, we also offer hydraulic builds to meet your design specs. Whatever your hydraulic needs are, you can trust the experts at Bernell Hydraulics.
If your hydraulic system is malfunctioning or has failed completely, call Bernell Hydraulics right away at 909-899-1751. Our technicians will come out as quickly as possible to diagnose the problem and work to get your equipment back up and running as quickly as possible. No matter what time of day or night it is, you can trust Bernell Hydraulics to get the done in a timely and efficient manner. If you are looking for the best hydraulic repair services in Riverside, CA and the surrounding areas, call Bernell Hydraulics right away!


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+ Added Recipes 4 years ago in Deserts / Sweets, Breakfast, Snacks, Pasta
Hello and welcome to Sweet and Savoury Pursuits, my creative place on the net! My name is Fida, and I have a passion for food. I think good food should be celebrated and I take joy in cooking for others and love making people happy through food.I started this blog about a year and a half ago.&...read more


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Babyliss 7860U Pro Beard Trimmer
Type: Beard Trimmers , Manufacturer: Babyliss
- Type
- Beard Trimmers
- Power Supply
- Battery
Important informationSpecifications are collected from official manufacturer websites. Please verify the specifications before proceeding with your final purchase. If you notice any problem you can report it here.
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Our client-vested approach ensures that we only offer the services that will best suit your business and your needs. We provide the following accounting related services to help your business achieve success. All services are available remotely.
Business Structure & Formation– Determining the appropriate legal structure for your business such as Limited Liability Company (LLC) or Corporation (S-Corp, C-Corp). Register business license, annual state filings, and registered agent services.
Bookkeeping– Invoices, receivables, payables, and bank reconciliations.
Outsource– CFO, controller, or full accounting department can be handled by our team. Reduce your overhead and operating costs. We provide a confidential consultation.
Taxes– Business and personal taxes.
QuickBooks – We specialize in QuickBooks and can manage your QuickBooks account from setup to monthly/quarterly accounting. Training can also be provided.
Payroll Services– Payroll, payroll taxes, payroll related forms & filings.
Financial Statement Preparation– Customized financial statements.
Financial & Business Analysis– Includes industry benchmark assessment.
Operational & Process Improvement- Develop efficiencies in business operations, provide deliverables, and cost reduction strategies.
Centralization– Entrepreneurs with multiples business can centralize these functions with us providing a one-stop shop and cost savings.
We work not only to see your business successful, but to see YOU successful! Each business owner is unique and deserves a customized solution for your needs. Schedule your free consultation to discuss the future of your success with us.


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Gothic Quater Cannabis Tour
|Posted on June 7, 2018 at 7:30 AM
For more than 2,000 years, the Gothic Quarter has been the spiritual and Buzzing center of the city. The Relics of ancient Roman buildings can still be found on the many historical buildings, However The Middle Ages are best represented into this quarter.
There are many masterpieces to be found in Gothic area and the beautiful architecture that lays in the narrow streets of this ancient quarter, We will visit the medieval cathedral that stands out in the city center en we will wander around aimlessly, but with a destination in mind to discover this magical and traffic-free medieval world of Barcelona's inner city.
We will explore around in a delightful maze of narrow cobblestone filled streets and her atmospheric and magical alleyways. We will Discover some of the picturesque and genuine old squares embedded by the sounds of people talking and laughing that echoes in to the Gothic Quarter's hidden corners.
During the tour will pass by and visit some of the many little cafés that are found in this area. With its inviting Attitude and our Cannabis friendly Mood, the little boutiques, restaurants, and museums Makes the Gothic Quarter the perfect place to come and explore with us.
We will show you all the nice areas that you can revisit by yourselves so you can benefit and enjoy your Trip from the first day until the last day. During this Cannabis Friendly tour we will Visit 2 Social-cannabis Locations where you obtain your membership for the rest of your holidays and we visit the Cannabis Museum.
For more information feel free to contact us directly or by email.
Smoke weed in Barcelona Now and let us be your Guide!
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Do you have a spare room? Would you like to help someone who is homeless? Hosting may suit you!
Action Housing provides accommodation for destitute asylum seekers with no recourse to public funds.
Action Language provides free English courses in Newcastle and Sunderland city centre.
LET is a pilot project to provide training and resources to enable small community organisations to start beginner English language sessions.
InterAction provides a friendly, accessible, interactive and supportive environment to help new and other migrants experiencing loneliness, isolation and feelings of disorientation, to connect with other people and cultures, local places and services.
Action Access offers a community-based alternative to clients who have been refused asylum and are currently held in detention. The project has been designed in partnership with the UNHCR and the Home Office.


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1935 (Oct 23) Boston, Mass - Macao, Portuguese Chinese Asia. 1c green stat card + adtl. Arrival cachet on front. Raarity destination. VF.
1933 (4 Febr) Portland, Oregon - MACAU (6 March) Portuguese China. 1c green stat card + 3 adtl + arrival on front. Lovely itemm
1894 (22 Feb). San Antonio, TX - Germany (9 March). 2c green stat env 2 adtls. Fine.
1894 (9 Jan). Schwenkvile / PA - NY. 3cts Colon issue stat env. Fine cancel.
1907. 2c red albino embossed stat env.
1930 (14 Feb). Baltimore, MD. 2c red stat env private print hold for postage + US Lincoln added on next day (15 Feb). Fine.
1920 (4 April). SF, CA - Australia, Sydney (25 May). 1c green stat card taxed arrival Aussi postage due 2d bicolor tied arrival cds v nice cond combination slogan cancel aux pmk join the Navy. Danish Consul.
1934 (15 MArch). Charleroi, PA - Macao, China (13 April). 1c green stat adtl with arrival cachet alongside. V rare dest.
1941 (9 April). Chicago, Ill - Macau, Asia (14 May 41). 1c green stat card + 2 adtl red Cross issue cds with arrival date cachet po reverse VF uncensored at WW II tiem post unususal.
1882 (24 Oct). Collegeville, Minnesota - Sweden, Stockholm (9 Nov). 5c blue stat env 3 adtls taxed Swedish pdue 40ore oval blue. Fine.
1926 (6 Oct). Sweden - New Haven Comm US 2cts red stat env sent back as reply service with adtl wedish stamps tied Stockholm. Fine comb.
1855 (7 Sept). Ottawa, Ill - Germany. Wuttemberg, Betra (28 Sept). US 3c red stat env on paid 30 (3cts stat) transatl usage. Via Liverpool - Aachen (25 Sept). Franco. V scarce so.
1869 (6 Oct). Detroit, Mich - Canada, Ontario, Sincor (8 Oct) arrival cds 3cts rose stat env cds 10 due charge. US pension agent leave cork cancel. Fine used to foreigh dest.
1870 (28 Feb). Tamola Station, Mississippi - Vermon, NJ 3 cts rose stat env used from rail station manuscript cancel date this was a PO between 1866-79. interesting. Nice item.
c.1874-5. chicago - Germany. 1c blue yellow stat env via England fancy town oval canel. Nice item.
c.1876. US 3cts grreen Centenary stat env uncirculated. VF (U 219O, complete and faultless.
1877 (16 Oct). St Louis, Mi - Switzerland, Airolo, Ticino (2 Nov) 1c US stat card 1c adtl blue arrival cds. Fine.
1879 (13 Dec). Phillipsburg - DC. Official War Dept 3c red state env. Fine. Appears fancy cancel.
c.1879. Wimington WC - Richmond, VA. 3c green stat env due 3c arrival postage due 3c tied fine early comb.
c.1882 (22 March). Nashville Tenn. Local official 6c PO stat env black on yellow. Fine used.


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I have a good deal for you Dove Body Lotion lovers. There's a new coupon available on Red Plum for$1.50 off one Dove Body Lotion. This is a Target coupon that can be combined with the manufacturer coupon available in the 10/4 RP insert to get $3 off one bottle. If you do not have this manufacturer coupon from the newspaper inserts, then you can use this printable coupon for $1 off one Dove Lotion.
Thanks KC Penny Pinching Mama!


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Do you have the skills, desire, willingness and expertise to build an exceptional sales organization?
- Do your sales leaders thrive on growing and developing people?
- Are they highly passionate about the team's success?
- Can they remove their egos and accept mini failures?
Great sales leaders understand their limitations, leverage their strengths, ask for help and find ways to implement other people's ideas.
The most common concern I hear is inconsistent execution. There are many causes of this, not the least of which is, do you have the right people in the right roles? Other issues include metrics, inspection, urgency and management skills. How effective is your sales leadership as coach, mentor, motivator, recruiter and driver of accountability and what are you doing to improve your salespeople's skills?
In almost every interview I conduct the candidate says, "I'm a consultative salesperson". The problem is they can't explain the "how" and most of the time they don't demonstrate they have the skills. What is your value proposition, can your sales team articulate it, is there a selling process, do they follow it and how effective are they at differentiating themselves from the competition and creating high value relationships?External influences impact our attitude, bravery and clarity. Salespeople react differently to this; some need close monitoring and others a kick in the back side. Sales leaders must know their people better than they know themselves. Are you a great role model for attitude, bravery and clarity? What causes you to loss focus and where do you go for help?


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Where does your donation go?
Learn more about how Team Adem helps others.
Team Adem ABN 26 901 199 294 is a registered charity through the Australian Charities and Not-for-profits Commission.Team Adem is also DGR approved having passed very stringent criteria.
All donations made to Team Adem of $2 and over are Tax Deductible.


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This article will give you the information you need to ensure all necessary steps are completed when getting your WinRed organization approved.
Note: Currently WinRed is only accessible to federal candidates and state parties. State-level candidates are welcome to use Revv to collect donations until WinRed becomes available.
Existing Revv Accounts
Reach out to our team at [email protected] and we'll be happy to assist in setting up a WinRed account for your organization.
New Accounts
1. Request an Account
The initial step in this process is to request a WinRed account by heading to https://winred.com/contact/. Fill out the required information and submit.
A member of our team will reach out with the Account Registration link when we are ready to onboard your committee.
2. Input your Information
The registration link will bring you to the registration portal where you will be asked to input your committee's information. We strongly recommend having everything ready before you begin to speed up your approval.
- Business Fill in your Business Name (must match the name on your IRS documentation), Website, Phone Number, and Tax ID Address.
- Account Owner: Fill in your First and Last Name, Email, Phone Number, Address, Date of Birth, Last 4 Digits of Social Security Number, and Front and back picture of your driver's license, passport or other government ID.
Click Submit Verification
Bank Account:
There is one more step you'll need to take in order to be approved: Fill in your Bank Account information. Once you submit verification, you will be directed to your WinRed dashboard. You can also login at app.revv.co and click the WinRed tab at the top of your screen. You must be on the WinRed side in order for this step to be considered complete. You should see this icon in the top left corner:
Click on your icon in the top right-hand corner and click Settings in the drop down menu.
Click on Bank Account on the left side of your screen, and click the green Add Account button. Fill in your Bank Account Name, Routing Number, and Account Number.
If you are unable to complete the above steps in one sitting, you can go back and finish. When you do, however, make sure you are on the WinRed side and not the Revv side. To confirm that you are on the WinRed side, the icon below should be in the top-left corner of your screen:
If you do not see this icon, and instead see the purple Revv icon (seen below), simply click the WinRed tab at the top of your screen to be taken to the WinRed side.
Head to your settings by clicking your icon in the top right-hand corner of your screen and clicking Settings in the drop down menu. Use the tabs on the left to complete your information.
3. Get Approved
Once you've submitted your verification and completed your Bank Account information, our team will be able to approve your organization and you can begin collecting donations.
Note: If you are working with a digital marketing agency please let us know as we are working very closely to get them trained up on the ins and outs of the platform. More than likely, we've already taught them the product, but let us know.
As always, reach out to our team at [email protected] if you have any questions on this process!


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Hypothesis and Theory ARTICLE
Soft TCPTP Agonism—Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine
- 1Department of Biomedical Sciences for Health, University of Milan, Milan, Italy
- 2Tixupharma, Milan, Italy
- 3Laboratory of Immunorheumatology and Tissue Regeneration, Rizzoli Orthopaedic Institute, Bologna, Italy
- 4Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna, Italy
A reparative approach of disrupted epithelium in obstructive airway diseases, namely asthma and chronic obstructive pulmonary disease (COPD), may afford protection and long-lasting results compared to conventional therapies, e.g., corticosteroids or immunosuppressant drugs. Here, we propose the polyamine spermidine as a novel therapeutic agent in airways diseases, based on a recently identified mode of action: T-cell protein tyrosine phosphatase (TCPTP) agonism. It may include and surpass single-inhibitors of stress and secondary growth factor pathway signaling, i.e., the new medicinal chemistry in lung diseases. Enhanced polyamine biosynthesis has been charged with aggravating prognosis by competing for L-arginine at detriment of nitric oxide (NO) synthesis with bronchoconstrictive effects. Although excess spermine, a higher polyamine, is harmful to airways physiology, spermidine can pivot the cell homeostasis during stress conditions by the activation of TCPTP. In fact, the dephosphorylating activity of TCPTP inhibits the signaling cascade that leads to the expression of genes involved in detachment and epithelial-to-mesenchymal transition (EMT), and increases the expression of adhesion and tight junction proteins, thereby enhancing the barrier functionality in inflammation-prone tissues. Moreover, a further beneficial effect of spermidine may derive from its ability to promote autophagy, possibly in a TCPTP-dependent way. Since doses of spermidine in the micromolar range are sufficient to activate TCPTP, low amounts of spermidine administered in sustained release modality may provide an optimal pharmacologic profile for the treatment of obstructive airway diseases.
Asthma and chronic obstructive pulmonary disease (COPD) are widespread respiratory disorders, both responding to bronchodilator drugs and to a reduction of toxic insults, e.g., smoking cessation. However, lung function is fully reversible only in subjects with asthma, whilst COPD shows a steady progress pattern. Both asthma and COPD are inflammatory disorders, but their inflammatory patterns differ: the mast-cell/CD4+ T cells/eosinophils axis prevails in asthma, whereas neutrophils/CD8+ T cells (Barnes, 2008) and premature lung senescence (Chilosi et al., 2013) associated to the activation of the receptors of advanced glycation end-products (RAGE; Robinson et al., 2012) fuel COPD. Indeed, most treatments for COPD and asthma are presently focused on suppressing the inflammatory cascades. However, the two conditions share other similarities, such as excessive mucus secretion, impaired epithelial barrier, and obstructive condition. Although the integrity of impaired airways is deemed to be a key player in initiating and determining the clinical outcomes in respiratory diseases (Tam et al., 2011), little attention is paid to its control.
The expression of genes that encode junctional proteins, such as claudin-1 and zona occludens (ZO)-1, and adherent junctional E-cadherin is repressed in "leaky" airways (Balda and Matter, 2009; Pohl et al., 2009; Shaykhiev et al., 2011). In turn, defective tight junctions allow the penetration of further toxic insults. The susceptibility to environmental injury leads to the persistent TH2-activation of lymphoid cells, whose interplay with myeloid cells results in the increase of circulating and intra-cellular levels of transforming growth factor (TGF)-β.
Stressor autocrine pathways induced by TGF-β upregulation lead to epithelial-to-mesenchymal transition (EMT; Grainge and Davies, 2013), i.e., the loss of epithelial phenotype and the acquisition of mesenchymal phenotype, which plays an important role in the airway remodeling that occurs in chronic bronchial diseases (Pain et al., 2014). Aberrant epithelium repair prompts the accumulation of fibroblasts which initiate the deposition of extracellular matrix (ECM), thus sustaining persistent bronchial obstruction.
Other cytokines, such as tumor necrosis factor (TNF)-α, via Src kinases activation, concur to determining the barrier dysfunction observed in chronic airways diseases (Hardyman et al., 2013). The consequence is a chronic, self-sustained wound scenario dominated by the secretion of secondary stress growth factors (Heijink et al., 2012; Gan et al., 2013; Hardyman et al., 2013) which fuel the progression of airway wall remodeling (Boxall et al., 2006).
Impaired airway epithelial integrity also parallels the higher expression of epidermal growth factor receptor (EGFR) in airway epithelia (Burgel and Nadel, 2008). Dimeric EGFR cooperates with TGF-β in activating one or more downstream effectors, including the MEK/ERK (MAPK kinase/extracellular signal-regulated kinase), PI3K/AKT (phosphatidylinositol-3-kinase/protein kinase B), and STAT (signal transducer and activator of transcription) pathways through receptor autophosphorylation and cytoplasmic protein binding (Jorissen et al., 2003). Therefore, EGFR and its downstream signaling effectors have been targeted by a number of new drug candidates (Figure 1).
Figure 1. Single-step pharmacologic strategies in chronic respiratory disease of approved and under-development EGFR pathway inhibitors compared to spermidine activity. Single step strategies: (i) anti-EGFR monoclonal antibodies that bind the extracellular domain of EGFR and prevent downstream activation (cetuximab, trastuzumab); (ii) tyrosine kinase inhibitors targeting the intracellular portion of EGF receptors to block autophosphorylation (erlotinib, gefitinib, lapatinib, HKI-272, BIBW-2948); (iii) Inhibitors of specific downstream EGFR signaling pathway (ARRY-142886, PX-866, ruxolitinib, perifosine, selumetinib, and sorafenib). Acting on TCPTP, spermidine can exert multiple direct and indirect effects.
Many metabolites are involved in airway remodeling in obstructive diseases, e.g., the natural polyamines (PA) spermidine and spermine, although their role as either worsening or alleviating factors is complex and still under debate (Hoet and Nemery, 2000; Erle and Sheppard, 2014). In fact, the EMT transition and barrier disruption that are present in airway diseases parallel PA/spermidine scarcity (Prunotto et al., 2010). Conversely, spermidine alone may be able to moderate most consequences triggered by EGFR and its downstream effectors via agonism on T-cell protein tyrosine phosphatase (TCPTP; Figure 1), as better detailed hereafter.
Role of Polyamines in Lung Cell Physiology
Spermidine and spermine are the most common and important members of PA, small organic polycations ubiquitously present in nature. Spermidine [NH2(CH2)4NH(CH2)3NH2] and spermine [NH2(CH2)3NH(CH2)4NH(CH2)3NH2] are sequentially derived from putrescine [NH2(CH2)4NH2]. Their biosynthesis requires decarboxylated S-adenosyl-L-methionine (DcAdoMet) as aminopropyl group donor and the enzymatic activity of spermidine synthase and spermine synthase, respectively. PA are essential for living cells, where they specifically interact with DNA, RNA, histones, and other proteins, thus affecting gene expression and biological protein activity (Pegg, 2009; Igarashi and Kashiwagi, 2010). Each PA has an array of distinctive properties. A unique role of spermidine in eukaryotes is the covalent modification of eukaryotic initiation factor 5A (eIF5A), resulting in an unusual amino acid, hypusine [Nϵ-(4-amino-2-hydroxybutyl)lysine] (Park, 2006). eIF5A and modified hypusine are necessary for the viability and growth of mammalian cells (Nishimura et al., 2012). PA are synthesized by a pathway involving L-arginine metabolism via arginase and ornithine decarboxylase (ODC) and their synthesis is regulated through a limitative feed-back control activated by increased PA level through antizyme (AZ)-mediated ODC down-regulation (Figure 2). However, L-arginine, via the nitric oxide synthase (NOS) pathway, also supports the formation of NO, which is key for the maintenance of airway tone (Ray et al., 2014). The balance between NOS isozymes and arginases regulates airways tone (Meurs et al., 2003). In particular, the constitutive neuronal and endothelial isoforms (nNOS and eNOS) support lung smooth muscle tone and relaxation. Noteworthy, NO scarcity may be compensated by the presence of inducible NOS (iNOS), which is upregulated in airway inflammation, e.g., in asthma (North et al., 2010; North and Scott, 2011). A negative role for PA in lung diseases was suggested by studies showing that an increase in the arginase/PA pathway at the expense of the NOS/NO output exacerbates COPD and asthma (Bergeron et al., 2007). Actually, enhanced arginase activity decreases L-arginine availability, thereby causing NO deficiency that contributes to airway hyper-responsiveness (Morris, 2013; North et al., 2013). Following the suggestion that the NOS-to-arginase shift may play a negative role in airway remodeling (Meurs et al., 2002, 2003), PA biosynthesis has been proposed as a new therapeutic target (Pera et al., 2014).
Figure 2. Competing pathways in L-arginine metabolism. Arginine is a substrate for both NOS, yielding NO and L-citrulline, and for arginase, to produce ornithine and urea. Ornithine is then metabolized by ODC to putrescine, or by OAT to provide proline, a major precursor for collagen biosynthesis. Putrescine supplies the building blocks of the higher polyamines spermidine and spermine, synthetized by SpdS and SpmS, respectively. ODC activity is controlled by AZ, which in turn accumulates via mechanisms triggered by increasing polyamine levels. The polyamine pool is also back-regulated via acetylation and degradation by SSAT and PAO. Both spermine and, to a lesser extent, spermidine, inhibit NOS activity. NOS inhibition can further derive from the feedback effect of NOHA, the intermediate in NO production. Arginine is also incorporated into proteins, where it can be mono- or di-methylated by PRMT to free MMA, ADMA, and SDMA upon proteolysis. ADMA acts as an endogenous inhibitor of NOS, and SDMA competes in L-arginine uptake. AZ, antizyme; NO, nitric oxide; NOS, nitric oxide synthase; NOHA, Nω-hydroxy-L-arginine; PRMT, protein arginine methyltransferases; Protein-R-(CH3)n, n-methyl-arginine-containing proteins; MMA, monomethyl arginine; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; P5C, L-pyrroline-5- carboxylate; P5CR, pyrroline-5-carboxylate reductase; SpdS, spermidine synthase; SpmS, spermine synthase; PAO, polyamine oxidase; SSAT, spermidine/spermine-N1-acetyltransferase; N1-AcSpd, N1-acetylspermidine; N1-AcSpm, N1-acetylspermine.
The hypothesis that envisages a pathogenic role for PA is also supported by the findings of a study describing the integrin-mediated localization of spermidine/spermine-N1-acetyltransferase (SSAT, the key PA catabolic enzyme) in close proximity to the lipid kinase phosphatidylinositol phosphate (PIP)5K1γ, the main source of phosphatidylinositol 2 phosphate in airway smooth muscle, whose activity is regulated by PA. It has been shown that proper assembly of the integrin-SSAT-PIP5K1γ complex results in efficient PA catabolism and airways contraction inhibition, whereas administration of the higher-order PA spermine enhanced contraction (Chen et al., 2004, 2012). However, it should be noted that these studies used exposure doses (100 μM) at least 10-fold higher than physiologic levels of PA. It is also important to consider that exogenously added PA—even at low doses—can actually inhibit intracellular PA biosynthesis by negative feed-back, as mentioned above, possibly resulting in increased availability of L-arginine and its shift toward NO formation.
A further element of complexity derives from the occurrence of L-arginine derivatives, such asymmetric and symmetric dimethyl arginine (ADMA and SDMA, respectively) which represent degradation products of proteins containing post-translationally methylated arginine residues (Scott et al., 2011, 2014; Holguin et al., 2013; Scott and Grasemann, 2013). The accumulation of both ADMA and SDMA can lead to reduced NOS activity. ADMA is a NOS endogenous inhibitor associated with lung disease (Zakrzewicz and Eickelberg, 2009), whereas SDMA contributes to the NO imbalance by competing with the cationic amino acid transporter-2 for L-arginine uptake (North and Scott, 2011). ADMA levels range between 50 and 75 nM, whereas its potency in NOS inhibition is 100-fold higher than that of spermine. Hence, lung diseases such as acute respiratory dysfunction triggered by NO scarcity might be the result of proteolytic waste of L-arginine and subsequent accumulation of these arginine derivatives, rather than derive from PA production favored by the NOS-to-arginase shift.
The Key Driving Force in Epithelial Stabilization: TCTPT Agonism
Although some of the studies presented above suggest a negative role for PA in airways diseases, we propose that one specific PA, i.e., spermidine, may play a positive (possibly therapeutic) role in pulmonary disarray by virtue of two mechanisms: interaction with TCPTP and activation of autophagy.
TCPTP (aka PTPN2), a phosphatase expressed in most mammalian cells, is an intracellular suppressor of metastatic and inflammatory signaling pathways. The two TCPTP isoforms, resulting from differential splicing, suppress a variety of signaling substrates including receptor tyrosine kinases (RTKs), such as EGFR, insulin growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGR), colony-stimulating factor-1 (CSF-1) receptor, and vascular endothelial growth factor receptor (VEGFR), as well as mitogen-activated protein kinases (MAPKs). Moreover, TCPTP quenches signaling mediated by nuclear (e.g., STAT-1, -3, -5, 6), and cytoplasmic proteins (e.g., JAK1, JAK2, p52-Shc, Src-tyrosine kinases; Mattila, 2009; Muppirala et al., 2013). The dephosphorylation of key targets in downstream signaling pathways leads to the inactivation of pro-inflammatory responses induced by IFN-γ, TNF-α, and IL-6. TCPTP proteins impact airway physiology in multifarious ways (Figure 3), mostly by quenching TGF-βR and EGFR (Tiganis et al., 1999; Mattila et al., 2005) and relevant post-receptor signaling intermediates, thus providing an anti-mitogenic effect (Chernoff, 1999; Rajeeve et al., 2013).
Figure 3. Mode of action of spermidine through TCPTP-activation. In the cytoplasm, activated TCPTP down-regulates signal transduction pathways downstream EGFR, TGF-βR, the Frizzled/Wnt/Lrc complex, and most of conjugated signaling effectors of EGF, IFN-γ, TNF-α, IL-6, and IL-1β/TGF-β. Inhibition occurs by dephoshorylation of key components of JAK1/jnk/jun/fos/p38, noncanonical Ras/Raf/MEK/ERK, and PI3K/PIP3/Akt pathways, as well as the nucleus-addressably proteins STATs and R-SMADs. In particular, dephosphorylated R-SMADs prevent coSMAD binding and the R-SMAD/coSMAD complexes to accumulate in the nucleus and act as transcription factors in TGF-β/Smads and p53 loci via Snail/Slug and Zeb1. Their inhibited access to chromatin represses detachment, migration, and EMT proteins like fibronectin, vimentin, MMP2/9, N-cadherin, and collagens I/III. Phenotypic involution toward EMT is prevented, whereas spermidine epigenetically favors the expression of c-fos, c-jun, and c-myc. Outcomes consist of the enhanced expression of tight junctional and adherens proteins: claudin-1, occludin, ZO-1/2/3, and E-cadherins (in bold). EGF, epidermal growth factor; EGFR, EGF receptor; TGF-β, transforming growth factor β; TGF-βR, TGF-β receptor, TGFβRI/II, TGFR tetramer; TAK1, TGFβ-activated kinase 1; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6;, IL-1β, interleukin-1β; Wnt/Lrc, ligand to Frizzled receptor; Jnk/Jun/fos/p38 signal transduction proteins; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol-3'- kinase; PIP3, phosphatidylinositol(3,4,5) trisphosphate; Akt, activator kinase; JAK, Janus kinase; R-SMAD and coSMAD, family of transcriptional smadx activator protein/cofactors; STATs, signal transducers/activators of transcription; GSK3, glycogen synthase kinase; ZO, zonula occludens; Snail/Slug, transcription factors; Zeb, zinc finger E box binding homeobox; TGFβ/Smads/p53/p21, genes for chemotaxis; c-fos/c-jun, gene expressing junctional proteins; MMP, matrix metalloproteinases; EMT, epithelial-to-mesenchymal transition.
Recently, a high-throughput assay aimed at selecting novel TCPTP agonists screened as many as 64,280 small molecules to identify finally two leads, mitoxantrone and spermidine, both active at low micromolar levels (Mattila et al., 2010). Modalities of action were elucidated: the former directly binds to TCPTP catalytic domain, whereas spermidine agonism does not require interference with TCPTP active center (Ylilauri et al., 2013).
This study showing a direct interaction between spermidine and TCPTP may provide a unifying explanatory view of a vast array of spermidine biological features, firstly described in human dermal fibroblasts and still lacking an explanation, such as the down-regulation of matrix metalloproteinase-2, histone acetyltransferase, p-ERK, p-JNK, and NF-κB activity and expression, and increase in histone deacetylase 1 and sirtuin 1 (Park and Kim, 2012).
Indeed, parallel studies confirmed that the increase of TCPTP level and enzymatic activity driven by spermidine were coupled with decreased phosphorylation of the downstream transducers in IFN-γ cascades (Penrose et al., 2013). TCPTP activation in response to stress factors was also observed in human THP-1 monocytes challenged with IFN-γ. Exogenous spermidine enhanced TCPTP level, reduced phosphorylation of STAT-1,3, p38-MAPK, and lowered secretion of ICAM-1, monocyte chemoattractant protein (MCP)-1, and IL-6 in a TCPTP-dependent manner, as the modulatory effects of spermidine against IFN-γ exposure were absent in TCPTP-knockdown cells (Moron et al., 2013).
Lessons from the Intestinal Epithelium
The connection of PA with intestinal repair, replacement and stabilization has been investigated for 40 years (Luk and Yang, 1987; Wang and Johnson, 1990, 1992; McCormack and Johnson, 1991; Johnson and McCormack, 1999; Timmons et al., 2012). Several studies have evaluated their effect on the growth capacity of the gastrointestinal mucosa, which has the highest turnover in human body (approximately every 78 h), and have shown a positive role for PA both in conditions of physiological growth and in mucosal repair. In fact, lack of PA stabilizes p53 and JunD, thereby inhibiting intestinal epithelial re-growth (Li et al., 2001, 2002). In addition, since appropriate PA levels are essential for the stability of occludin and adherens junctions (Guo et al., 2003, 2005), as well as for c-myc-mediated E-cadherin transcription (Liu et al., 2005, 2009), PA might have a beneficial effect in maintaining the intestinal epithelial barrier function. This "improved barrier" theory has a sound background since PA, and particularly spermidine, assist cell-cell interactions by promoting the expression of adherens and tight junction proteins in the intestine (Wang, 2005).
Another mode of action of PA in ensuring mucosal integrity was revealed by a study showing that PA are needed for microtubule (MT) formation during gastric mucosal healing (Banan et al., 1998). These results are in line with the essential role of PA in cytoskeleton assembly (Anderson et al., 1985). In this context, the facilitated tubulin attraction, MT nucleation, and elongation promoted by PA, and particularly by spermidine, occur through plain electrostatic mechanism (Mechulam et al., 2009; Hamon et al., 2011). Indeed, PA depletion causes the disappearance of actin filaments and MT in CHO cells (Pohjanpelto et al., 1981), while PA was found to associate to anionic MT via promoted formation of MAP4 (MT-associated protein 4) and redistribution of EB1 (end-binding protein 1) and MT plus-end-binding protein (Savarin et al., 2010). Therefore, as gap junctions require extended MT up to cell periphery (Shaw et al., 2007), it is possible to envisage a process in which PA-induced MT elongation and modulation sustain epithelial integrity.
In these settings, TCPTP agonism enacted by spermidine matches 40 years of research on bowel homeostasis, and also explains the molecular mechanisms underlying the contribution of spermidine to the alleviation of pro-inflammatory signals and intestinal barrier dysfunction. In fact, on a functional level, it has been shown that spermidine in co-incubation experiments with IFN-α protects intestinal epithelial barrier functionality in the setting of inflammation, by limiting the increase of epithelial permeability (Penrose et al., 2013). Moreover, spermidine administration decreased mucosal damage and weight loss in a mouse model of colitis (Moron et al., 2013).
Finally, the anti-"inflammaging" action and barrier reinforcement triggered by TCPTP agonism (or autophagy, see below) may account for the positive effects of enhanced spermidine/PA production in the luminal tracts on longevity (Matsumoto et al., 2011; Kibe et al., 2014) and tumorigenesis (Soda et al., 2013).
Autophagy: Promotion by Spermidine and Role in Lung Disorders
Autophagy is a self-cleaning process that represents an adaptive response elicited by "anti-nutrient" signals and starvation, and contributes to longevity (Eisenberg et al., 2009). Its role in COPD and asthma is complex and not fully clear (Poon A., et al., 2012; Poon A. H., et al., 2012; Jyothula and Eissa, 2013). Among the factors involved, oxidative stress may act both as autophagy promoter via AMP-activated protein kinase (AMPK) signaling (Wu et al., 2014), and repressor, in analogy with cytokine effectors that dock EGFR and TFG-βR (Khan et al., 2008; Tripathi et al., 2013). Conversely, particulate matter 2.5 (PM2.5), an aggravating factor for most obstructive airways diseases, seems to favor autophagy, as it inhibits the PI3K/AKT/ mammalian target of rapamycin type 1 (mTOR) pathway (Liu et al., 2015). Overall, the overactive immune responses present in airway pathologies display an array of divergent pro- and anti-autophagy effects (Figure 4).
Figure 4. Role of Spermidine-TCPTP in the complex dynamics of pro- and anti-autophagic factors. Autophagy requires the coordinated activation of multiple factors. Several Atg proteins are recruited within the ULK1 complex during vesicle nucleation and within the ubiquitin-like conjugation systems Atg12-UBL and LC3-UBL during phagophore elongation, and completion. The main autophagy repressor, mTORC1, exerts its inhibitory action by phosphorylating ULK1. mTOR interacts with the proline-rich Akt, newly synthesized in the PI3K/PI3P/Akt pathway, downstream of EGFR, and TGF-βR signaling, which in turns responds (inter-alia) to cytokines such as IFN-γ, TNF-α, IL-6, and IL-1β/TGF-β. A mTORC1 antagonist and autophagy promoter is AMPK. Upon activation from non-immune stress factors including starvation, hypoxia, ROS (e.g., , H2O2, OH.), and RNS (e.g., peroxynitrite) via ATM/LKB1, AMPK prompts autophagy. AMPK is directly involved in the formation of ULK1 complex and cooperates with Becl 1-VPS34 complexes, pro-autophagy adapters involved in nucleation of autophagosome membrane. While ROS simulate EGFR activation, other environmental stressors (e.g., MP2.5) have an opposite effect, by inhibiting PI3K/PI3P/Akt. Agonistic activity of spermidine on TCPTP would down-regulate both EGFR and TGF-βR and their downstream effectors. Yet, the prevalent, established mode of action of spermidine in autophagy entails the inhibition of EP300, leading to reduced histone acetylation (particularly histone-3), and, consequently, to Atg (particularly Atg7) expression and autophagosome elongation and closure. Spermidine activity on EP300 is similar to that exerted by low cytoplasmatic levels of Ac-CoA due to scarce nutrient availability, a situation that also activates AMPK. Ac-CoA, acetyl-coenzyme A; ROS, reactive oxygen species; RNS, reactive nitrogen species; superoxide anion; H2O2; hydrogen peroxide; OH., hydroxyl radical; PM2.5, particulate matter 2.5; IFN-γ, interferon-γ; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6;, IL-1β, interleukin-1β; EGFR, epidermal growth factor receptor; TGF-βR, transforming growth factor β receptor; ULK1/2, UNC-51-like kinases 1/2; ATM ataxia telangiectasia mutated kinase; LKB1, upstream kinase of AMPK; AMPK adenosine monophosphate-activated protein kinase; TSC2, tuberous sclerosis 2; mTORC1, mammalian target of rapamycin type-1 complex; EP300, E1A-binding protein p300; Beclin 1-VPS34 complex, Beclin 1-ATG14L; VPS34, class III phosphatidylinositol 3-kinase; Atg autophagy related proteins; ULK1-complex, Atg13-FIP200-Atg101-Atg14L conjugation system; FIP200, FAK-family interacting protein of 200 kDa; Atg12-UBL system, Atg5-Atg12-Atg10-Atg16-Atg7 pathway; LC3-UBL system, LC3-Atg3-Atg4-Atg7-PE pathway; LC3, microtubule-associated protein light chain 3; PE, phosphatidylethanolamine; HAT, histone acetyltrasferase.
As noted by Mercado et al. (2015), current information about the role of autophagy in bronchial and alveolar epithelial cells in lungs of patients with active COPD is divided into evidence showing that excessive autophagy and mitophagy results in cell death, and proof that pathological changes are the consequence of an underlying defective autophagy. Insufficient autophagy is thought to lead to involution patterns including stem cells depletion, reduced antioxidant responses, and defective mitochondrial function, which may contribute to progressive cell senescence (Fujii et al., 2012; Ito et al., 2012; Takasaka et al., 2014). Conversely, excess autophagy leads to shortening and, finally, total consumption of cilia components, called "ciliophagy" (Lam et al., 2013). Epithelial cells from mice lacking LC3B, which is required for autophagosome formation, are protected from cigarette-smoke (CS)-induced autophagy and resist emphysema. However, the effect of CS-promoted ciliophagy on mucociliary clearance is reversible and only lately associated with the increased apoptosis of ciliated cells that occurs when accumulation of CS-denatured protein exceeds cellular degradative capacity. This suggests that ciliophagy only partially contributes to airway dysfunction (Cloonan et al., 2014) and might be a regulatory pattern in ciliogenesis through the control of ciliary proteins (Orhon et al., 2015).
In clinical specimens of COPD patients, autophagy markers are upregulated at early stages of disease progression whereas caspases are activated later on. This might indicate that autophagy either precedes apoptosis in response to disease stress on lung, or is an attempt to self-protect from CS stress (Ryter et al., 2009). Autophagy may altogether act as a clearance mechanism, since macrophages in lung biopsies from non-COPD smokers showed impaired autophagic efficiency and debris accumulation (Monick et al., 2010).
With regard to the protective role of spermidine in airway diseases, it is of interest to underline that the promotion of authophagy is another key aspect of spermidine activity (Eisenberg et al., 2009; Morselli et al., 2011; Minois, 2014). The beneficial impact exerted by spermidine in several model systems, including improved fertility (Bauer et al., 2013) and retained memory in aging (Gupta et al., 2013) or reverted age-dependent arterial degeneration in murine arteriosclerosis (LaRocca et al., 2013), correlates with the activity of autophagy promotion. Outcomes are comparable to starvation and caloric restriction mimetics, a new class of autophagy-inducing therapeutics with potential clinical utility in diabetes, metabolic syndrome, and cardiovascular diseases (Madeo et al., 2014). Autophagy is repressed by mTOR complex (mTORC1), but is activated by pathways involving the "longevity factor" Sirt1 or the "energy sensor" AMPK (Cetrullo et al., 2015). However, spermidine appears to function as an autophagy inducer not by directly affecting the mTOR pathway, but rather by inducing a status of protein hypoacetylation via inhibition of histone acetylases (Morselli et al., 2009), such as EP300 acetyltransferase, an endogenous repressor of autophagy (Pietrocola et al., 2015).
As EP300 is strictly regulated by cytoplasmatic acetyl-coenzyme A levels (Marino et al., 2014b) and nutrient availability, this confirms the "starvation-mimicking" property of spermidine (Figure 4).
In line with the proposed role of the crosstalk between spermidine and TCPTP (see above), recent studies have demonstrated that TCPTP also regulates autophagy in human intestinal cells and other cell types (Scharl and Rogler, 2012; Scharl et al., 2012). TCPTP dysfunction correlated with impaired autophagosome formation and defective autophagy, thus contributing to the development and perpetuation of chronic inflammatory disorders, such as inflammatory bowel diseases (Spalinger et al., 2015). Conversely, high TCPTP levels were found to increase autophagy (Aradi et al., 2015). Therefore, it may be speculated that spermidine can promote the autophagic process at least in part via TCPTP; however, the role of TCPTP in spermidine-induced autophagy remains to be explored, together with a careful examination of the effective dose. Indeed, manipulation of autophagy is a delicate matter, as this process can generally antagonize degenerative processes, but its excessive induction may result in maladaptive tissue remodeling (Fimia and Piacentini, 2010; Marino et al., 2014a; Filomeni et al., 2015; Maiuri and Kroemer, 2015). Thus, it is important to monitor carefully basal levels of autophagy and its flux as a function of time and dosage of stimulant. This can be carried out with experimental models usually employed to study chronic epithelial disorders, such as in vitro cultures of immortalized human bronchial epithelial cell line (BEAS 2B cells) or primary cells from specimens obtained from donors for lung transplantation (Kinnula et al., 1994). Genetic deletion of specific autophagy proteins in animal models (Cann et al., 2008) may also be useful to study the involvement of this process in airway disorders and the possible modulation by spermidine treatment. Keeping in mind the complexity of the autophagic process, multiple, short-term and long-term assays are recommended to monitor autophagy, including flux measurements in normal, and blocked turnover conditions. Indeed, autophagy flux is the entire process, from autophagosome biogenesis to cargo digestion by lysosomes, and macromolecule release back into the cytosol. Therefore, the evaluation of lysosomal competence as the final stage in this process is essential for a correct interpretation of the results. Finally, human lung specimens from patients can be analyzed for autophagy markers, such as increased autophagosome numbers and increased expression of LC3B-II, the active form of LC3B, or assayed by transmission electronic microscopy to monitor autophagic structures. This issue, i.e., the different outcome of autophagy as a function of its level, introduces dose-finding concepts in pursuit of the therapeutic use of exogenous spermidine, and underscores the need for fine-tuning the dose of spermidine in clinical settings, as further detailed in the next paragraph.
Therapeutic Applications
Spermidine is currently used as dietary supplements and topical hair-growth promoter, with proven efficacy in vitro (Ramot et al., 2011) and in clinical settings (Rinaldi et al., 2004); therefore, the molecule has a proven history of safe long-term use in consumer products.
Over the past 5 years our group has set up a therapeutic platform based on spermidine. We have focused on the preparation and set up of a variety of supramolecular complexes (SMC) in which spermidine was bound to different polymers, and our studies showed that sustained delivery from SMC is able to optimize the effect of spermidine on fibroblast viability (Ghisalberti et al., 2013). The sustained-release efficiency is unrelated to the matrix polymer, since a variety of anionic or SMC-forming carriers (e.g., spermidine-hyaluronate, Spd-HA) afforded similar results.
A pilot study on vestibulitis, in which pain relief was assessed in terms of absolute and relative effect size compared to present and purported drugs for vulvodynia (Murina et al., unpublished), showed that a regimen of 1 μmole of spermidine (administered as gels containing Spd-HA) at 3 doses/week for 1 month, followed by 2 doses/week for another month was effective in blunting pain, assessed by the visual analogic score, and improving dyspareunia, thus indicating that spermidine may be envisaged as a new paradigm in widespread female disorders. Indeed, another pilot study in post-menopausal vaginal atrophy showed that a similar regimen, but by intravaginal application, resulted in clinical outcomes close to those of estrogenic and pro-estrogenic therapies (Carena Maini et al., unpublished), yet avoiding estrogenic risks while retaining comparable efficacy and duration. Other exploratory trials to evaluate the efficacy of spermidine complexed to SMC for atrophic or degenerative disorders, such as periodontitis and osteoarthritis are ongoing, following a series of studies on joint metabolism and mesenchymal differentiation (Borzi et al., 2014).
The question of "how a common metabolite at sub-micromolar dose becomes of therapeutic relevance" may have an answer in the localization of spermidine, which is 90% bound to intracellular anionic moieties (Watanabe et al., 1991; Matthews, 1993). Therefore, even a low dose in the micromolar range can lead to a meaningful increase, actually doubling the free pool and thereby impacting on overall cell physiology. Hence, spermidine "therapeutic window" may reside at sub-micromolar level (Figure 5). In addition, this dosage prevents plain toxicity (Foster et al., 1990) while impeding apoptotic/inflammatory cascades to prevail (Silva et al., 2011).
Figure 5. Effects of unbound spermidine on cellular fate. The multimodal activity of spermidine primarily relies on local concentration, recognized as a critical factor. The polycationic spermidine tends to saturate anionic molecules, partly in the phospholipid (PL) bilayer within cell membranes, partly coupling phosphate and adenosine triphosphate (ATP); yet, it is mostly associated with the core anionic biopolymers, i.e., RNA, but also DNA and histones, therefore modifying chromatin conformation. Ultralow doses of exogenous spermidine double the content of free (unbound) spermidine. Excess and paucity levels are closer than previously deemed, as a change in the micromolar range may switch a trophic (or growth promoting) activated state pattern into an inflammatory-apoptotic pattern. Concentrations are just indicatory, as levels of spermidine in cytosol and nucleus differ from tissues to tissues and during lifespan.
Interestingly, it has been shown that low doses of spermidine in the micromolar range are sufficient to activate TCPTP (Mattila et al., 2010), thereby triggering the downstream signaling pathways discussed above, and also activating autophagy, which has been shown to be induced by low doses of spermidine (Eisenberg et al., 2009). Activated TCPTP moderates most destabilizing processes set in motion by secondary growth and stress factors in asthma and COPD airways, while sustaining epithelial barrier repair and reinforcement. Moreover, the addition of spermidine should contrast increasing polyamine biosynthesis, which may negatively impact on NO formation. Although only preliminary data are presently available, it can be predicted and experimentally tested that sub-micromolar doses of spermidine in SMC, possibly delivered using an inhalator, may be beneficial in bronchial repair to improve patient outcomes in chronic lung diseases, including asthma and COPD.
A reparative approach of disrupted epithelium in obstructive airway diseases may afford protection and more lasting results than therapies such as corticosteroids or immunosuppressant drugs. PA have been proposed as potential therapy in airway diseases. However, it has also been claimed that increased PA biosynthesis exacerbates lung diseases, as the PA synthetic pathway competes for L-arginine at the expenses of NO production, thus resulting in bronchoconstriction. Moreover, the pathways involved in the beneficial effect of PA in airway diseases are still poorly defined and controversial. A recent study showing a direct interaction between spermidine and TCPTP may place the beneficial role of PA in airway epithelial integrity in a coherent framework. In fact, the dephosphorylating activity of TCPTP represses the signaling cascade that activates genes favoring cell detachment and epithelial-to-mesenchimal transition, while increasing the expression of adhesion and tight junction proteins. Moreover, the protective effect of spermidine may also depend on the capacity of TCPTP to trigger autophagy. As such, TCPTP agonism may provide a unifying explanatory view of a vast array of biological features of spermidine and foster a mechanism-guided application of PA in COPD. Since, doses of spermidine in the micromolar range are sufficient to activate TCPTP and downstream signaling pathways, the sustained delivery of very low amounts of spermidine from SMC may provide an optimal pharmacologic profile for the treatment of obstructive airway diseases while avoiding potential harmful effects of excess PA, as suggested by preliminary evidence obtained in vitro and in vivo. Therefore, we propose spermidine in a sustained release modality as a novel therapeutic approach for airway barrier remodeling, based on a novel mode of action: TCPTP agonism. The overall features of inhaled spermidine (Figure 6) appear promising in order to provide hope for chronic lung sufferers, who are still in need of novel therapies providing more appropriate, long-lasting benefits to effectively treat chronic lung diseases.
Figure 6. Exogenous spermidine may interrupt the vicious cycling in chronic airways diseases. Locally delivered spermidine works both as a TCPTP agonist and stimulator of the expression of genes coding for adherens and tight junction proteins. The former effect down-regulates the secondary growth factor signaling pathways, such as epidermal growth factor (EGF) and transforming growth factor-β (TGF-β) receptors and relevant pathways. This repression prevents the epithelial-to-mesenchymal transition to occur, which would further impair the epithelial impermeability and contribute to chronic remodeling and fibrosis. Strengthening the airways epithelial barrier translates into a reduced exposure to allergens, stressor proteins, and reactive oxygen species.
Author Contributions
CAG conceived the review and co-wrote the paper; GC co-wrote the paper; SC, FF, and RMB substantially contributed to editing and writing the paper.
This work was supported by grants from Ministero dell'Istruzione, dell'Università e della Ricerca, Italy, (PRIN and FIRB RBAP10KCNS), University of Bologna (RFO), and Ministero della Salute (Fondi cinque per mille).
CAG owns shares in Tixupharma.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Keywords: TCPTP, spermidine, chronic airway disorders, FGF, TGF, stress growth factors, epithelial to mesenchymal transition, autophagy
Citation: Ghisalberti CA, Borzì RM, Cetrullo S, Flamigni F and Cairo G (2016) Soft TCPTP Agonism—Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine. Front. Pharmacol. 7:147. doi: 10.3389/fphar.2016.00147
Received: 29 February 2016; Accepted: 19 May 2016;
Published: 03 June 2016.
Edited by:Ajay Sharma, Chapman University School of Pharmacy, USA
Reviewed by:Istvan Lekli, University of Debrecen, Hungary
Emilia Maellaro, University of Siena, Italy
Copyright © 2016 Ghisalberti, Borzì, Cetrullo, Flamigni and Cairo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Gaetano Cairo, email@example.com