text:
Q: Module run inside module returns - TypeError: 'numpy.float64' object cannot be interpreted as an integer I am running a function inside another function in a loop and having a problem when the first module (Mod1) uses values defined in the second module (Mod2) to generate the result for the equation called Overall.
The error returned is thus:  TypeError: 'numpy.float64' object cannot be interpreted as an integer
The code is as follows
def Mod1(A,
       B,
       C):
    As = pd.read_csv('AProps.csv',index_col='AName')
    As = As.dropna(0,how='all',thresh=None,subset=None,inplace=False)
    AvailAs = As.index.tolist()

    Bs = pd.read_csv('BProps.csv',index_col='BName')
    Bs = Matrices.dropna(0,how='all',thresh=None, subset=None,inplace=False)
    AvailBs = Bs.index.tolist()

    Prop1_A = As['Prop1'][A]
    Prop2_A = As['Prop2'][A]
    Prop3_A = As['Prop3'][A]
    Prop4_A = Prop1_A/(2*(1+Prop3_A))
    Prop1_B = Bs['Prop1'][B]
    Prop3_B = Bs['Prop3'][B]
    Prop4 = Prop1_B/(2*(1+Prop3_B)

    Overall = Prop1_A*C+Prop1_B*(1-C)

    Return Overall

def Mod2(NumItems):
    A_List = []
    B_List = []
    C_List = []

    for i in range(NumItems):

        A_List.append(input('Choose the A_Input for item {0}:  '.format(i+1)))
        B_List.append(input('Choose the B_input for item {0}:  '.format(i+1)))
        C_List.append(input('Choose the C_input for item {0}:  '.format(i+1)))

    for i in range (NumLams):
        Func1(A_List[i],B_List[i],C_List[i])

Just to be clear, the intention of the code is that Mod2 creates lists of inputs which are then used within Mod1.  In Mod1 inputs A and B are used to pull outputs from csv files which are read into As and Bs.  Input C is used in functions within Mod1, ie Overall.
When I run Mod1 manually, there are no issues at all.  But when run in conjunction with Mod2 as outlined above...
The code in Mod1 runs through using the values Prop1_A and Prop1_B pulled from As and Bs without issue, but when it comes to run the function for Overall the aforementioned error is returned within Python.
I am certain that the issue is with the way the value for C is interpreted, ie as a float64, but don't understand why python might be expecting to see an integer there.
I am using Spyder 3.2.6 as installed with the Anaconda package.
Any help given is gratefully received.

A: I solved the problem by explicitly defining all the selections from the csv, Prop_1 etc, in Mod1 as floats.  A simple answer really.


Do you share your favourite book with a top CEO?
The books you are drawn to can say a lot about your personality and passions.
Here's an infographic from ceo.com showing the favourite books of 22 of the top CEO's (past and present).
Well worth a read, you might just find something inspiring in them too!


text:
10 Most-Appealing US State Welcome Signs
World of Signs
Carefully designed to portray every state's uniqueness.
From sea to shining sea, America is covered in road signs. Touring the country by road, you'll see that a sign that welcomes the visitor marks each state-line. Today we're looking at ten of the most artistically appealing US state welcome signs. No standard informational white text on a bland blue or green background here, these signs are carefully designed to portray the state's unique geography, history, or culture in a fun or evocative way.
10. Montana
This sign breaks from traditional geometric shapes to reflect the actual boundaries of the state. We think this is perfect for the big sky country state; the last outpost of the Wild West, and definitely a place for those who don't fit into the box.
9. Kentucky
Sure it's blue and white… but the design is anything but boring. Linking Kentucky's long history of horse-racing (Kentucky Derby, people!) and the state motto "Unbridled Spirit" the only thing this sign is missing is a bottle of bourbon.
This cheerful sign welcomes you to "the Sunflower State" with a reminder that every fall the Kansas prairie fills with wild sunflowers that smile out at travelers, filling the countryside with a golden glow. We love that the petals look like the rays of the sun, and the happy yellow K that looks like it's ready to kick up its heels and dance!
7. Alaska
Alaska isn't going to do things the same as they do in those pansy states down south. No steel and paint for the "last frontier" state that shares more in common with Siberia than the Mid-West. Alaska's all-natural wooden welcome sign is just as natural, rugged, and beautiful as the state itself. The use of wood supports the state's logging industry, and the carved map includes free-roaming Alaskan moose – the state animal. The only thing missing is a waving Sasquatch… and maybe Sarah Palin waiting on the side of the road to personally greet each and every traveler?
6. South Dakota
What is the most famous attraction in South Dakota? An attraction so important it gives the state its nickname? Mount Rushmore, of course, that American-sized tribute to four Presidents who shaped the nation: Washington, Roosevelt, Jefferson, and Lincoln. On this sign, the Presidents solemnly welcome you, while the tag "Great Faces, Great Places" is a cute pun on the carved attraction and the traditional Mid-Western friendliness of the state's inhabitants.
Colorado literally translates from Spanish as "red," and this appropriate welcome sign shows snow-capped, ruddy mountains against the brilliant red sky of a Colorado sunset. Add the yellow text, and this sign is bright enough to warm the heart of even the most reluctant visitor. We don't think the caption "Mountains and Much More!" really adds anything, though. Much more what…? These days "Mountains and Legal Cannabis!" would probably be more descriptive, and maybe a bright green 7-pointed leaf to add contrast?
As simple, bright, and pretty as the land that welcomed the first settlers to the New World, this sign shows the state bird –the Cardinal – perched on the state flower – the Dogwood. Primary colors – blue, green, red, yellow and white – and a classic, elegant design catch the eye and make this one of our very favorite welcome signs.
3. Wyoming
Landmark volcanic outcropping Devil's Tower and a bucking bronco in silhouette symbolize Wyoming's unique landscape and cowboy culture. Absolutely the most artistically detailed of the signs in this list, the photo-realistic background of a blue sky with wispy clouds is layered with cartoon-like elements in a totally non-realistic scale – just look how big that horse and rider are over those teeny tiny trees! However, just like with the state itself, the extremes meld to create a beautiful whole.
2. Nevada
At first glance the mountains and sunset sky of this sign reminded us a little of Colorado, but the seated miner, stoically gazing out over black mountains, is a clear nod to Nevada's long-links with silver mining. The dark color scheme of this sign may not be as welcoming and friendly as other state's signs, but they definitely reflect Nevada's arid geography. A sign that brings a sense of vague uneasiness and a reminder that in Nevada, you might oughta watch your back when you're gamblin' in the saloon.
Our number one sign has a dinosaur hooking its tail through the blocky U of Utah, a wacky welcome that grabs your attention with a nod to Utah's dinosaur residents, whose bones are visible at the Dinosaur National Monument Park. The slogan invites you to "Life Elevated," a vague hint at the high standard of living enjoyed by Utah residents, the high altitude of the state's famous snow covered slopes, and maybe even the high calling of the state's Mormon Elders. Shame Utah nabbed this slogan before Colorado thought of it… in that state living life elevated could mean something else altogether!
alaska, colorado, kansas, kentucky, montana, nevada, south dakota, utah, virginia, wyoming
Sign Shop:
Welcome to Nev...
Welcome to Ala...
Welcome to Col...
Welcome to Kan...
Welcome to Ken...
Getting Lost in Beijing
The Ins and Outs of Sexhow Lane, UK
Don't Jump Ou...
Vaci utca
Calle Habana
10 Most Satanic Road Signs in the US
10 Top Shortest Place Names in the World
10 Most Beautiful Drives in the U.S.
10 Top European Countries Reduced to Ame...
Honey I'm So Hungry I'm Tuckered Out
A Hippie Intersection
The Other Standing Mammal Inhabiting Aus...
10 Rarest Animals Featured on Road Signs
Lewis and Clark Trail
Luxury Shopping in Europe
As Ragged Ass Broke as a Mine Ever Was
Perfect If You Don't Know Where Yo...
Fancy a Nibble on a Hoecake?
Well Endowed Ladies Welcome
Tuck That Thought Away
The Honest (Rather Boring) Truth
Not Quite a Pink Wedding Yet
Needless by Name but Not by Nature
Specific Signs
How Considerate of You
So Where to from Here?
A Quiet Moment in Beijing
Please Dispose of Bicycles Here

redpajama_set_name: RedPajamaCommonCrawl


Welcome to South East Retina Center –Clinical Trial Research Division!
Southeast Retina Center Clinical Trial Division has brought cutting edge therapies to our community and patients. Drs. Marcus and Lalane have been clinical research investigators for many of the clinical trials investigating new state-of-the-art therapies for retinal diseases. Several of these therapies, such as anti-vascular endothelial growth factor (anti-VEGF) injections were evaluated in our office as part of clinical trials. Patients participating in clinical trials at Southeast Retina Center had the opportunity to receive therapies prior to their availability. Many treatments such as anti-VEGF injections were subsequently FDA approved and are now are standard treatment for many blinding retinal disorders such as wet age-related macular degeneration, diabetic retinopathy and retinal vein occlusion. Our clinical trial research division continues to study and evaluate new preventative and therapeutic treatments as we participate in numerous clinical trials and studies ranging from Phase I through Phase IV.
Our Physicians are national leaders in clinical research as they continue to offer our patients cutting edge therapies in trials sponsored by the National Eye Institute and leading pharmaceutical companies. Our practice has participated in the internationally recognized, National Eye Institute-sponsored Diabetic Retinopathy Clinical Research (DRCR) Network which is a leading entity for vital research for our diabetic patients. Dr. Marcus has served in several leadership roles for the Network including the DRCR executive committee, protocol chairman, DRCR writing committees and protocol development committees. Dr. Marcus is national protocol chairman for the DRCR Protocol AA evaluating wide field imaging of the retina in diabetic retinopathy. Wide field imaging may provide a more comfortable way for patients undergoing photography of their retinas. Dr. Lalane has published a comprehensive review in a medical journal discussing this new technology of wide field imaging. Dr. Marcus has served and continues to serve in a leadership position for several pharmaceutical study steering committees including evaluation of a surgically implanted device which slowly releases anti-VEGF medicine in the eye. This device may potentially limit the number of eye injections a patient needs to have. Dr. Marcus has helped developing and revising the surgical technique for device placement and in study protocol development and oversight. Our physicians also have devised unique approaches to the surgical removal of blood in diabetic eyes and have coined the term "endolaserless" surgical vitrectomy. Drs. Marcus and Lalane continue to study this technique within our clinical research unit as part of their clinical trial, known as the LASER LESS Trial. With this technique they are studying whether omitting laser during blood removal in a diabetic patient can lead to better vision while saving patients from the side effects of laser.
Our dedicated Certified Research staff brings extensive experience in quality data capture, visual function examinations, ophthalmic photography, OCT and OCT angiography, wide field retinal imaging, retinal visual field and microperimetry testing , retinal electrophysiology and ultrasonography. Our staff has cross-certification for various studies, strict protocol adherence and good clinical practice guidelines. Our site had been recognized as "Clinical Research Site of the Month" by the DRCR network.
Our imaging section performs anterior and posterior segment photography/fluorescein and indocyanine green angiography, posterior segment OCT (Cirrus, Heidelberg Spectralis ), HRT III, Corneal Topography (, microperimetry, and diagnostic and biometric ultrasonography .The visual field section employs Humphrey perimeters and on-site electrophysiology testing. Biologic specimens are collected, centrifuged, stored (-20F and -86F freezers), packaged under CLIA accreditation and shipped by EHSO certified personnel.
This extensive experience in well-designed, controlled clinical trials has made us one of today's nationally recognized leaders in retina based clinical research. Our doctors take pride in offering the most promising vision-saving treatment to our patients.
Why consider us for as your next Research Site?
If you are a Sponsor /CRO please contact the Clinical Research Manager- Christina Rex at 706-650-0061 , if you are interested in working with us as a site.
Please visit our Services page to find out more information on the services we provide.
Contact us today to find our more about our services or to arrange an appointment time. We have 13 locations to help serve you better.


Misty can host up to 3 dogs per night.
Misty can host up to 4 dogs per day for day care.
Misty was great! She stayed in contact, sent great pictures and made the whole experience easy! Highly recommend Misty!
Olive loves Misty and crew! She is always thrilled to hang out and go on adventures with the pups. Misty was super flexible with pick up and drop off and even took Olive to the beach on the way home. What a day!
Misty helped us out on short notice, picked up and dropped off our pup, and took great care of her. We truly appreciate the flexibility she offered as well as the kindness and open communication. It was easy and efficient.
Misty was a huge life saver when I had to have knee surgery. She took Olive last minute and really helped out with pickup, drop off, lots of updates and a bath for Olive. I don't know what we would have done without her. I know she will be a huge support over the next couple of recovery months as well.
Misty was so great! My dog has some fear issues and Misty was so patient working with her. Rainy loved her stay and came back so happy and tired. I loved getting the text and picture updates as well! This was definitely our best experience with dog sitting to date, thanks so much Misty!
Misty was great! We did a test weekend and he came back super happy. She was attentive, very communicative (including pictures), and an all-around nice person. Our dog needs a lot of exercise, which he got! We could tell he really liked her, after only spending 2 days!
I was completely confident in Misty's ability to watch Khan from start to finish.
From our initial meeting, Misty showed she was knowledgeable and attentive to the needs and energy levels of different dogs, asked all the right questions to learn about my pup Khan, and was very easy to coordinate/communicate with. During Khan's stay, Misty clearly made every effort to make sure Khan was comfortable in her home. She provided plenty of updates on Khan and pictures. I could tell that he was getting lots of play time, both outdoors and indoors, and that his play was both individualized as well as with other dogs for variety.
When Misty dropped Khan off, Khan was excited to see me, but sad to see Misty go! I've never had a situation where Khan has wanted to run back to his sitter, so I know he absolutely loved his stay. We look forward to booking with Misty again soon and cannot recommend her enough.
Bonus: An unexpected perk was Misty's optional pickup/drop off service! After a five-day trip and a sleepless overnight flight, the last thing I wanted to do was drive out to pick him up. Misty was gracious enough to have a pickup/drop off service for a very reasonable fee.
Misty was great with Olive and gave use the best updates. Olive is always thrilled to go to Misty's and comes home in perfect shape. We are so grateful.
Misty is an absolute champion. She took outstanding care of Miss Stella together with her own two Shiblings over the Holidays. I got regular updates, pictures, stories, and action shots of Stella playing, hiking, and even unwrapping presents - Stella was happy and healthy when I got home. I was returning from a super long flight overseas and Misty even took the time before my arrival to take Stella on a long hike to get her lots of exercise. So, when I arrived Stella was excited, but then slept like a baby through the entire night, which I was so grateful for - a tired Stella is a happy Stella. :) Misty is a true professional and excellent with dogs - I would strongly recommend her and thank her so much for making Stella happy and comfortable, and keeping her engaged with lots of activities over the Holidays!
Now offering roundtrip transportation for my clients to and from Seatac Airport. Why not save money and enjoy having a few more minutes with your furbabies. $35 includes it all. I drive a Subaru Outback: heated leather seats in the back too. Fully insured and always hands free.
To all my repeat clients and new ones searching for a loyal and devoted pet sitter, I am still actively pet/house sitting in the Seattle city neighborhoods! Boarding your sweet babies just got better. We have relocated south off I5/99. But please don't worry about the drive as I am offering round trip pick up and drop offs ($15). My townhouse was strategically positioned between Grandview dog park and the Green river in a small gated community which also happens to be close to the Seat-Tac airport. Which makes your life easier too if you would like to park your car in the garage. I have walking paths and trails outside my door to the river and park. Inside we have 3 levels of all access furbaby fun! The lower level has no stair access from the garage with a window, couch, TV, radio, toys, blankets and more. If your poor pet is having issues we can accommodate. The main level has direct access out to the patio and a 2nd potty area. We baby gate door access areas too. Still not convinced your dog should be in a huge house outside the city for fear it will escape? I have tile sports that I can attach to their collars and track them. :) I can and will always provide the best experience you and your pets need.
I understand how important special diets and medications are and how to keep their current schedule. If you're looking for a new sitter then please consider me as I am home full time. I can provide additional training as I've raised my dogs from pups and have had new born pups along with rescued 1 week old kittens. If you are working on socializing, then we can have some dog park time or if you prefer your dog to be alone then a quiet neighborhood walk can be accommodated. I want to provide a stress free environment with maximum amount of fun and a positive interaction. If you have any concerns or questions please ask.
Many people that reach out ask about up-charges. I will not up charge for dogs that pull on leashes. I do not add additional costs for walking during a stay or anything. Accidents happen and not all pups had the same from the start training. I love rescues! If you request your dogs to be the only additions to my home and I have no other bookings then I will accommodate that. If your pup needs alone time then I will provide that too. My two dogs are super chill and they also sometimes disappear to their own safe/quiet space. I do allow pets to sleep with me. If I sense they are homesick then I will lay wherever they prefer and help alleviate their symptoms with all the distractions as possible.
During our meet and greet I will go over many things so we have the chance to get to know each other and feel comfortable and confident going forward. Here is a list of just some of the questions I will ask.
Everything and anything you want me to know.
9 years, 8 mo. old, 23 lbs.
9 years, 8 mo. old, 35 lbs.
On leashed walks I will take your dogs through the small community, on quiet trails connected to the community, by the green river. For active dogs that need more and are social. I have a 34 acre dog park across the street from my neighborhood. See photos! Let me know if your dog likes adventures and car rides. While you're away I promise your pets will miss you but not too much.


text:
91 Wangarra Road Frankston	VIC 3199
Loads of Potential in a Prime Location!
Situated in a prime Frankston location stands this fantastic chance to buy an incredible value property that could return outstanding dividends. Providing a rare opportunity to be either a future family home or a profitable investment, this home confidently presents a prospect that is not so often found.
Designed with functionality in mind, whilst still maintaining the spacious open feel buyers frequently request, this properties floorplan is a lesson in practicality. Positioned upon entry is the first living area that features a gas fireplace & receives plenty of natural light whilst overlooking the front gardens.
Flowing on from the living is the spacious kitchen, boasting sleek stainless-steel appliances, plenty of bench space & an abundance of cabinetry. Being centrally placed allows for ease of entertaining & overall practicality.
Impressively large & conveniently open, the second living area sits at the opposite end of the home. Catering to the larger of families, the second living zone spills off the guest bedrooms & also offers French door access out to the courtyard.
Additional highlights include:
–	Garden shed
–	Off street parking for up to 4 cars
–	Gas wall furnace
–	Stainless-steel appliances
Grasp the chance to establish yourself within a growing suburb, filled with potential & overflowing with opportunity. Being only moments from schools, Frankston CBD & award-winning beaches this property is convenient in the lifestyle it provides. Containing the foundations of an outstanding family home, this properties location adds to its current value & is sure to be a rising asset over time.
Fast Facts about Frankston
Area Size 20.8 km2 (8.0 Sq Mi)
Location 41 km (25 mi) from Melbourne city centre
LGA Frankston City
Known infamously as the 'Gateway to the Mornington Peninsula', Frankston is indisputably one of the best beachside suburbs in Victoria. Offering access to the best schools the Peninsula has to offer, combined with its award winning beach and waterfront precinct, make this constantly evolving city the ideal place to live.
European settlement of Frankston began immediately after the foundation of Melbourne in 1835, as an unofficial fishing village serving the early Melbourne township.
Prior to its settlement, the Frankston area was populated primarily by the Mayone-bulluk clan of the Bunurong people of the Kulin nations.
Frankston has been a popular seaside destination of Melbourne since the early 1880s.
Frankston Beach is still one of the most popular in Victoria and is recognised as one of the cleanest in Australia.
Olivers Hill was named after local fisherman, James Oliver, who built a cottage atop the hill from where he kept an eye out for fish in the waters below.
The first official land sales in the area were held in 1853, and Frank Liardet (the eldest son of prominent settler, hotelier and descendant of French nobility, Wilbraham Liardet), established the "allam Ballam" estate in 1854. The estate was the earliest officially recorded settlement in Frankston, and was located to the east of Port Phillip, in what is now known as the locality of Karingal. Liardet's original homestead "Ballam Park" remains today, and is now heritage listed.
Daniel Robinson
Licensed Estate Agent
91 Wangarra Road, Frankston VIC 3199


The long, gently meandering character of the clouds in this view is a tracer of the generally stable flow of Saturn's atmosphere. The flow is disrupted at turbulent belt-zone boundaries and, here and there, by eddies and vortices.
The image was taken with the Cassini spacecraft narrow-angle camera on May 11, 2006 at a distance of approximately 2.9 million kilometers (1.8 million miles) from Saturn and at a Sun-Saturn-spacecraft, or phase, angle of 147 degrees. The image was obtained using a spectral filter sensitive to wavelengths of infrared light centered at 938 nanometers. Image scale is 17 kilometers (11 miles) per pixel.


Online retailing performance is under intense scrutiny this fall (Amazon's guidance is already down for Q4). How will others fare? One way to unpack performance is by how well firms generate attract, engage, and convert shoppers, especially as compared with competitors.
Displayed below (minus labels for now) is a visualization that does this. Each bubble represents a firm whose online revenues in 2007 ranked in the top 100 of all online retailers, according to Internet Retailer's "Top 500 Guide" (2008 edition). The size of each bubble is a relative indication of the number of monthly unique visitors to that firm's online retailing sites. The horizontal dimension measures the ratio of the number of monthly visits to the number of monthly unique visitors -- the higher the ratio, the greater the presumed "engagement" with that retailer. The vertical dimension represents Internet Retailer's estimate of the conversion rate (orders/visits, as IR calculates things) across each firm's online retail properties. I plotted the horizontal and vertical dimensions on a log-log basis, to help you see individual points a bit better. I'm sure you can guess the identity of at least one data point.
Inquiries very welcome from potential clients interested in the analysis and its implications. There are many interesting and useful variants to it as well. I'm planning to do it again with newer data soon, and maybe even work in time (with motion) as a fourth dimension.


text:
Elektrická jednotka řady ABe 8/12 je třídílná elektrická jednotka, vyrobená společností Stadler Rail pro Rhétskou dráhu (RhB) ve Švýcarsku v letech 2009–2010. Celkem bylo vyrobeno 15 kusů. Jednotka je schopna provozu na střídavé napájecí soustavě 11 kV 16,7 Hz i stejnosměrné soustavě 1 000 V a je určena pro dopravu všech druhů vlaků, včetně nákladních.

Jednotka je široce známá pod názvem Allegra, kterým byla výrobcem a provozovatelem pokřtěna. Allegra je tradiční rétorománský pozdrav.

V roce 2011 byly následně vyrobeny úzce příbuzné jednotky pro S-Bahn Chur řady ABe 4/16. Tyto jednotky jsou určeny pouze pro střídavou napájecí soustavu, mají pouze jeden hnací vůz, dva střední vozy a řídicí vůz.

Vývoj
Po přípravě koncepce vozového parku na rok 2005 vypsala Rhétská dráha výběrové řízení na dodávku dvou typů vozidel:

 15 třívozových dvousystémových jednotek pro hlavní síť a trať Bernina,
 5 jednosystémových vícedílných jednotek pro hlavní síť s nejméně 180 místy.
Specifikace vyžadovaly modulární konstrukci, pohodlné nastupování, bezpečné přechody mezi vozy a elegantní a harmonický design. Kromě toho byl ve vlaku vyžadován prostor pro cestující s omezenou schopností pohybu a orientace.

Společnosti Bombardier Transportation a Stadler Rail předložily své nabídky v rámci výběrového řízení. Společnost Stadler nakonec získala zakázku díky nižší ceně. Stadler také navrhl vybavit vlaky osmi trakčními motory namísto čtyř.

Pro jednotky Allegra platí tyto tabulkové normativy dopravované zátěže:

Provozní koncepce

Třídílné jednotky jsou určeny pro použití na nejstrmějších tratích RhB, kterými jsou:

 trať Bernina (maximální sklon 70 ‰, stejnosměrná napájecí soustava 1 000 V),
 trať do Arosy (maximální sklon 60 ‰, střídavá napájecí soustava 11 kV 16,7 Hz),
 trať do Davosu (maximální sklon 45 ‰, střídavá napájecí soustava 11 kV 16,7 Hz).
Vzhledem k tomu, že na trati Bernina je největší provoz v létě a na ostatních dvou tratích v zimě, byly univerzální jednotky pro všechny tři tratě navrženy tak, aby byly dvousystémové pro střídavý proud 11 kV o frekvenci 16,7 Hz i pro stejnosměrný proud 1 000 V. To umožnilo vyrobit velkou, jednotnou řadu a vedlo k co největší flexibilitě provozu.

Koncepce vozidla vyplývající ze zadání byla obtížná kvůli kombinaci vysokého trakčního výkonu, schopnosti používat dva systémy, dvojitého brzdového zařízení, bezbariérové konstrukce ve stísněných prostorových podmínkách metrového rozchodu a náročných provozních podmínek na trati Bernina. Souprava se skládá ze dvou stejných koncových vozů a částečně nízkopodlažního prostředního vloženého vozu. Rozměry vozidla jsou dány zejména tratí Bernina s malými poloměry oblouků 45 m; vlak byl navržen primárně pro trať Bernina. Bylo možné použít pouze krátké karoserie, což vyžadovalo důmyslné rozdělení hnacího ústrojí. Aby bylo dosaženo co největší adhezní hmotnosti, bylo hnací zařízení soustředěno na dva koncové vozy. Tím se také předchází riziku vykolejení lehkých vozidel při vysoké vrstvě sněhu. Dva hlavní transformátory a čtyři trakční měniče jsou umístěny pod podlahou vozu; klimatizace, brzdové odporníky a vysokonapěťové zařízení jsou umístěny na střeše. Uvnitř vozu musely být umístěny pouze dva kompresory a vývěva. To umožnilo vytvořit 12 míst v první třídě a 24 míst ve druhé třídě v každém koncovém voze.

Koncepce vozidla umožňuje dopravu i těžkých nákladních vlaků. To také vysvětluje poměrně velký trvalý výkon 2,3 MW pro elektrickou úzkorozchodnou jednotku. Nejvýkonnější typ lokomotivy RhB, Ge 4/4III, má trvalý výkon 2,4 MW. Rozjezdová tažná síla jednotky Allegra je dokonce o 30 % vyšší než u lokomotivy Ge 4/4III, a to 280 kN, zejména díky rozložení hnací síly na osm náprav. To je nezbytné pro tažení těžkých vlaků na rampách tratí Bernina a Arosa. Po uvedení do provozu se maximální přípustná hmotnost vlaku na trati Bernina pro tyto jednotky zvýšila ze 140 na 160 tun.

Trakční model požadovaný ve specifikacích s hmotností vlaku 70 t na sklonu 70 ‰ by vyžadoval zatížení náprav 14 t na čtyři dvojkolí a vedl by k velkému opotřebení kol a kolejnic. Realizovaná vícedílná jednotka s osmi hnacími nápravami má tlak na nápravu pouhých 11 t, a přesto utáhne vlak o hmotnosti až 140 t.

Každá jednotka stála přibližně 10 milionů švýcarských franků.

V roce 2011 následovala výroba úzce příbuzných jednotek pro S-Bahn Chur řady ABe 4/16. Tyto jednotky jsou určeny pouze pro střídavou napájecí soustavu, mají pouze jeden koncový motorový vůz, dva střední vozy a řídicí vůz. Primárně jsou nasazeny na osobních vlacích na trase Schiers – Chur – Rhäzüns.

Technický popis

Mechanická část
Každý ze tří vozů jednotky je posazen na dvou dvounápravových podvozcích. Základní koncepce podvozků byla převzata z panoramatických vozů pro trať Bernina. Jízdní vlastnosti, zejména středových vozů a při vyšších rychlostech, vedly ke stížnostem. Na trati údolím Prättigau a na trati Vereina je totiž ve vybraných úsecích povolena rychlost 100 km/h. Tyto problémy si vyžádaly přepracování ze strany výrobce. Výběr a nastavení vypružení, které musí vyhovovat v úzkých obloucích i na vysokorychlostních úsecích, byly náročné a vedly ke zpoždění dodávky jednotek. Vozy jsou navzájem mezi sebou spřaženy krátkými spřáhly.

Celá jednotka je pevně spojena jako třídílná; jednotlivé vozy lze oddělit pouze při údržbě v depu. Přechody mezi vozy jsou chráněny měchy a jsou bezpečné pro průchod cestujících i za jízdy. Neexistují však žádné možnosti přechodu do přípojných vozů. Střední vůz bez pohonu má sníženou podlahu mezi podvozky s nízkopodlažními vstupy na obou stranách a posuvnými schůdky, které zajišťují bezbariérový vstup pro osoby se sníženou pohyblivostí. Za každou ze dvou kabin strojvedoucího se nachází oddíl první třídy. Dělicí stěna do kabiny strojvedoucího je prosklená a nabízí cestujícím výhled na trať přes rameno strojvedoucího sedícího v ose vozidla. V těchto jednotkách byl poprvé u RhB řídící pult strojvedoucího umístěn uprostřed a nikoli vpravo.

Jednotka nabízí celkem 24 míst k sezení v první třídě a 76 míst k sezení ve druhé třídě, 14 sklopných sedadel a místo pro dva invalidní vozíky. Toaleta pro tělesně postižené se nachází v nízkopodlažní části prostředního vozu. Prostory pro cestující a kabiny strojvedoucího jsou klimatizované.

Vozové skříně jsou navrženy pro podélnou tlakovou sílu 800 kN a mají svařovanou hliníkovou integrální konstrukci. Většina potřebných lisovaných profilů byla vyvinuta speciálně pro jednotku Allegra. Skříň středního vozu je konstruována podle stejných konstrukčních zásad jako koncové vozy. Charakteristický tvar přední části jednotky umožňuje lepená přední kapota z plastu vyztuženého skelnými vlákny.

Elektrická část

Každý z koncových vozů má jednoramenný sběrač se třemi ližinami pro stejnosměrný proud, prostřední vůz má jeden sběrač pro střídavý proud. V případě poruchy a při posunu však může být každý sběrač použit při obou napětích. Všechny tři sběrače proudu jsou vzájemně propojeny vysokonapěťovým střešním vedením; proudový systém je automaticky rozpoznán vozidlem, když strojvedoucí vydá povel "zapnout hlavní vypínač". Při změně stávajícího systému musí strojvedoucí lokomotivy spustit všechny sběrače, když vozidlo stojí nebo je v pohybu. Jakmile se sběrač opět zvedne, vozidlo rozpozná současný proudový systém. Při rychlostech nad 15 km/h se používá lehké elektrické brzdění se spuštěnými pantografy. Takto získaná energie se využívá k nepřetržitému napájení pomocných systémů (nouzová brzda).

Při provozu pod střídavým napájecím systémem se energie dostává přes vysokonapěťové trolejové vedení, vakuový hlavní vypínač, trakční transformátor a měnič energie do trakčních motorů. Ve stejnosměrném režimu proudí energie z vysokonapěťového trolejového vedení přes stejnosměrný odpojovač, stejnosměrný rychlý spínač a síťovou tlumivku do stejnosměrného článku měniče. Při střídavém provozu odpojovač stejnosměrného proudu přeruší stejnosměrné zařízení, které je izolováno pouze na 1 000 V, od vysokonapěťového střešního vedení. Řadové tlumivky jsou umístěny v tělese transformátoru, a proto jsou chlazeny olejem.

Trakční transformátory mají přídavná vinutí 320 a 1 000 V pro vlakovou přípojku. Na tratích se střídavou trakční soustavou je vlaková přípojka napájena napětím 320 V. Pro napájení dlouhého osobního vlaku energií pro klimatizační a topné systémy je zapotřebí až 1 300 A. Vinutí 1 000 V napájí chladiče v multifunkční jednotce. Allegra je již připravena na možné zvýšení napětí hlavní vlakové přípojky na 1 000 V. Na trati Bernina jsou vlaková přípojka a chladiče soupravy napájeny přímo z trakčního vedení o napětí 1 000 V.

Vozidla RhB stojí v zimě přes noc se spuštěnými sběrači, aby se sběrač pod tíhou sněhu pomalu nesnižoval a nezpůsobil elektrický oblouk, který by mohl poškodit trakční vedení. Aby se zabránilo nočnímu ochlazování souprav ABe 8/12, jsou napájeny prostřednictvím tzv. topného stojanu a vlakové přípojky, které chladiče využívají k udržování teploty v prostoru pro cestující a strojvedoucího na zvolené úrovni. Kromě toho je napájena nabíječka baterií, která zajišťuje napájení spotřebičů 36 V.

ABe 8/12 mají dálkové ovládání pro provoz s řídicími vozy, tedy v režimu push-pull. Provádí se prostřednictvím 25 + 2 × 4pólového ovládacího kabelu LBT. V dlouhodobém horizontu se počítá s dálkovým a vícenásobným ovládáním všech trakčních jednotek RhB prostřednictvím tohoto kabelu, který pak převezme i funkce LBT (ovládání reproduktorů, osvětlení a dveří). Se stávajícími hnacími vozidly RhB nelze vlaky Allegra provozovat ve vícenásobné trakci.

Mnohé komponenty jsou technicky úzce spjaty se souběžně vyvíjenou soupravou řady RABe 4/12 "NExT" pro regionální dopravu Bern–Solothurn (RBS). Trakční motory jsou čtyřpólové asynchronní motory s nucenou ventilací. Stejné motory se používají nejen v "NExT", ale také ve vlacích Stadler-Diamant a RBe 4/8 na trati Chemin de fer Lausanne-Echallens-Bercher. Diagnostický systém je z velké části shodný s diagnostickými systémy jiných vozidel Stadler, jako je Flirt nebo GTW.

Provoz

Na trati Bernina jsou jednotky Allegra od roku 2011 nejdůležitějším hnacím vozidlem: vozí regionální vlaky, Bernina Express, složený pouze z panoramatických vozů, i nákladní vlaky. Bernina Express je z Churu do Tirana tažen jednotkou Allegra; výměna hnacího vozidla v Pontresině při přechodu ze střídavé na stejnosměrnou trakční soustavu díky tomu již není nutná.

Na trati do Arosy je většina osobních vlaků tažena vlaky Allegra. V méně vytížených časech dne jsou provozovány samostatně. Před uvedením jednotek Capricorn do provozu jezdily Allegry také na trati Landquart–Davos a po zastávce trvající něco málo přes půl hodiny pokračovaly po trati do Filisuru. Přitom byly využívány ve vratných soupravách řídicí vozy Bt 52801 až 52808, vyrobené v roce 2018.

V případě potřeby lze použít jednotky v celé síti Rhétské dráhy. Denní, týdenní a měsíční kontroly a drobné opravy se provádějí v depech v Landquartu, Samedanu a Poschiavu. Výměna střešního zařízení a kompletních podlahových sestav je možná pouze v Poschiavu a Landquartu. Aby se více jednotek nemuselo kvůli údržbě rozdělovat, bylo nutné v Landquartu postavit novou halu dlouhou asi 100 m.

Rychlostní rekord
20. prosince 2010 bylo při zvláštní jízdě dosaženo v tunelu Vereina s jednotkou číslo 3502 rychlosti 145 km/h a ustaven tak nový rychlostní rekord vozidel metrového rozchodu. Předtím bylo při zkušebních jízdách 5. prosince 2009 dosaženo rychlosti 139 km/h. Předchozí rekord pro metrové vozy 134 km/h byl stanoven v červnu 2009 společností Regionalverkehr Bern–Solothurn s vozem RABe 4/12 "NExT", rovněž vyrobeným společností Stadler. Na RhB byla dříve zaznamenána maximální rychlost 110 km/h.

V noci z 8. na 9. července 2022 pak byl tento rychlostní rekord překonán (opět v tunelu Vereina na Rhétské dráze) s jednotkou Capricorn čísla 3113. Nová jednotka dosáhla při zvláštní jízdě rychlosti 163 km/h.

Jména jednotek
První čtyři jednotky byly pokřtěny 1. května 2010. Jako jména nových jednotek byla vybrána jména významných osobností Graubündenu.

Seznam jednotek ABe 8/12

Příbuzné typy
 RhB řada ABe 4/16 3101–3105 – jednotka Allegra pro příměstskou dopravu na linkách S-Bahn Chur

Odkazy

Reference

Literatura

Externí odkazy

ABe 8/12
ABe 8/12
Železniční vozidla Stadler

redpajama_set_name: RedPajamaWikipedia


text:
Ninja's 10-in-1 multicooker is 41 percent off at Amazon
Jon Fingas
·Weekend Editor
November 30, 2021, 9:14 a.m. ·1 min read
Don't worry if you missed out on Black Friday multicooker deals — there are still big bargains available. Ninja's OS301 Foodi 10-in-1 cooker is back on sale at Amazon for $119, or 41 percent off its usual price. As with a similar deal in October, this might be the best value-for-money sale for one of Ninja's do-everything devices.
Buy OS301 Foodi 10-in-1 at Amazon - $119
This Foodi model remains an easy pick. It offers the usual multicooker features like pressure cooking, slow cooking and and steaming, but adds air frying and other perks you don't always see. You might also appreciate a 6.5-quart capacity that's ever-so-slightly larger than the typical 6qt Instant Pot size.
Once gain, the main catch is simply the ecosystem. Ninja has its supporters, but Instant Pot's fan base offers plenty of recipes and other advice you won't necessarily find for competing cookers. If that's no issue, though, Ninja's machine is a safe pick at this price given the broad feature set.
Get the latest Black Friday and Cyber Monday offers by visiting our deals homepage and following @EngadgetDeals on Twitter.
Tom Brady loses all-pro tackle, who hasn't missed a snap all season, on first series
Six-year-old Wichita girl killed, two others hurt in Friday crash on Turnpike: KHP
Officials say the SUV struck the rear of a semi truck.
HALIFAX — As students prepare to return to in-person classes on Monday, the president of the Nova Scotia teachers union says he has "severe doubts" whether schools will remain open until the end of the week. Paul Wozney says he believes the governmetn has been "overselling" its plan to help schools remain open as the Omicron variant of COVID-19 continues to sweep through the province. Wozney says schools had to be closed earlier than anticipated before the holiday break when staffing levels coul
After eight years of trying to stick around, Ocon finally has a foothold on a lasting F1 career.
Oldest remains of modern humans are much older than previously thought, researchers say
Remains in Ethiopia were originally thought to be around 200,000 years ago. Scientists now say they are at least 230,000 years ago.
Take 25% off Everlane jeans, leggings, shoes, outerwear, joggers and more during the brand's 48-hour sale.
NEW YORK (AP) — After a month at no. 1, "Spider-Man: No Way Home" has finally been overtaken at the box office. Paramount Pictures' "Scream" reboot debuted with $30.6 million in ticket sales over the weekend, according to studio estimates Sunday. "Scream," a self-described "requel" that is both the fifth film in the franchise and a reboot introducing a new, younger cast, led all releases over the Martin Luther King Jr. holiday weekend. Paramount forecasts that it will total $35 million including
All four hostages were released after the hours-long stand-off
Heat waives Marcus Garrett, expected to use open two-way contract spot to sign Kyle Guy
It appears that Kyle Guy will stick with the Miami Heat.
COLLEYVILLE, Texas (AP) — Authorities on Sunday identified a 44-year-old British national as the man who took four people hostage at a Texas synagogue for 10 hours before an FBI SWAT team stormed the building, ending a tense standoff that President Joe Biden called "an act of terror." Malik Faisal Akram was shot and killed after the last of the hostages got out at around 9 p.m. Saturday at Congregation Beth Israel near Fort Worth. In a statement, the FBI said there was no indication that anyone
Prime minister will overhaul way his top team works, Tory chairman says
Cashmyer and her boyfriend were featured on the MTV reality show in 2014


text:
Reminiscences of the Swami Vivekananda
Constance Towne
On one occasion there was an all-star cast in "Faust" at the Metropolitan Opera, on a Monday night when all society appeared to sit in their boxes and show their anatomy covered with jewels: to gossip, to visit, to come in late and be observed of all observers, and to do everything but listen to the opera. There was Melba in her prime, and de Reszkes, and Bauermeister. The Swami had never been to the opera, and our subscription seats were in a conspicuous part of the orchestra. I had suggested that the Swami be invited to accompany us. Mama said to him, "But you are black. What will the world say?" To which he laughed and said. "I will sit beside my sister. She does not mind. I know." He never looked more handsome. Everyone about us was so wrapped up in him that I am sure they did not listen to the opera at all that night.
I tried to explain the story of "Faust" to Vivekananda. Mama, hearing me. said, "Heavens! you, a young girl, should not tell this awful story to a man,"
"Then why do you make her come herself, if it is not good?" said the Swami.
"Well," replied Mama, "it is the thing to do to go to the opera. All the plots are bad; but one need not discuss the plot."
Alas for poor, vapid humanity and its foolishness! Later on during the performance the Swami said, "My sister, the gentleman who is making love to the beautiful lady in song, is he really in love with her?"
"Oh, yes, Swami."
"But he has wronged her, and makes her sad."
"Yes," I said humbly.
"Oh, now I see," said the Swami. "He is not in love with the handsome lady, he is in love with the handsome gentleman in red with the tail — what do you call him? — the Devil." Thus that pure mind reasoned out, weighed and found wanting both, the opera and the audience.
One of society's pets, a very young girl, came down between the acts to Mama and said, "Mama is consumed with curiosity lo know who the elegant man is in the yellow dressing gown."
Ours was a great friendship, and I fancy the only one that remains unpublished to the world. It was purely of the spirit, absolutely apart from the material loves and hates. He spoke always of when and what and where our souls would be ultimately, where in that other realm. He never spoke of me to anyone, nor mentioned my name. It was a friendship of Spirit. It still is. He taught me much of the philosophy he preached and wrote about, how to meditate, and what a power it would he against the hurts of life; what force of purpose it would attain for the preservation of the body, for logical thought, for self-control, for ecstasy, for the attraction of others; its power tor good, its knowing how to read others and their needs; not to dull the edge of your sword, to be moderate in one's consumption of food, to know what one's own body needs to make it live well; of chastity, tolerance, purity of thought, and love for the world — not of one person but of everyone and of all created things.
And now, forty years later, he has released me from the long silence and has demanded and commanded certain things he wishes done....
How liberal he was, how understanding of others' points of view! He went to Mass with me at St. Leo's Church, the little one on Twenty-eighth Street, where all was beauty, and the old priest, Father Ducey, such an artist. There he knelt at high noon at the canon of the Mass. A ray of light falling from the stained-glass window — blue, red, and gold — lit his white turban and outlined his beautiful profile against the marble walls. A great, gorgeous spot of living fire his orange robe made on the marble pavement, and the dear face was rapt in prayer. As the bell rang at the consecration and all heads were bowed in adoration of the presence of Christ on the altar, his hand touched mine, and he whispered, "It is the same God and Lord we both worship."
(Prabuddha Bharata, January 1934)
The main theme of my life is to take the message of Sanatana Dharma to every home and pave the way for launching, in a big way, the man-making programme preached and envisaged by great seers like Swami Vivekananda. - Mananeeya Eknathji
विवेकानन्द केन्द्र कन्याकुमारी (Vivekananda Kendra Kanyakumari)
Vivekananda Rock Memorial & Vivekananda Kendra : http://www.vivekanandakendra.org
Read Article, Magazine, Book @ http://eshop.vivekanandakendra.org/e-granthalaya
Cell : +91-941-801-5995, Landline : +91-177-283-5995
. . . Are you Strong? Do you feel Strength? — for I know it is Truth alone that gives Strength. Strength is the medicine for the world's disease . . .
This is the great fact: "Strength is LIFE; Weakness is Death."
Follow us on blog twitter youtube facebook g+ delicious rss Donate Online
Swami Vivekananda in the memory of Ida Ansell - 11
Swami Vivekananda in the memory of Ida Ansell - 9
Swami Vivekananda in the memory of Ida Ansell
Swami Vivekananda in memories of Christina Albers
Swami Vivekananda in the memory of Reeves Calkins - 3
Swami Vivekananda in the memory of Roxanne Blodgett
One Day When I Decided To Quit
Swami Vivekananda in the memory of Viraja Devi
In the Memory of Jamini Roy (11 April 1887 – 24 Ap...
Serve Man Serve God
The Monk Who Saw Religion Through The Prism Of Sci...
An article by Sri David Frawley - 2
An article by Sri David Frawley -1
Nation - An Extension of Man
Nation -an extension of Man


text:
Q: How to select everything but a specific pattern I am building a quick hack in sublimetext (yes, I know this is not optimal):
I have the following regex that selects all code within <script></script> tags:
<script[\s\S]*?\/script>

How can I select everything but this, i.e. the inverse of this / all code OUTSIDE script tags?
\\\code i want to select
 <script> code i don't weant to select</script>
/// code i want to select

I'd really appreciate examples both incluisve and exclusive of the script tags themselves.

A: Turn on DOTALL mode (?s) to make dot to match newline characters also.
(?s)^.*?(?=<script>)|(?<=<\/script>).*$

DEMO

A: There are many pitfalls using regex for html manipulation.
Consider for example this case:
<input type="text" value="<script> code i don't weant to select</script>" />

How do you handle this with a regex?
Therefore I would recommend to use an external library doing the parsing job for you. Google which ones are available in your programming language. Here are some I can recommend :

*

*Java: jsoup

*Python: BeautifulSoup

redpajama_set_name: RedPajamaStackExchange


Cocoa pod is an outer part of cocoa fruits being discarded during cocoa bean processing. Authors found out that data on its usage in literature as cosmetic materials was not recorded in vast. In this study, cocoa pod extract was investigated for its potential as a cosmetic ingredient.
Cocoa pod extract (CPE) composition was accomplished using UHPLC. The antioxidant capacity were measured using scavenging assay of 1,2-diphenyl-2-picrylhydrazyl (DPPH), β-carotene bleaching assay (BCB) and ferric reducing antioxidant power (FRAP). Inhibiting effect on skin degradation enzymes was carried out using elastase and collagenase assays. The skin whitening effect of CPE was determined based on mushroom tyrosinase assay and sun screening effect (UV-absorbance at 200-400 nm wavelength).
LC-MS/MS data showed the presence of carboxylic acid, phenolic acid, fatty acid, flavonoids (flavonol and flavones), stilbenoids and terpenoids in CPE. Results for antioxidant activity exhibited that CPE possessed good antioxidant activity, based on the mechanism of the assays compared with ascorbic acid (AA) and standardized pine bark extract (PBE); DPPH: AA > CPE > PBE; FRAP: PBE > CPE > AA; and BCB: BHT > CPE > PBE. Cocoa pod extract showed better action against elastase and collagenase enzymes in comparison with PBE and AA. Higher inhibition towards tyrosinase enzyme was exhibited by CPE than kojic acid and AA, although lower than PBE. CPE induced proliferation when tested on human fibroblast cell at low concentration. CPE also exhibited a potential as UVB sunscreen despite its low performance as a UVA sunscreen agent.
Therefore, the CPE has high potential as a cosmetic ingredient due to its anti-wrinkle, skin whitening, and sunscreen effects.
Skin wrinkles form as a result of natural aging process and the presence of excessive amount of reactive oxygen species (ROS). Although skin has self-defence system to deal with ROS, excessive and chronic exposure to UV can overwhelm the condition leading to oxidative stress and damage resulting premature aging. An imbalance of ROS on skin is due to factors such as overexposure to sunlight and lack of essential nutrient intake. In normal condition, skin produces enzymes such elastase and collagenase, at similar rate as aging process occurs and age increases. However, with overexposure to sunlight (UVA and UVB), the presence of excessive ROS and smoking habit, the enzymes are produced at a faster rate resulting in faster degradation of elastin and collagen which are the main foundation of extracellular matrix (ECM) of the dermis. Additionally, excessive exposure to sunlight, induced production of melanin in the skin layer. Tyrosinase is the responsible enzyme that initiates skin pigmentation. Potentially, plant extracts can suspend the skin aging and pigmentation process by encountering the ROS and protecting the skin from UV. The extracts protect the skin in various ways, including scavenging the ROS, reducing the ROS reactivity, inhibiting the oxidation, absorbing the UV light, suppressing the enzymes, thus reducing the risk of wrinkle formation and protecting the skin from aging. Examples of compounds that have attracted researchers to topical skin applications are polyphenols, triterpenes and stillbenoids. Polyphenols have a phenol ring with at least one hydroxyl substituent that enables scavenging of ROS, reducing metal ions, modulating protein phosphorylation (related to inhibition of enzymes activity), and inhibiting lipid peroxidation.
Cocoa pods are agricultural waste abundantly produced in cocoa plantations during the extraction of cocoa beans for cocoa processing. As discarded materials, cocoa pods can initiate the black pod disease by making the ground susceptible to the growth of fungi, which is typical in hot and humid climate. The pods vary in color (from maroon to green) and thickness when ripe depending on their clone. In Malaysia, multi clones of cocoa are planted and when discarded, the pods are thrown away in bulk regardless of the clones. Cocoa pods are used as fertilizers and animal feed. It is also being used as a source of activated carbon (unpublished data), potash, colorant, gum and anti hypercholesterolemia supplement. Previously, total phenolic content of cocoa pod husk was determined at 45.6-46.4 mg gallic acid equivalent of soluble phenolic while 32.3% carbohydrate, 21.44% lignin, 19.2% sugars, 8.6% protein and 27.7% minerals were reported. Higher level of antioxidant in cocoa pod extract by total phenolic content (TPC) was reported at 49.54 ± 3.39 mg gallic acid equivalent (GAE) per gram sample and total flavonoid content (TFC) at 22.42 + 0.98 mg rutin equivalent per gram sample (unpublished data). Sartini et al. also reported higher TPC value of cocoa pod husk at 56.5 ± 0.57 mg GAE/g. Scientific publication on identification of polyphenol compounds in a cocoa pod is rare, necessitating further research. Therefore, the present experiment was carried out to determine the cocoa pods compounds, which are rich in antioxidant activity and can be turned into high end-value products. In this paper, we investigated the anti-wrinkle, skin whitening and potential UV-protecting properties of cocoa pod extract using in-vitro assays and identified the potential of this extract as a new active ingredient for cosmetic.
Reagents used were of analytical grade purchased from suppliers such as Sigma-Aldrich for 1,2-diphenyl-2-picrylhydrazyl (DPPH), L-(+)-ascorbic acid (AA), Linoleic acid, Kojic acid (KA), Tyrosinase (from mushroom) and L-3,4-dihydroxyphenyl-alanine methyl ester hydrochloride (L-DOPA); Merck for acetonitrile, ammonium formate, formic acid, dimethylsulfoxide (DMSO), hydrochloric acid (HCl), chloroform and Tween-20; Fluka for 2,4,6-tripyridyl-s-triazine (TPTZ); Qrec for ferric chloride (FeCl3), Friendemann Schmidt for ferrous sulfate (FeSO4.7H2O), GmbH for ethanol and methanol; and MP Biomedicals for β-carotene powder. Test kits purchased from Enzo Life Sciences, Inc. Standardized Pine Bark extract (PBE) sample was provided by DKSH Malaysia Sdn Bhd (sample from Horphag, Switzerland) and used as a positive control in addition to ascorbic acid, and butylated hydroxytoluene (BHT, Sigma-Aldrich) in β-carotene bleaching assay. Human dermal fibroblast, adult (HDFa) was acquired from Life Technologies Corporation (GIBCO, catalogue number C-013-5C; Lot number 1378119) as well as Dulbecco's modified Eagle's medium (DMEM) and Trypsin (Trypsin LE™). Fetal bovine serum (FBS) and Streptomycin/Penicillin (antibiotics) obtained from Biowest. MTT or 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide supplied by Bio Basic Canada Inc. Phosphate Buffer Saline (PBS, 10X) purchased from R&M Chemicals, UK.
Discarded cocoa pods collected from a cocoa fermentation site at cocoa plantation in Cocoa Development and Research Centre, Jengka, Pahang, Malaysia. After rinsed with tap water, the pods were chopped using mechanical fruit slicer (FC-312, Zhaoqing Fengxiang Food Machinery, China) for better water removal when drying in high-performance dryer (FD-825, Protech, Malaysia). The dried pods were ground into powder at 1.0 mm size using a grinding machine with a sieve (Automatic Hammer Mill Grinder, China). The cocoa pod powder was kept in tight containers at room temperature (not more than two weeks) until extraction procedure adjourned.
Twenty millilitres of aqueous ethanol (80%) were poured into a conical flask containing one gram of cocoa pod powder and shaken in water bath shaker (BS-21, Lab Companion, Korea) at 120 rpm, 40°C, for 30 minutes to prepare the extract. Soluble portion filtered (using Whattman No.1), evaporated to dryness (using vacuum rotary evaporator; IKA, Germany) and re-dissolved with 5 ml ethanol after recording the weight. Insoluble material was filtered out using filter paper. The extracted sample was stored in air-tight vial at -10°C until evaluated. We used AA and PBE as the positive control in the study, due to its effective role in maintaining cellular function[13, 14] which affects the skin condition.
LC-MS/MS analysis was performed using a Perkin Elmer Flexar FX-15 ultra-high performance liquid chromatography (Perkin Elmer, USA). It equipped with a reversed-phase C18 analytical column of 50 mm × 2.1 mm × 1.9 μm particle size (Perkin Elmer, USA). The column oven temperature was set at 35°C, and the flow rate was 250 μl/min. Mobile phases A and B were water and acetonitrile, respectively, each containing 5 mM ammonium formate and 0.1% formic acid. The linear gradient programme was set as follows: 0–0.1 min, 90% A/10% B; 0.1-8 min, 10% A/90% B; 8–10 min, 10% A/90% B; 10–15 min, 90% A/10% B. The injection volume was 20 μL with a run time of 15 minutes. The UHPLC was hyphenated to a triple quadrupole mass spectrometer 3200 QTrap (ABSciex) equipped with an electrospray ionization interface set at negative mode. The interface heater held at the temperature of 500°C and an ion-spray (IS) voltage of -4500 eV. The nebulising gas (GS1), heating gas (GS2) and curtain gas pressures set at 40, 40 and 10 psi, respectively during the whole analysis. Nitrogen was used as collision and spray gas. Full scan data acquisition was performed, scanning from m/z 5 to 1500 in enhanced MS IDA EPI mode. Analyst software version 1.5.2 was used for method development, data acquisition and data processing.
Antioxidant activities were evaluated using three different assays; i.e., 1,2-diphenyl-2-picrylhydrazyl (DPPH), Ferric Reducing Antioxidant Power (FRAP) and β-carotene bleaching assay (BCB). Methods of preparation of DPPH[2, 15, 16], FRAP[17–22] and BCB solutions and calculation were adopted with some modifications. Briefly, DPPH solution was prepared by diluting 1.2 ml of DPPH stock solution (0.2 M DPPH in ethanol) with 3 ml ethanol and 0.5 ml DMSO. In a 96-well microplate, 270 μl of the solution was added to 30 μl of tested sample in a dilution series of 7.8-1000 μg/ml, and absorbance measured at 550 nm. Scavenging effect was calculated by the percentage of faded purple DPPH solution color into yellow by the tested sample against the control (DPPH solution only). The EC50 of DPPH assay represents the concentration of the tested sample needed to reduce the DPPH by 50% where the value obtained from linear regression graph.
For FRAP assay, two solutions; i.e., ferric chloride solution (3 mM in 5 mM citric acid) and TPTZ solution (2,4,6-tripyridyl-s-triazine; 1 mM in 0.05 M hydrochloric acid) were prepared. Fifteen μl of the tested sample was added to 270 μl TPTZ solution and measurement at 620 nm was carried out immediately after addition of 15 μl of ferric chloride solution. The absorbance data calculated against serial dilution of Ferrous sulfate (FeSO4.7H2O) and recorded as equivalent to μM Fe2+. Effective concentration at 50% (EC50) of FRAP value is the sample concentration required to reduce 0.5 mol of Fe3+ to Fe2+.
Two milligrams of β-carotene powder was dissolved in 0.2 ml chloroform followed by adding 0.2 ml linoleic acid, 2 ml Tween20 and 100 ml of distilled water to make β-carotene solution. It was mixed until almost transparent solution obtained. Two hundred μl of the solution was added to 20 μl of the tested solution and measurement at 450 nm was carried out after incubation for 20 minutes at 50°C. Measurement was monitored for 2 hours at 30-minute intervals. Calculation of antioxidant activity percentage obtained by the difference of degradation rate of tested sample to the degradation rate of control (β-carotene solution only). Effective concentration at 50% (EC50) was determined to represent the ability of the sample to protect the β-carotene solution from degradation, thus indicating high antioxidant activity with low concentration of the tested sample. Instead of AA, BHT was used as a positive control in this assay. BHT is well-known antioxidant specifically in protecting lipid oxidation and stronger antioxidant compound compared with AA.
Elastase and collagenase inhibition measurements were carried out using drug discovery kits (Neutrophil Elastase Colorimetric and MMP-1 Colorimetric, respectively) following protocols as in Enzo Life Science[27, 28]. For elastase inhibition assay, 20 μl of tested sample was diluted with 65 μl buffer-solution containing 100 mM HEPES, 500 mM NaCl and 0.05% Tween20 in DMSO in a 96-well plate. Elastatinal (100 μM) used as the control inhibitor. The neutrophil elastase enzyme (purified human neutrophil elastase, 2.2 μU/μl) at 10 μl was added to the diluted tested sample and incubated for 10 minutes at 37°C. Later, 5 μl substrate (MeOSuc-Ala-Ala-Pro-Val-pNA, 100 μM) was added to each well and absorbance at 405 nm was monitored for 10 minutes.
Briefly for collagenase inhibition assay, 20 μl of tested sample was diluted with 50 μl buffer-solution (50 mM HEPES, 10 mM CaCl2, 0.05% Brij-35 and 1 mM DTNB in DMSO). Twenty microlitres of MMP-1 enzyme (E.coli recombinant human MMP-1 catalytic domain, 153 mU/μl) was added to each well prior to incubation at 37°C for 30 minutes. Control inhibitor, NNGH (N-Isobutyl-N-(4-methoxyphenylsulfonyl) glycylhydroxamic acid; 1.3 μM), was used for comparison. Substrate (thiopeptide, Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5; 100 μM) at 10 μl was added to each well and absorbance at 410 nm was monitored for 10 minutes.
For both protocols, slope of remaining activity for the tested sample against the control (without sample) was calculated in percentage and inhibition percentage was obtained by subtracting the obtained value from 100. Inhibition concentration of 50% (IC50) is the concentration of the tested sample that can inhibit the enzymes activities to 50%.
Skin whitening effect was evaluated based on inhibition of mushroom tyrosinase by the tested sample with L-DOPA as substrate using a method described by Chiari et al.. The tested solution was diluted in series (1000-250 μg/ml) using DMSO and 20 μl was pipetted into a 96-well microplate, followed by addition of 138 μl PBS (phosphate buffer solution) and 2 μl mushroom tyrosine solution (2500 U/ml, in PBS). After incubation at 37°C for 90 minutes, 40 μl of L-DOPA (2.5 mM in PBS) was added, and measurement at 450 nm monitored for 20 minutes. Kojic acid was used for comparison.
For UV sunscreen potential activity, the tested sample was dissolved in ethanol (ratio 8:125). Same solvent was used as blank for baseline correction so that the results obtained could be compared with each other. Samples were scanned at 200-400 nm wavelength using dual beam UV-Spectrophotometer (Cary 60, US). Absorbance of tested samples at critical wavelengths (290, 308, 330 and 350 nm) were selected for comparison. Higher value of absorbance indicates better potential as UV-sunscreen agent, in-vitro. Two broad spectrums of commercial sunscreens; i.e. Avobenzone and Octylmethoxycinnamate (OMC) were used for comparison. The results obtained were only for screening purposes; therefore, a subsequent experimental study using non-invasive method had to be carried out to determine the Sun Protecting Factor (SPF) value prior to using the extracts in formulation.
Healthy cells were initiated from cryopreserved HDFa in a 25 cm2 tissue culture flask in DMEM containing 10% FBS and 1% antibiotics. The cells were incubated at 37°C, 95% humidity and 5% CO2 until confluent. The growth media was refreshed every two days until at least 80% confluence was achieved, and was trypsinized with Trypsin LE™ to passage for not more than eight times for cell viability study. The cells were seeded into a 96-well microplate at density of 1×105 per well and incubated for 24 hours. Serial dilution of CPE, AA and PBE were added to the well, respectively, after removal of the spent media and incubated for another 24 hour. Forty microliters (40 μL) of MTT in PBS (2.5 mg/mL) was added to each well and incubated for 4 hours before the absorbance was measured at 570 with 630 nm as reference wavelength. A hundred microlitres (100 μL) of DMSO was used to dissolve the dye crystals. The percentage of cell viability was calculated based on the optical density of each well against the control (cell without any treatment). Inhibition concentration of 90% (IC90) is the sample concentration that enables 90% of cells survived after treatment with the tested sample which metabolized the MTT salts to formazan.
The results were presented as mean ± standard deviation determined in triplicates of two independent samples. Comparison was made using two samples T-test by Minitab Software version 14.12.0 (US Inc.). Results were significantly different when p-value was less than 0.05 (p < 0.05).
Crude CPE was analyzed by LC/MS/MS in negative mode. We identified each compound based on the literature search in established spectrum databases[34, 35]. Product ion spectrum of CPE was compared with the product ion spectrum obtained from the search and was listed in Table 1[34–54] according to its retention time. We considered the compound as its derivatives, if the product ion spectrum did not hit any of the product ion m/z in the search but hit only the first and one or more of the product ion m/z in the spectrum. PBE, known to contain a wide range of polyphenol compounds, was also analyzed for comparison purposes. Compounds that are similarly available in PBE also were noted at the end column of Table 1. Additionally, compounds that appear only in PBE were listed in Table 2[34, 35, 55–57]. CPE and PBE contained almost similar compounds, including polyphenols, carboxylic acid (citric acid and malic acid) and sugars (gluconic acid sodium salt). Polyphenolic compounds in CPE included phenolic acids (protocatechuic acid, p-hydroxybenzoic acid and salicyclic acid), flavonols (kaempferol) and flavones (linarin), which also were detected in PBE. Resveratrol, a stilbenoids compound that was detected in wine, was also found in CPE but not in PBE. CPE also was detected with tartaric acid, apigenin and luteoin (flavone), crysoplenol (terpenoids), linoleic acid and oleic acid (fatty acids) which were not available in PBE. Nevertheless, gallic acid, catechin, quercetin and caffeic acid were found in PBE but were absent in CPE.
Absorbance at 550 nm of cocoa pod extract was measured after at least 10 minutes of incubation at room temperature by DPPH assay to ensure complete reaction. The incubation time was determined previously (data not included) by a method as described by Marxen et al.. CPE achieved optimum value of scavenging percentage by this assay at 62.5 μg/ml, which was lower than PBE and AA at 125 μg/ml (Table 3). At this optimum value, CPE showed significantly higher antioxidant activity (87.07 ± 3.09%; 62.5 μg/ml) compared with AA (72.21 ± 2.21%; 125 μg/ml) and PBE (67.43 ± 1.71%; 125 μg/ml). The effective concentration to reduce the DPPH radical to 50% (EC50) was determined by plotting linear regression curve of DPPH activity versus ratio of sample concentration to DPPH as previously reported. The tested sample with EC50 value lower than 30 μg/ml by DPPH assay, has high efficiency as free radical scavenger, specifically against superoxide anion radicals, as exhibited by CPE, which was two times higher antioxidant activity than standardized PBE, although it indicated a two-fold decrease in comparison with AA. The presence of more than one carboxylic acid such as citric acid and malic acid, besides phenolic acid and other polyphenols in CPE is suggested to contribute to high antioxidant value by DPPH assay, compared with AA, which contained a single compound. In addition, higher antioxidant activity values by DPPH assay for CPE than PBE is also attributed to the presence of terpenoid and resveratrol compounds.
a b c dDifferent alphabet in column means significantly different within sample for each assay.
α β γ δ ε € £different symbol means significantly different within concentration for each sample in row for each assay.
The ability of the tested solution to deviate the mechanism of Fenton reaction by chelating the metal ions, such as Fe2+ and Cu2+, which is responsible to convert the hydrogen peroxide to hydroxyl radical (ROS) on the skin, can be measured using FRAP assay. We conducted a kinetic study for the tested sample at 620 nm (data not published) measured after five minutes of reaction. CPE had similar antioxidant activity to AA when measured using FRAP assay except at 250 μg/ml, where the FRAP value of CPE was significantly higher than AA and PBE. Antioxidant activity of PBE was proportional to the tested concentration and showed significantly higher activity when compared with EC50 value of the FRAP assay. Although CPE had three times higher metal reducing ion potential than AA, its performance was 1.4 times lower than that of PBE. The presence of chlorogenic acid derivatives in CPE is likely to contribute to its higher FRAP value than that of AA since this compound is metal chelating ion. Other than that, flavonol derivatives are also known to chelate metal ions, such as rhamnetin and kaempferol derivatives in CPE. Flavanol compounds, such as catechin and quercetin also contribute to the chelating action on metal ion, which are available in PBE but absent in CPE, resulting in higher FRAP value for PBE. Phenolics with ion reducing ability diminish the possibility of hydroxyl radical's formation path from superoxide anion radicals and additionally inhibit enzymes due to their abilities to chelate copper at the active site.
β-carotene in BCB assay serves as an indicator that degrades during the oxidation process when linoleic acid turns to hydroperoxides at high incubation temperature. The presence of antioxidant eliminates or reduces the action of this radical species to β-carotene as measured by the absorbance at 450 nm and calculation of degradation rate. High value of antioxidant activity by BCB assay exhibits the tested solution is a good antioxidant agent due to the presence of linoleic acid that acts as pro-oxidant. CPE achieved optimum value of antioxidant activity at concentration of 250 μg/ml (81.55 ± 1.08%), which was higher than standardized PBE and BHT at 125 μg/ml (40.71 ± 8.66% and 91.72 ± 2.51%, respectively). Both CPE and PBE had lower antioxidant activity than BHT, but based on EC50 value; antioxidant activity of CPE was four times lower than that of BHT while PBE showed the lowest activity. Higher antioxidant activity of CPE in comparison with PBE can be attributed to the terpenoid and resveratrol compounds that solely indicate better protection for lipid oxidation[62, 63]. Plant extracts with lipid protection properties can protect the skin from lipid peroxyl radicals that attack stratum corneum layer.
Determination of elastase inhibition at higher concentration (1000 μg/ml) of CPE and standardized PBE resulted in a negative slope, which was also observed for 500 μg/ml of AA, indicating no activity of elastase either for inhibition or activation. This situation should be taken into consideration to avoid false positive results, where the inhibition detects at certain concentration range of extracts or control inhibitors; i.e. elastatinal (100 μM) as in Enzo Life Science Manual as well as the concentration of enzyme (neutrophil elastase enzyme, 100 μM) used in this study. Table 4 summarizes the result of elastase inhibition for CPE and standardized PBE at concentrations of 500, 100 and 10 μg/ml. At high concentrations (500 and 100 μg/ml), standardized PBE exhibited higher activity than control inhibitor, which was in agreement with a previous report by Grimm et al.. Catechin, a flavanol was reported to have an inhibitory effect on elastase enzyme[65, 66], which is also present in PBE. Catechin was also reported to inhibit binding activity of transcription factors and kinases (AP1 and NF-κB), therefore lowering the activity of MMPs for collagen degradation and inflammation. CPE has lower elastase inhibition activity in comparison with standardized PBE at 100 μg/ml but similar to AA and control inhibitor. Terpenoid compounds were previously suggested as elastase inhibitors, is present in CPE since flavones and flavonol components are not efficient inhibitors of elastase activity. The inhibition of elastase by CPE was much lower at lower concentration (10 μg/ml) in comparison with AA although higher than standardized PBE. Therefore, CPE has low activity to inhibit the elastase enzyme which is responsible for degradation of elastin fibrous structure in a dermal matrix.
a b c dDifferent alphabet in column means significantly different within sample.
α β γ δ ε € £different symbol means significantly different within concentration for each sample in row.
CPE exhibited almost two times better collagenase inhibition activity than AA and three times better activity than standardized PBE based on IC50 (Table 5). Increasing the concentration of the tested sample to more than 1000 μg/ml (data not shown) did not affect the collagenase inhibition activity, as exhibited by standardized PBE. Research by Lim et al. indicated that kaempferol and quercetin were strong inhibitors of collagenase while apigenin, a flavone compound, weakly inhibited collagenase activity. Flavonols had better inhibition activity towards collagenase enzyme than flavones compounds. Resveratrol, a stilbenoid compound, was patented for its effective inhibition against the collagenase effectively in reducing wrinkles. CPE showed significantly higher collagenase inhibition activity than standardized PBE at 1000 μg/ml, although the activity was lower than that of AA and control inhibitor. The higher inhibition by CPE can be attributed to the presence of more than one flavonol compound, whereas PBE has only one identified flavonol compound. In addition, CPE was detected with two types of resveratrol compared with PBE. Therefore, the presence of various polyphenolic compounds in CPE contributed to its potential in maintaining collagen longevity in the skin layer.
The capability of CPE to inhibit tyrosinase activity can be translated to its potential as skin whitening agent. When tyrosinase enzyme activity is inhibited, melanin production is reduced, resulting in a fairer skin. Inhibition of tyrosinase activity was significantly higher in CPE than standardized PBE at high concentration (1000 μg/ml) but showed similar activity at lower concentration (125 μg/ml). CPE was also found to be a better inhibitor of tyrosinase activity than kojic acid and ascorbic acid (Table 6), although not as good as PBE based on the IC50 value. CPE and PBE contained several compounds, such as flavonols, flavanols, stilbenoids and phenolic acid, reported to inhibit tyrosinase activity. Quercetin, a flavonol compound, inhibits tyrosinase enzyme better than kaempferol suggesting higher inhibition activity by PBE than CPE. Resveratrol in CPE contributed to the inhibition of the enzyme; however, this contribution was less than that of oxyresveratrol. Fatty acids, such as linoleic acid and oleic acid, which were detected in the CPE too, were also reported to have skin whitening properties without adverse effects.
a b c dDifferent alphabet in column means significantly different within concentration.
Absorbance at UVC spectrum (200-290 nm) is not discussed as the radiation is filtered by the atmosphere. Meanwhile, absorbance of the tested samples at the other two spectrums; i.e., UVB (290-315 nm) and UVA (315-400 nm), are discussed. There are two ways in which sunscreen agent in cosmetic products protect the skin: (i) by scattering the UV-rays (titanium and zinc oxide) or (ii) by absorbing the light before reaching the skin. Plant extract, especially with flavones and pigments, can absorb UV light. CPE showed potentially good UV absorbance at UVB range wavelength, which was even better than the commercially used UV-protecting agent in this study; i.e. Avobenzone and Octylmethoxycinnamate (OMC), and significantly better than standardized PBE at 290 nm (Table 7). Higher sun-protecting action in CPE could be caused by flavone derivatives such as luteolin at UVA wavelength (330 nm), which were absent in PBE. The presence of methyl salicylate compound in CPE also enhanced the absorbance. CPE also showed similar absorbance potential to that of commercial UV-sunscreen agent, OMC, at 308 nm, the peak of erythema (sunburn) action spectrum. The extract had significantly better absorbance than Avobenzone at this wavelength.
a b c dDifferent alphabet in column means significantly different within sample at each wavelength.
CPE had moderate absorbance of UVA (315-400 nm) as illustrated in Figure 1, which was significantly better than that of standardized PBE (Table 7). Therefore, CPE has better potential as UVB sunscreen agent for cosmetic formulation and can be recommended in combination with other sunscreen agents with higher UVA absorbance properties. All plant extracts with pigment and color can absorb UVA and or UVB as CPE does. Extracts with color might not be possible for white cream formulation; however, for color cosmetic products; i.e. foundation and lipstick, colored extracts could be added as sunscreen agents.
Absorbance of tested samples compared with commercial UV-sunscreen agent (Avobenzone and Octylmethoxycinnamate) at 200–400 wavelength.
In this study we calculated the IC90, the concentration of the tested solution to maintain the cell population up to 90%. High value of IC90 indicated that high concentration of the tested solution is needed to maintain the population of fibroblast cell. Table 8 shows that CPE had the lowest value indicating low concentration of the extract was able to maintain the cell growth at 90%. The highest value obtained by PBE, which was almost two times more than that of AA. Almost no toxicity was exhibited by CPE at the tested concentration since the cell viability percentage was more than 50%, in comparison with AA and PBE. There could be possible cell death at low concentrations for AA at 62.5 μg/ml and PBE at 31.3 μg/ml, in contrast to the results of a study conducted by Kim et al. in which PBE showed no toxicity to B16 melanoma cell, a type of cell different from the one used in this study. At high concentrations of AA and PBE, the cells proliferated as shown by the increasing percentage of more than 100%, the highest percentage being achieved at 500 μg/ml. Contrary to CPE, at low concentration of the tested solution (3.9 μg/ml), the percentage of cell viability was the highest, thus low concentration of CPE was needed to induce proliferation of fibroblast cell.
We concluded that cocoa pod extract exhibits better antioxidant activities potential than standardized pine bark extract as measured using DPPH scavenging and β-carotene bleaching assays, but lower activities using FRAP assay, based on EC50. CPE inhibited collagenase and elastase enzymes better than PBE, although second to PBE in inhibiting the tyrosinase enzyme. Cocoa pod extract also possesses high potential as UVB sunscreen agent although with lower performance in UVA range wavelength than that of the commercial sunscreen agent. However, the extract can be formulated with other UVA protecting agents to work synergistically for a broad spectrum of UV exposure. We, therefore, recommend a possible use of CPE as ingredient for functional cosmetic products specifically for anti-wrinkles as well as skin whitening or sunscreen products in combination with natural plant extracts to widen the spectrum of protecting from sun-rays.
Azila Abdul Karim, Azrina Azlan contributed equally to this work.
The authors would like to thank Analytical Service Laboratory of Malaysian Cocoa Board for using laboratory facilities. Special gratitude extended to Malaysian Cocoa Board for funding the research. Authors would like to acknowledge Universiti Putra Malaysia for consultation and expertise advice to complete the research work.
AAK conceived of the study, participated in its experimentation and coordination, performed the statistical analysis and drafted the manuscript. AA involved in the design of the study and helped to draft the manuscript. AI designed the study and supervised the experimental workflow. PH drafted the experimental flow and helped to draft the manuscript. SSAG involved in data arrangement and help to draft the manuscript. BHZ carried out the LCMS/MS experimental work, performed the LCMS analysis and drafted this section. NAA performed the antioxidant assay and helped to draft the manuscript. All authors read and approved the final manuscript.


text:
Famous DNA
Archaeological Ruins
Famous & Infamous People
COV-19 Italy ReportLocal Informations
Did you know all Jewish Cohanim descend from a common ancestor?
Did you know the warrior gene is linked to aggression?
Did you know descendants of Prince Branciforte Barresi refused to give DNA samples to authenticate his remains?
Did you know DNA analyses finally identified the true identity of Anna Anderson?
Did you know the eruption of Mount Vesuvius froze Pompeii at 79 AD?
COV-19 Italy ReportLocal Coronavirus Informations
Famous DNARoyalty
Did you know DNA analyses were used to accurately identify the Romanov remains?
Formal photograph of the Romanovs
One night, while they were under the imprisonment of the Bolshevik secret police, the entire Romanov family simply vanished. Amid rumors and conflicting information released by the Soviet leadership, the fate of this Russian imperial family remained unknown.
More than 70 years after their disappearance, a mass grave was discovered near where they were held captive. And genetic analyses confirmed what had long been feared – Tsar Nicholas, Tsarina Alexandra and at least three of their children were brutally executed on that fateful night in July 1918.
Who were the Romanovs?
Tsar Nicholas II
The House of Romanov was the second dynasty to rule Russia. Tsar Nicholas II, the last ruler from this imperial family, was the eldest son of Emperor Alexander III and Maria Feodorovna of Denmark.
He married Princess Alix of Hess and by Rhine, one of Queen Victoria's granddaughters. Princess Alix was given the name Tsarina Alexandra Feodorovna when she was received into the Russian Orthodox Church upon her marriage to the Tsar.
The couple had four daughters (Olga, Tatiana, Maria and Anastasia) and one son (Alexei). Alexei suffered from haemophilia B, an inherited blood clotting disorder caused by mutations in the F9 gene. It was known as the 'royal disease' at the time, because the mutation was passed down through two of Queen Victoria's daughters to various royal families across Europe.
The execution of Tsar Nicholas II
Tsarina Alexandra Feodorovna, Alix of Hesse
At the end of the February Revolution (the first of the two Russian revolutions in 1917), Tsar Nicholas was forced to abdicate. In March 1917, the entire Romanov family and their loyal servants were put under house arrest, before they were evacuated to Tobolsk.
When the Bolsheviks came to power later that year, the Romanov's imprisonment conditions grew stricter. By May 1918, the Romanov family, plus three servants and the family doctor, had been moved to Ipatiev House at Ekaterinburg, in the Urals of Central Russia.
Then the Bolsheviks announced the execution of Tsar Nicholas in July 1918. According to the official press release "Nicholas Romanov's wife and son have been sent to a secure place."
What happened to the rest of the Romanovs?
In the following years, the Soviet leadership continued to release conflicting information regarding the fate of the remaining Romanovs. Some reports stated they were all murdered, while others outright denied that they had killed the other family members.
Finally in 1926, the Bolsheviks acknowledged the murders of the Romanov family. However, they still denied responsibility and claimed that the bodies had been destroyed. Numerous people came forward claiming to be members of the Romanov family in the ensuing years, including Anna Anderson, the most famous Romanov imposter.
Discovery of the remains
The fate of the Romanovs remained a mystery until 1991, when nine human skeletons were discovered in a shallow pit about 20 miles from Ekaterinburg. Historians speculated that the remains belonged to the Romanov family.
DNA testing was performed in an attempt to identify the remains. Three types of DNA tests were used to confirm the identity of the Ekaterinburg remains – gender determination, relationship testing and mitochondrial DNA analysis.
The science behind the genetic analyses
Gender determination:
The gender of each of the nine skeletal remains were confirmed by analyzing a region of the amelogenin gene. This is a robust technique for gender analyses of unknown remains, because the X and Y chromosome have different versions of the amelogenin gene. This allows researchers to distinguish between female remains (with 2 X chromosomes) and male remains (with one X and one Y chromosome).
Relationship testing:
Autosomal STR (short tandem repeat) markers were used to determine whether the nine skeletons were immediate family members. This technique is useful for looking at ancient and degraded DNA samples, because only a small region of DNA is required for analysis.
We inherit one copy of each autosomal chromosome from each parent. This means that for each STR, a child will have one marker that matches one parent, and another marker that will match to the other parent.
Autosomal STR analysis can be used for relationship testing in both males and females in recent samples (e.g. paternity and maternity testing), and during the identification of old skeletons.
Mitochondrial DNA analysis:
Regions of mtDNA amenable to DNA analysis
Mitochondrial DNA (mtDNA) is different from autosomal DNA, because it's strictly maternally inherited (passed from mother to child).
Both males and females inherit mtDNA from their mother, so both males and females can take the mtDNA test. However, only females will pass the mtDNA down to the next generation, providing a way of tracing maternal ancestry.
Also, there are hundreds of copies of the mtDNA DNA in each of our cells. This makes it very useful for the analysis of ancient samples.
What did the researchers find out?
The amelogenin gene analyses showed that there were five female and four male skeletons in the grave. Autosomal STR analyses indicated that five of these skeletons were from the same family (two parents and three female children). The other four were unrelated individuals.
Identifying remains through mtDNA analyses relies on access to DNA from maternal relatives, so the unknown samples can be compared to the reference. Luckily, the family trees of the European royal families are well documented. So it was a simple task to find maternal relatives of the Romanovs.
Identifying Tsarina Alexandra's remains
Tsarina Alexandra was the maternal granddaughter of Queen Victoria. This means she has the same mtDNA profile as Queen Victoria. Prince Philip is a great-great-grandchild (on the maternal side) of Queen Victoria. Therefore, he also carries the same mtDNA as Queen Victoria and Tsarina Alexandra.
Family tree showing the maternal relationship between the Romanovs and Prince Philip, Duke of Edinburgh
The mtDNA profiles generated from the putative skeletons of Tsarina Alexandra and her three children, matched to the mtDNA profile of Prince Philip.
This provided very strong evidence that the skeletal remains were maternal relatives of Prince Philip, and likely the remains of the missing Romanov family.
The remains of the Tsar
To confirm the identity of Tsar Nicholas, mtDNA profile from his putative skeleton was compared to mtDNA profiles obtained from two living maternal relatives.
Family tree showing the maternal relationship between the Tsar and the two living maternal relatives
The mtDNA profile from the putative skeleton of Tsar Nicolas matched the mtDNA profile of the two living maternal relatives, except at position 16169. It first appeared that Tsar Nicholas had a C nucleotide at position 16169, differing from the T nucleotide found in the reference samples.
However, closer examination revealed that Tsar Nicholas actually had a mixed mtDNA profile. This is known as heteroplasmy, where some of his mtDNA was 16169 C, while some was 16169T.
Heteroplasmy is very rare. So it immediately raised questions about the authenticity of the genetic analyses. Some groups claimed it was due to contamination with modern DNA samples.
A later study from the skeletal remains of Georgij Romanov (Tsar's Nicholas' brother) determined that Georgij's mtDNA profile was a perfect match to the Tsar's profile. It even included the same heteroplasmy at position 16169.
These studies provided very strong evidence that researchers had accurately identified the remains of Tsar Nicholas.
Church on Blood in Honour of All Saints. It stands on the site of Ipatiev house where Czar Nicholas II and his family were killed in 1918
The mtDNA profiles of members of the Romanov family have now been defined. Both the Tsar's and Tsarina's lineages include many notable historical figures who would share the same maternal mtDNA markers as members of the Romanov family.
If you have taken the DNA Maternal Ancestry Test, you can compare your mtDNA against members of this notable family to see if you may have also descended from royalty.
The skeletons of the Tsar, Tsarina, and three of their daughters were reburied with honours in the imperial-era capital of St. Petersburg. But what happened to the remaining daughter and their son, Alexei?
Had two Romanov children really escaped from the execution? Or were all the family members killed, as per the memoirs of Yurovsky, the Bolshevik officer in charge of the Romanov's captivity? Continue reading here to follow the remainder of this fascinating story.
Gill P et al. (1994) Identification of the remains of the Romanov family by DNA analysis. Nat Genet. 6:130-135.
Ivanov PL et al. (1996) Mitochondrial DNA sequence heteroplasmy in the Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nat Genet. 12:417-420.
Knight A et al. (2004) Molecular, forensic and haplotypic inconsistencies regarding the identity of the Ekaterinburg remains. Ann Hum Biol. 31(2):129-38.
Stone R. (2004) Buried, Recovered, Lost Again? The Romanovs May Never Rest. Science. 303(5659):753.
Famous DNA Editor - 0
There are some among the Jews who holds special status. These Jewish 'aristocrats' are known as the Cohanim. They belong to the Jewish priestly...
Famous DNA Genovate Laboratories - 0
Are your related to a Sicilian arts patron and benefactor?
When the Russian imperial family vanished one night, imposters embraced the opportunity to pretend to be royalty. This is the story of the DNA...
Weight LossGenovate Laboratories - 0
Did you know DNA affects your exercise motivation?
Moving, grooving, exercising and genes: The secret behind your love of exercise
Did you know DNA influences your risk of vitamin B6 deficiency?
Genovate Laboratories - 0
Vitamin B6, morning sickness and genetics
Did you know beta thal genetic variants protect against malaria?
DNA In the News - 0
Beta thalassemia is a blood disorder characterized by the reduced production of hemoglobin
Did you know "Y-chromosomal Adam" is the ancient father of all people living today?
Tracing the SNP variation in your Y-DNA
Did you know DNA affects your risk of soft tissue injury?
Staying off the bench: Soft tissue injuries and the genes behind them
Did you know your DNA type affects your cardiovascular health?
Healthy hearts - genetic clues to reducing the risk of a heart attack
Did you know DNA can protect you from diabetes and help at high altitude?
The bright side of mutations - protection from diabetes and advantages at high altitudes
COV-19 Italy Report
We provide you with news about DNA.
Did you know DNA determines your risk of lactose intolerance?
Food Sensitivities Editor - 0
Growing up, milk and butter were staples. In college, you feasted on Kraft dinner and extra cheesy pizza. But now, eating plain old yogurt...
Did you know DNA influences your response to alcohol?
Food Sensitivities Genovate Laboratories - 0
Genetic variation determines how efficiently you are able to metabolize alcohol
Did you know DNA determines your risk of celiac disease?
Food Sensitivities DNA In the News - 0
Genetic changes explain why some of us are more sensitive to gluten than others
Did you know your DNA influences your skin health?
Skin Health Genovate Laboratories - 0
Skin health and genetics - Loving the skin you are in
Did you know your risk of getting sunburnt is controlled by your DNA?
Genetic variants determine if you will go bright red or develop a nice even tan
Did you know your risk of eczema is affected by your DNA?
Living with eczema - how to cope when the FLG gene fails you
Famous DNA44
Metabolic Health18
Skin Health16
General Health10
© DNA in the News