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Q: CPU Speed stuck at 0.78 GHz, How do I increase it to its base speed of 2.30 GHz? My 1yr old laptop, HP Pavilion 15 has it's CPU, Intel i5-8300H stuck at 0.78 GHz whenever it's plugged and charging, and the moment I remove the charger plug or switch it off, it increases suddenly and keep fluctuating freely, with good utilisation percentage more than 1-2GHz or more as I experience Thermal Throttling.
I tried sneaking into the BIOS to see if I can do anything, but found nothing. I also downloaded Intel Extreme Tuning Utility (XTU) but couldn't control it.
My question is how do I increase the speed to say atleast 2 GHz (fixed, not fluctuating) when it's charging, irrespective of getting thermal throttle, and also maybe even control the fan speed if it does get hot?

A: This is a known bug with Windows 10 20H2.
You will either need to rollback your latest update, figure out a way to install KB4580364, or wait for Microsoft to give you a fix using the regular update mechanism.
Here is a page confirming the problem:
https://windowsreport.com/windows-10-slow-cpu-frequency/
Here is the Microsoft page that shows ways to get the patch:
https://support.microsoft.com/en-us/help/4580364/windows-10-update-kb4580364
PLEASE NOTE that this fix is still BETA (preview) software.  You need to ask yourself if you want to install another guess at a fix or roll back.

A: I have had a similar problem the last 2 times I upgraded windows 10 on my Lenovo X1 Carbon. The CPU is stuck at 0.78 Ghz.
This is the way I have fixed it these 2 times. (Last upgrade was to 20H2) I got this fix somewhere on the net. Don't know why it works.

*

*Unplug the power.

*Shut down the PC.

*Restart the PC on battery power.

*Plug the power back in.

After that, the CPU happily speeded up past the base frequency of 2.69 GHz, to 3 GHz and beyond as needed. This seems (for me) to be a permanent fix until the next Windows upgrade.

A: I was suffering this issue with my HP laptop.
What I did to fix it was by making sure I was updated to the latest BIOS on the HP website for my product, then resetting the BIOS to default settings.
If you are already on the latest BIOS for your laptop, then just go into the BIOS and reset settings to default.
Also, make sure to check for updates on Windows Update and HP Support Assistant. This issue seems to be quite a common bug with some HP laptops.

A: I have 2 identical dell laptops with identical specs (i3-4005u, 4gb ddr3)
I noticed one day that whatever happened, the cpu clocks on one laptop won't go above 798 Mhz,
I tested both machines on different OSs for months without battery, so it wasn't a battery problem huh.
Then I tried switching the power brick from the one that worked fine to the one whose cpu won't ramp up, and it worked.
So I concluded that it was due to a powerbrick limitation it didn't work well. Both powerbricks for both laptops were bought from 3rd party vendors.
I hope this is helpful for anyone facing the same issue on laptop.
There are some other solutions you should try first,
Remove the battery and start on charger power and restart and install battery.
Also try disconnecting  the charger and reconnecting it, that worked too when I had installed battery in the laptop which had its cpu clocks stuck at 798 Mhz

A: I just encountered the exact same problem with Windows 10 and Windows 11 on a Dell XPS 13 Ultrabook. After Upgrading the BIOS to the newest version, some new options appeared. One was related to the power state of the CPU. This specific BIOS option allowed the Operating System to regulate the CPUs power states instead of the chipset. After disabling this option, the Processor worked just fine, turboing to over 3GHz and 100% CPU utilization when needed. For some reason, Windows seems to mess with the power states of specific notebook configurations.

A: Update your system BIOS.
I had a similar issue with my Lenovo E580 laptop, running Windows 11 (22H2). Task Manager always reported CPU speed at 0.8 GHz. Changing power options had no impact.
Lenovo automatic update tool found a newer BIOS. After installing it (no reconfiguration) the clock speed behaved as it used to: It varies between 0.8 and 3.4 GHz, depending on system load.

A: I'm not sure if you have tried this, but:

*

*Right Click on the battery icon

*Go to Power Options

*Click on Change plan settings for the High Performance plan (if you don't have one, you can create one using the Create a power plan option on the left)

*Under Processor power management:
4.1. Set the Minimum processor state to 100%
4.2. Set the Maximum processor state to 100%
4.3. Set the System cooling policy to Active

A: I had the same issue on Windows 11.

Thanks to other answers I found a solution that hopefully might help you as well.

Go to Control Panel > Hardware and Sound > Power Options and switch from Ultimate Performance to Balanced. This did the trick for me.

redpajama_set_name: RedPajamaStackExchange


Multiple sclerosis (MS) is an autoimmune disease that affects the brain, optic nerve, and spinal cord. It primarily targets the CNS (central nervous system).
An autoimmune disease is a type of disease where the body's immune system mistakes a normal tissue or cell for a pathogen and attacks it.
MS is caused when the protective covering surrounding nerve cells called myelin sheaths are damaged. When the myelin sheath is damaged, nerve signals travelling through the nerve cells slow down or just stop.
A new study shows that taking part in a stress management program may help people with multiple sclerosis (MS) prevent new disease activity. The study is published in the July 11, 2012, online issue of Neurology®, the medical journal of the American Academy of Neurology.
The study involved 121 people with MS. Half received the stress management program, meeting with a therapist for 16 individual 50-minute sessions over five to six months. They learned about problem-solving skills, relaxation, increasing positive activities, and enhancing their social support. They could also choose optional sessions on topics such as fatigue management, anxiety reduction, pain management and insomnia treatment. After the treatment ended, the participants were followed for another five to six months. The remaining participants were put on a waiting list as a control group. After 10 months, they attended a five-hour workshop on stress management.
A total of 77 percent of those receiving stress management training were free of new lesions, or brain damage that indicates disease activity, during the treatment period, compared to 55 percent of those in the control group.
"The size of the effect is similar to other recent phase II trials of new drug therapies for MS," said study author David C. Mohr, PhD, of Northwestern University Feinberg School of Medicine in Chicago. "While it's premature to make any specific recommendations about using this type of stress management training to manage MS disease activity, it will be important to conduct more research to identify specifically how this treatment is benefiting people with MS."
However, the positive effects of the training did not continue after the treatment period. "This was unexpected," Mohr said. "It's possible that people were not able to sustain their new coping skills once the support ended, or that some aspect of the treatment other than stress management skills, such as the social support, was the most beneficial part of the treatment."


text:
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Min Beds 1 bed 2 beds 3 beds 4 beds 5 beds +
For Sale To Rent
Price desc Price asc Most recent first
A 700-year-old manor house in the Cotswolds with its own moat, secret gardens and royal connections
Toby Keel June 27, 2020
Bolehyde Manor in Allington, near Chippenham. Credit: Savills
Bolehyde Manor, a few miles from Chippenham, is a glorious, large house with wonderful gardens.
Any property that survives for more than 700 years will have plenty of stories to tell. In the case of Bolehyde Manor, in the village of Allington not far from Chippenham, several of those stories revolve around the Royal Family over the past few years. This was the home of the Duchess of Cornwall while she was still Camilla Parker Bowles and married to her first husband, Andrew.
In 1986 it came to the market just as Prince Andrew was about to marry Sarah Ferguson, and was rumoured as a possible family home for the new royal couple (as was every other property within on the market near of Prince Charles's home at Highgrove and Princess Anne's house, Gatcombe). A new royal connection was rumoured in 2011 when it was reported that the Duchess of Cambridge's parents, the Middletons, were tipped as its next owners. Needless to say, neither of these potential sales went ahead.
Bolehyde Manor sits in 80 acres of land in Allington, near Chippenham.
Today, Bolehyde Manor is up for sale again, with Savills quoting a guide price of £3.25 million. That's £2.65 million more than was being asked in 1986 and (if tediously gossipy reports such as this one are to be believed) £1.5 million less than was reportedly being asked nine years ago. As the pictures on this page show, there's clearly a bit of TLC required here and there, but this will only add to the possibilities and potential for a new owner.
The building itself has an equal number of stories to tell. Back in 1948 Country Life's erstwhile architecture editor Christopher Hussey paid a visit to write a two-part article in which, after an opening description, he wrote:
This cursory view establishes that it is a very old site and the house, of various dates, shaped like a U lying on its side of which the lower stroke, forming the south entrance front, is longer than the upper; but that it was formerly approached from the east. The gables, crowned with little gablets, might be 16th-century; the curious east lodges to the courtyard are possibly older; and the later gate-piers, gazebos and porch are evidently of late 17th-century date. The archway connecting chapel and house, though of Tudorish character, is in fact modern. What do we make of it all?
Bolehyde Manor in 1948. ©Country Life Picture Library
Hussey's conclusion was that it's essentially a medieval house greatly expanded in the 17th century, with several additions made since then. Exactly what was built when is hard to pin down, however, since patchy records remain from the tenure of the Gales, who owned Boleyhyde for three centuries. 'History is not helpful,' Hussey added, somewhat ruefully.
We can speak for certain as to how the house stands today, however: it's a Grade II*-listed manor house with four reception rooms, eight bedrooms, described by the agents as a house 'with the history of the ages knitted into its very fabric but with a heart that is unreservedly designed around both family living and formal entertaining.'
In 1948 the house had a four-poster bed for the dog… we can't confirm that it's still there! ©Country Life Picture Library
Period features abound from the moment you open the heavy oak front door and step into a flag stone floor hallway. There are open fires, stone mullioned windows and oak panelling, plus an ornately carved staircase.
A courtyard accessed via the reception hall or kitchen is an ideal spot for al fresco dining.
Beyond that there are simply magnificent grounds ('a veritable succession of secret gardens' as the colourful agents details note), stables, outbuildings and 80-odd acres of arable land.
All this and we've not yet mentioned the moat, swimming pool and tennis court. Bolehyde has all three.
As well as the main house there is also a three-bedroom annexe and three cottages — truly a small estate rather than just a large house. Given its easily-accessible Cotswolds location and huge potential for improvement, as well as its fascinating history, we'd foresee an awful lot of interest.
Bolehyde Manor is for sale via Savills at £3.25 million — see more pictures and details.
Thyme Walk and Highgrove House. Credit: Highgrove / GAP Photos / A. Butler
The Prince of Wales's garden at Highgrove: Flowers galore, a wildflower meadow and a treehouse once used by Prince William and Prince Harry
HRH The Prince of Wales's famed garden is open to the public now until October. Country Life's gardens editor Tiffany
Credit: Humberts
A magnificent lakeside home near Princess Anne's Cotswolds estate
This beautiful 19th century house lies on Gatcombe Water, just around the corner from HRH Princess Anne's Gloucestershire home.
Princess Anne's guest-edited issue of Country Life: What you'll find inside
HRH The Princess Royal has guest edited the July 29 issue of Country Life magazine.
Peter Phillips at Aston Farm, Cherington, near Tetbury. ©Millie Pilkington / Country Life Picture Library Credit: ©Millie Pilkington / Country Life Picture Library
Peter Phillips: 'People go to things because they're an "event" and that's the point – it's what you create around a sport that matters'
The son of HRH The Princess Royal and Capt Mark Phillips is now running the Festival of British Eventing, started
An entire village on the edge of the Snowdonia National Park up for sale at just over £1 million
An extravagant Surrey mansion with a helipad, an American diner and a colourful history of presidents and parties
A private island for sale in Weybridge with its own bridge, three acres of gardens and a rushing weir to sing you to sleep

redpajama_set_name: RedPajamaCommonCrawl


The Autism Society of the Mahoning Valley was founded by local families in 1989 to support families in Mahoning, Trumbull and Columbiana counties. Its mission is to improve the quality of life for families living with an ASD diagnosis, and to bring about community change and opportunities for individuals working through life with this disability. Our volunteers are dedicated to accomplishing this through information and referral, family support, advocacy, professional development, raising awareness and resource development. From the newly diagnosed child to the school aged children to the severely disabled autistic adult…ALL of the more than 6,000 families in the tri-county affected by ASD are Autism Society families and receive our help!


text:
WWI ARGUS ARCHIVE: British approach Baghdad
British soldirs guard Turkish prisoners captured in Mesopotamia in 1915
Great success in Gulf of Persia
Swamps take toll on health
WHILE awaiting the next report from Sir John French - nothing had come to hand at the necessarily early hours at which this summary is commenced - it may be well to glance at the progress of the operations in certain theatres of war from which greater events elsewhere have lately diverted attention.
What the old lady called "that blessed word Mesopotamia" has again been mentioned, for example, and we realise once more the number and magnitude of our commitments, the length of our arms and the wonderful adaptability of our troops.
For many months a mixed British and Indian force has been operating along the great rivers at the head of the Persian Gulf and under Sir John Nixon has carried the British flag into one of the few corners of the earth where our victorious banners have never before been seen.
Its record, it was pointed out the other day, is one of which the British Regulars who are taking part in the operations may well feel proud.
It is one which this morning may be quoted with peculiar appropriateness. It captured the important city of Basra, one of the greatest of Turkish seaports. It established itself, after two partially successful attempts, at the confluence of the Rivers Tigria as Euphrates.
It advanced so far up the Tigris that months ago a civil post office was actually established at Amara, a long stride along the road to Baghdad.
It penetrated over 100 miles along the swamps and shallow lakes of the Lower Euphrates, and, after a month of almost incredible toil, drove a fresh Turkish concentration headlong and established a new base.
The advance which ended in the battle of Nasiriyeh involved difficulties greater than those experienced by the late Lord Wolseley in the earliest efforts to reach Khartoum.
Another section of the force pushed up the River Karun to Altwaz in Persian territory in order to protect the all-important Anglo-Persian oil pipe line against tribal Attacks.
The troops engaged in some of these operations were occasionally in great peril. They repeatedly had to face surprise attacks. They endured great privations for the whole region with the summer one of the hottest places on earth.
The degree of sickness in the midst of the insect-haunted swamps in which they have often fought, has been necessarily high.
They faced all trials without complaint and whenever they came into touch with the enemy they have won in the end.
Of all the campaigns now in progress, that in Mesopotamia (one authority stated the other day) is the only one in which the Allies can claim continual success from the outset, unmarried by a single failure.
The area of Turkish territory conquered is already very great and includes large portions of one of the most fertile regions on earth.
Not only was the pipe line secured, the large Turkish forces absorbed by neither the European nor Caucasian theatre kept fully engaged, and numerous Arab tribes weaned from supporting the Sultan-great, as these achievements are-the crowning feat, so far, is that which formed the basis of the report submitted by the House of Commons by Mr Austen Chamberlain last night.


text:
Задарский мир — подписанный 18 февраля 1358 г. в городе Задар мирный договор между Венгрией и Венецианской республикой, по которому последняя теряла владения в Далмации.

Предыстория
В 1301 г. правившая Венгрией и Хорватией династия Арпадов прекратила существование, и после короткого перерыва была заменена Анжуйской. Первым анжуйским королем был Карл Роберт, правивший с 1312 по 1342 гг. Его поддерживал самый могущественный хорватский дворянин Павел Шубич, князь Брибира и бан Хорватии, правитель прибрежных городов Сплит, Трогир и Шибеник. Статус бана Хорватии дал ему многие полномочия  монарха, включая чеканку монет, предоставление городам хартий и взимание налогов.

Действия Павла привели к восстанию среди хорватской знати, которая обратилась к королю. Однако в обмен хорватская знать была вынуждена заявить о своей верности венгерской монархии, что подготовило почву для венгерских попыток изгнать Венецианскую республику с побережья Далмации. В то время как другие города Далмации страдали от борьбы между венецианцами, венграми и хорватами, принадлежавшая Венеции Рагуза стала крупным экономическим центром за счёт своего положения между западом и богатыми полезными ископаемыми Сербией и Боснией, а также между Европой и Левантом.

В 1350-х годах королю Людовику I удалось собрать войско в 50 тыс. человек, объединив свои силы с подкреплениями, присланными герцогом Австрийским Альбрехтом II, графами Горицы, сеньором Падуи Франческо I да Каррара и патриарх Аквилеи Николаем Люксембургским. В 1356 году коалиция осадила венецианцев в Азоло, Конельяно, Сенеде и крепости Тревизо. В то же время на побережье Далмации армия атаковала города Задар, Трогир, Сплит и Рагузу, а также другие более мелкие города, которые довольно быстро сдались.

Разбитые чередой военных неудач, понесенных на подконтрольной им территории, венецианцы смирились с невыгодными условиями, оговоренными в Задарском договоре, который был подписан 18 февраля 1358 г.

Последствия
Договор был подписан в монастыре Святого Франциска, и по условиям договора Дубровник и Задар перешли под власть Венгрии  и Хорватии. Это ознаменовало усиление Дубровника как независимого государства, что нельзя сказать о Задаре, поскольку позже он был продан Венеции Владиславом Неаполитанским.

В результате мирного договора Венеция должна была отдать королю все свои владения в Далмации, от Кварнера до Которского залива, но могла сохранить побережье Истрии и район Тревизо. Он также был вынужден отменить в титуле своего дожа любое упоминание о Далмации. Однако договор сохранил военно-морское превосходство Венеции в Адриатическом море, поскольку король Людовик согласился не строить собственный флот.

Людовик и его армия с триумфом вошли в Задар в 1358 г., предоставив обширные привилегии задарской знати и перенеся туда столицу Далмации.

Примечания

Ссылки
 Zadarski list Kako je i zašto Ladislav prodao Dalmaciju, June 7, 2008

Международные договоры Хорватии
Международные договоры Венгрии
История Задара
Международные договоры Венецианской республики
История Далмации
1358 год

redpajama_set_name: RedPajamaWikipedia


Need a Sci-fi kit? Well I got 'em... purging a few kits from my overflowing collection. Trying to focus on specific subjects and eliminate duplicates. All prices include lower 48 shipping - combined shipping available.
Are the Star Trek kits the 1/1000 size, or a mix of scales?
1/1000 scale... that's whats nice about Polar Lights... same scale for all.


Just the Vax is recommending a post by Joel Harrison, PhD, MPH has published on Science Based Medicine. Enjoy.
Mark Sawyer (2017 Oct 5). Immunizations stop infectious outbreaks. San Diego Union-Tribune, page B21-22.
Terry Roark (2017 Oct 5). Exemptions should be option for parents. San Diego Union-Tribune, page B21-22.
Mark Sawyer, MD is a professor of clinical pediatrics in the Division of Infectious Diseases in the UCSD Medical School. Terry Roark is the advocacy director in California for the nonprofit National Vaccine Information Center.
Each piece was given equal length, giving the impression that each side represented a legitimate position, creating a false balance. In all fairness, the UT has posted articles and editorials on other occasions that clearly supported vaccines and the science behind them, e.g., "In a win for science and for student safety, school vaccination rates are the highest they have been in California in at least 15 years" (The San Diego Union-Tribune Editorial Board, 2017). The problem is that people often have short term memories, may not have read the previous pro-vaccine articles in the UT, or, seeing two articles, were influenced more by the one than the other.
As will be shown in this paper, the article by Terry Roark is full of inaccuracies, falsehoods, and misrepresentations, cultivating readers to form an opinion that is invalid. Like Roark, the vast majority of antivaccinationists lack the basic understanding of the science underlying vaccines, view the world as all or none, subscribe to paranoid conspiracy theories, and their certainty of the rightness of their opinion clearly reflects research that shows once people form opinions, they seldom change them and, remarkably, when confronted with evidence that they are wrong, they often embrace their point of view even more tenaciously (Tavris, 2007). Some of this can be attributed to the Dunning-Kruger effect, which posits that people often fail to grasp their level of competence (or more to the point, incompetence), which precludes their ability to form credible opinions.
In publishing Roark's opinion piece, in my opinion, The San Diego Union-Tribune is guilty of creating a false balance. My intent is NOT to single out the UT as this is a problem belonging to our mass media in general. However, as the UT is my hometown newspaper, it provided an excellent example.
As I've demonstrated in this article, not one point made by Roark is valid. The underlying premise she is coming from is based on a lack of the basics of science, thus, a bogus opinion on vaccines, a lack of understanding of the economics of vaccine production, and a misleading quote, taken out of context, from a Supreme Court decision. Her affiliation with the National Vaccine Information Center and its Co-Founder and President, Barbara Loe Fisher, as shown by Fischer's statement, leaves little doubt that besides a lack of scientific knowledge, antivaccinationists suffer from paranoid delusions of conspiracy theories, project their own, often vicious, attacks on the integrity and honesty of those promoting vaccines, including threatening statements, and see the world in terms of the Nirvana Fallacy (for more on Barbara Loe Fisher as the main spokesperson for the National Vaccine Information Center, see Orac, 2017). Attacks on the integrity and honesty of scientists says more about the attackers than the scientists. Such attacks indicate that antivaccinationists are incapable of systematically and validly using microbiology, immunology, epidemiology, biostatistics, and the history of infectious diseases to make their case and display, in my opinion, a crass lack of integrity and decency, basically anything and everything goes to influence people's opinions, regardless of how unscientific, illogical, and including threats and character assassination.
Given that The San Diego Union-Tribune has, on the whole, been supportive of vaccines, it would behoove them, as well as our mass media in general, to re-evaluate their choice of opinion pieces and even letters that clearly do not reflect, have NO validity, on other issues as well. The UT's policy regarding Letters to the Editor, "It is also our policy to attempt to publish letters supporting or opposing a particular issue in a ratio reflecting the number received on each side," when one side is clearly taking an unscientific, invalid position, promotes false balance and is a disservice to readers (The San Diego Union Tribune. Letters and Commentaries).
Joel A. Harrison (2018 Feb). The So-Called Vaccine Debate, False Balance in the Mass Media, and the Risk to Public Health: Terry Roark's Opinion Piece in the San Diego Union-Tribune (Oct 5, 2017) , "Exemptions should be option for parents". Science-Based Medicine.
Joel A. Harrison, PhD, MPH is a retired epidemiologist who has been writing articles over the past years supporting vaccinations for Every Child By Two, an excellent non-profit founded in 1991. Every Child By Two has changed to Vaccinate Your Family, expanding its mission to include vaccines for people of all ages. You can find Executive Summaries of his previous ECBT articles that hyperlink to the complete articles as well as his brief biography on the archived ECBT Expert Commentaries page. Dr. Harrison has studied and worked in several countries, including Sweden (where he earned his doctorate) and Canada (where he earned a Masters degree). Having experienced both the Swedish socialized health care system and the Canadian non-profit single-payer system, over the past 30 years he has devoted considerable time to studying health economics and health care systems, concluding that, though the Swedish system is excellent, given American culture, he believes that a non-profit single-payer system would be best option for the United States. Dr. Harrison is a long time member of Physicians for a National Health Program.


Learn how to set up Kashoo to accept online payments.
The easiest way to accept credit card payments on invoices you email out to clients.
Payments is a way to receive credit card payments from your clients using Kashoo. By enabling Payments, you can email your invoice to clients with a link that allows them pay you online via Visa, MasterCard, or American Express. Payments are automatically logged in Kashoo, and when the money is transferred to your bank account that Transfer transaction will also be created in Kashoo, along with any associated "Payments" fees.
3. Confirm the contact information displayed and agree to the 'terms of service' and then click Start Accepting Payments. Your account is immediately ready to send out your first invoice and accept payments via credit card. However, your payment portal still needs to be verified in the next step.
4. To access your payment portal and setup how you want your money deposited, check your email for log in instructions from WePay. You can also find the Log Into WePay button located under the Account Mappings area.
Note: Please verify your account as soon as possible to ensure all funds are deposited to you without delay.
Once you have set up your first credit card processing account you will now have the option to set up a Payment Notification Email. This notification will email you every time your customers pay. You don't need to do anything because the payment will automatically show up in Kashoo. But this notification lets you know in real-time who has paid you. Additionally, Kashoo will notify you if there is ever a problem when trying to process a payment.
In the Clients section (accessible on left sidebar), you'll have the option to choose your default Credit Card Processor to use for a specific Client. This will save you steps when you create an Invoice.
Select appropriate Client, Project (if applicable), Dates, and input Line Items, Prices, and Taxes (if applicable).
4. Set the Receivable Account to Unpaid Accounts Receivable*. If the Invoice is marked as "Paid" you will NOT be able to use Credit Card Processing for that Invoice.
*This can be "Due on Receipt", "30 days", "60 days" etc. as long as it is Unpaid Accounts Receivable.
5. Once all desired fields are populated, click "Add Invoice". An Invoice cannot be emailed if it has not been created. Once an Invoice has been created a dialogue will appear (this is a temporary dialogue box).
6. Click Email to email the Invoice to the Client. There will be a message on the bottom of the Email pop-up letting you know which processor is associated with that Invoice. Enter in any additional information you want into the body of the email and click Send.
1. Your customer will receive an email that contains a link, allowing them to pay the invoice online using their credit card.
1. To initiate a refund to a Client who used Payments, you must first refund from the Payment Processor.
2. Next, in Kashoo, select the Invoice that needs to be refunded and follow the Kashoo Refund Help Article for Refund options.* NOTE: Issuing a refund in Kashoo will NOT refund a payment made via Payments.


text:
using System;
using System.Collections.Generic;
using System.Linq;
using System.Reflection;
using System.Text.RegularExpressions;
using System.Threading.Tasks;

using ASC.Common.Logging;

using Telegram.Bot;
using Telegram.Bot.Types;

namespace ASC.TelegramService.Core
{
    public class TelegramCommand
    {
        public string CommandName { get; private set; }
        public string[] Args { get; private set; }

        public Message Message { get; private set; }
        public User User { get { return Message.From; } }
        public Chat Chat { get { return Message.Chat; } }

        public TelegramCommand(Message msg, string cmdName, string[] args = null)
        {
            Message = msg;
            CommandName = cmdName;
            Args = args;
        }
    }

    public class CommandModule
    {
        private readonly ILog log;

        private static readonly Regex cmdReg = new Regex(@"^\/([^\s]+)\s?(.*)");
        private static readonly Regex argsReg = new Regex(@"[^""\s]\S*|"".+?""");

        private readonly Dictionary<string, MethodInfo> commands = new Dictionary<string, MethodInfo>();
        private readonly Dictionary<string, Type> contexts = new Dictionary<string, Type>();
        private readonly Dictionary<Type, ParamParser> parsers = new Dictionary<Type, ParamParser>();

        public CommandModule(ILog log)
        {
            this.log = log;

            var assembly = Assembly.GetExecutingAssembly();

            foreach (var t in assembly.GetExportedTypes())
            {
                if (t.IsAbstract) continue;

                if (t.IsSubclassOf(typeof(CommandContext)))
                {
                    foreach (var method in t.GetRuntimeMethods())
                    {
                        if (method.IsPublic && Attribute.IsDefined(method, typeof(CommandAttribute)))
                        {
                            var attr = method.GetCustomAttribute<CommandAttribute>();
                            commands.Add(attr.Name, method);
                            contexts.Add(attr.Name, t);
                        }
                    }
                }

                if (t.IsSubclassOf(typeof(ParamParser)) && Attribute.IsDefined(t, typeof(ParamParserAttribute)))
                {
                    parsers.Add(t.GetCustomAttribute<ParamParserAttribute>().Type, (ParamParser)Activator.CreateInstance(t));
                }
            }
        }

        private TelegramCommand ParseCommand(Message msg)
        {
            var reg = cmdReg.Match(msg.Text);
            var args = argsReg.Matches(reg.Groups[2].Value);

            return new TelegramCommand(msg, reg.Groups[1].Value.ToLowerInvariant(), args.Count > 0 ? args.Cast<Match>().Select(a => a.Value).ToArray() : null);
        }

        private object[] ParseParams(MethodInfo cmd, string[] args)
        {
            List<object> parsedParams = new List<object>();

            var cmdArgs = cmd.GetParameters();

            if (cmdArgs.Any() && args == null || cmdArgs.Count() != args.Count()) throw new Exception("Wrong parameters count");

            for (var i = 0; i < cmdArgs.Count(); i++)
            {
                var type = cmdArgs[i].ParameterType;

                if (type == typeof(string))
                {
                    parsedParams.Add(args[i]);
                    continue;
                }

                if (!parsers.ContainsKey(type)) throw new Exception(string.Format("No parser found for type '{0}'", type));

                parsedParams.Add(parsers[cmdArgs[i].ParameterType].FromString(args[i]));
            }

            return parsedParams.ToArray();
        }

        public async Task HandleCommand(Message msg, ITelegramBotClient client, int tenantId)
        {
            try
            {
                var cmd = ParseCommand(msg);

                if (!commands.ContainsKey(cmd.CommandName)) throw new Exception(string.Format("No handler found for command '{0}'", cmd.CommandName));

                var command = commands[cmd.CommandName];
                var context = (CommandContext)Activator.CreateInstance(contexts[cmd.CommandName]);

                var param = ParseParams(command, cmd.Args);

                context.Context = cmd;
                context.Client = client;
                context.TenantId = tenantId;
                await Task.FromResult(command.Invoke(context, param));
            }
            catch (Exception ex)
            {
                log.DebugFormat("Couldn't handle ({0}) message ({1})", msg.Text, ex.Message);
            }
        }
    }

    public abstract class CommandContext
    {
        public ITelegramBotClient Client { get; set; }
        public TelegramCommand Context { get; set; }
        public int TenantId { get; set; }

        protected async Task ReplyAsync(string message)
        {
            await Client.SendTextMessageAsync(Context.Chat, message);
        }
    }

    public abstract class ParamParser
    {
        protected Type type;

        protected ParamParser(Type type)
        {
            this.type = type;
        }

        public abstract object FromString(string arg);
        public abstract string ToString(object arg);
    }

    public abstract class ParamParser<T> : ParamParser
    {
        public ParamParser() : base(typeof(T)) { }

        public override abstract object FromString(string arg);
        public override abstract string ToString(object arg);
    }
}

redpajama_set_name: RedPajamaGithub


text:
About Events Publications Multimedia
Acton Institute Powerblog
connecting good intentions with sound economics
The Importance of the Pastoral Ministry
July 07, 2005 • by Jordan J. Ballor
In the words of puritan Samuel Hieron, in the care of a bad Miller we loose but our meale, of the Farrier but our horse, of the Taylour but our garment, of the Lawyer but our money, of the Physitian but our bodyes: but in the hands of an vnfaithfull minister a man looseth his soule and his everlasting portion in heaven. Continue Reading...
'For God's Sake, Please Just Stop!'
A fascinating interview with Kenyan economist James Shikwati in the July 4 edition of Der Spiegel: SPIEGEL: Mr. Shikwati, the G8 summit at Gleneagles is about to beef up the development aid for Africa… Shikwati: … for God's sake, please just stop. Continue Reading...
One of the free downloads offered today in the iTunes music store (click here to access it via iTunes) is an interview with Jack and Meg White of the White Stripes. Continue Reading...
'The Warning to Rich Oppressors'
The Acton Institute has announced the honorees for the 2005 Homiletics Award, on the text of James 5:1-6, "The Warning to Rich Oppressors." In first place ($2,000) is Earl Eckbold, a Master of Divinity student at Lutheran Theological Seminary, Philadelphia. Continue Reading...
The Late(st) Great G8 Debate
From an interview on Zenit.org with Ettore Gotti Tedeschi, the president of the Italian division of Santander Central Hispanic Bank and a professor of the Catholic University of Milan, discussing African debt relief: "What should have been done was to put these countries in a condition of being able to pay the debts, even if in 1,000 years, helping them to create the necessary wealth for their own survival, as well as for their own dignity as human beings, who do not want to feel incapable and failures, in need of non-repayable charity…the G-8 members should listen to the suggestions of Pope Benedict XVI, who shows himself to be the most concrete "statesman," inviting them to take concrete measures to help Africa, appealing for the just distribution of the goods of the earth. Continue Reading...
The Unity of Faith and Politics, More or Less
The legitimization of so-called same-sex marriage in Spanish law has not surprisingly elicited a strong response from Christians around the world. This particular disagreement is often cast by proponents of change as a matter of Religion trying to encroach on Politics. Continue Reading...
Summer Olympics in London, 2012
London has been awarded the 2012 Summer Olympics, beating out Paris, New York, Madrid, and Moscow. According to a report, "The victory means that London will play host to the world's premier sporting event in seven years' time with a specially-built stadium and village rising from what is now an urban wasteland in the east of the city." And PM Tony Blair pledged full support for the games, "My promise to you is we will be your very best partners," Blair said. Continue Reading...
Africa Needs More than Foreign Aid
So says Dr. George Ayittey, a professor of economics at American University and founder of the Free Africa Foundation, in an interview on today's Morning Edition from NPR. Ayittey argues in part that after the African nations gained independence, they rejected the market system out of hand as a Western innovation, to the detriment of their societies. Continue Reading...
Why Not Give Yourself?
A question over at the ONE Campaign blog: Why don't these celebrities cough up their own money and stop asking for mine? Answer: First off, they are. Most of the celebs involved in the campaign give hundreds of thousands, if not millions to charity. Continue Reading...
Bush Says Abortion Not A Limtus Test For High Court
President Bush, on his way to the G-8 Summit, said that views like abortion or gay marriage will not serve as litmus tests for selecting a Supreme Court nominee. "I'll pick people who, one, can do the job, and people who are honest, people who are bright and people who will strictly interpret the Constitution and not use the bench to legislate from," Bush said. Continue Reading...
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text:
Research ArticleAngiogenesis Free access | 10.1172/JCI33493
Multipotential stem cells recapitulate human infantile hemangioma in immunodeficient mice
Zia A. Khan,1 Elisa Boscolo,1 Arnaud Picard,1 Sarah Psutka,1 Juan M. Melero-Martin,1 Tatianna C. Bartch,1 John B. Mulliken,2 and Joyce Bischoff1
1Vascular Biology Program and Department of Surgery, and 2Department of Plastic Surgery, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Joyce Bischoff, Vascular Biology Program and Department of Surgery, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, USA. Phone: (617) 919-2192; Fax: (617) 730-0231; E-mail: joyce.bischoff@childrens.harvard.edu.
Find articles by Khan, Z. in: JCI | PubMed | Google Scholar
Find articles by Boscolo, E. in: JCI | PubMed | Google Scholar
Find articles by Picard, A. in: JCI | PubMed | Google Scholar
Find articles by Psutka, S. in: JCI | PubMed | Google Scholar
Find articles by Melero-Martin, J. in: JCI | PubMed | Google Scholar
Find articles by Bartch, T. in: JCI | PubMed | Google Scholar
Find articles by Mulliken, J. in: JCI | PubMed | Google Scholar
Find articles by Bischoff, J. in: JCI | PubMed | Google Scholar
Published June 5, 2008 - More info
Published in Volume 118, Issue 7 on July 1, 2008
J Clin Invest. 2008;118(7):2592–2599. https://doi.org/10.1172/JCI33493.
© 2008 The American Society for Clinical Investigation
Published June 5, 2008 - Version history
Received: August 2, 2007; Accepted: April 23, 2008
Infantile hemangioma is a benign endothelial tumor composed of disorganized blood vessels. It exhibits a unique life cycle of rapid postnatal growth followed by slow regression to a fibrofatty residuum. Here, we have reported the isolation of multipotential stem cells from hemangioma tissue that give rise to hemangioma-like lesions in immunodeficient mice. Cells were isolated based on expression of the stem cell marker CD133 and expanded from single cells as clonal populations. The CD133-selected cells generated human blood vessels 7 days after implantation in immunodeficient mice. Cell retrieval experiments showed the cells could again form vessels when transplanted into secondary recipients. The human vessels expressed GLUT-1 and merosin, immunodiagnostic markers for infantile hemangioma. Two months after implantation, the number of blood vessels diminished and human adipocytes became evident. Lentiviral expression of GFP was used to confirm that the hemangioma-derived cells formed the blood vessels and adipocytes in the immunodeficient mice. Thus, when transplanted into immunodeficient mice, hemangioma-derived cells recapitulated the unique evolution of infantile hemangioma — the formation of blood vessels followed by involution to fatty tissue. In summary, this study identifies a stem cell as the cellular origin of infantile hemangioma and describes for what we believe is the first time an animal model for this common tumor of infancy.
Infantile hemangioma (IH) is a vascular tumor that occurs in 5%–10% of infants of mixed European descent (1). A hallmark of IH is its life cycle, which is divided into 3 stages. The proliferating phase spans the first year of postnatal life and is characterized by cellular masses of endothelium without a defined vascular architecture. The involuting phase begins around 1 year of age and continues for 3–5 years as blood vessels become more obvious and even enlarged. The involuted phase is reached by 5–8 years of age, at which point blood vessels are replaced with a fibrofatty residuum and capillary-sized channels. Intrinsic (2, 3) and extrinsic (4, 5) factors have been hypothesized to contribute to the rapid growth and the slow, yet spontaneous regression. Nevertheless, the cellular origin of IH and the biologic signals for the uncontrolled growth remain elusive.
Histological analyses of IH in the early proliferating phase have generated a number of developmental theories suggesting an embryonic or primitive cell origin of IH. Virchow (6), Pack and Miller (7), and Malan (8) described IH as sequestered embryonic mesoderm and/or activated dormant angioblasts. The presence of CD34-positive cells in the interstitial compartment of IH led Smoller and Apfelberg to postulate a primitive vascular progenitor cell giving rise to both ECs and pericytes in the tumor (9). In vitro studies have suggested that ECs isolated from IH have an immature fetal phenotype (10). We have shown that hemangioma ECs (HemECs) are clonal (3), indicating that the HemECs might originate from a single stem or progenitor cell. We also used human stem cell (SC) marker CD133 (also known as AC133 and prominin-1) (11) to detect (12) and isolate (13) hemangioma endothelial progenitor cells (HemEPCs). Our demonstration that HemECs and HemEPCs are more similar to normal human cord blood EPCs (cbEPCs) than mature human dermal microvascular ECs (HDMECs) (13) provides further support to the earlier histological studies that first suggested hemangioma is composed of immature vascular progenitor cells.
Herein we report on less-differentiated SCs in hemangioma specimens — referred to as hemangioma-derived SCs (HemSCs). The HemSCs exhibited robust proliferative and clonogenic capacity and differentiated into cells of multiple lineages. Importantly, clonal HemSC populations expanded in vitro from single cells produced human GLUT-1–positive microvessels in vivo. Over time, the human blood vessels diminished and human adipocytes became evident, reminiscent of the involuted phase of IH. In summary, our findings provide evidence for a SC origin of IH, which is in contrast to the long-held view that IH arises from ECs (14).
Isolation and characterization of HemSCs. Anti-CD133–coated magnetic beads have been used to isolate stem/progenitor cells from blood (15) and from human tumors (16, 17). We isolated CD133+ cells from proliferating phase IH using this methodology and found that approximately 0.2% of the hemangioma cells were selected with anti-CD133–coated beads (n = 28; age 11 ± 1.47 months, CD133+ cells, 0.2% ± 0.027%). The CD133-selected cells, referred to hereafter as HemSCs, were expanded in an endothelial growth medium (endothelial basal medium 2 [EBM-2]/20% FBS). Human umbilical cbEPCs (18), HDMECs, BM–mesenchymal stromal cells (BM-MSCs), and neonatal human dermal fibroblasts (NHDFs) were cultured under identical conditions and used for comparison. Flow cytometric analyses for endothelial and mesenchymal cell-surface markers showed that HemSCs were most similar to BM-MSCs and NHDFs (Figure 1A). HemSCs expressed CD90, a mesenchymal cell marker, and proteins that are displayed on ECs, such as VEGF-R1, VEGF-R2, neuropilin-1 (NRP-1), and CD146, but none of these are exclusive to the endothelial lineage. Hence, the cell-surface markers on HemSCs indicate a mesodermal or mesenchymal cell phenotype. Phase contrast images of HemSCs in culture are shown in Supplemental Figure 1 (supplemental material available online with this article; doi: 10.1172/JCI33493DS1).
HemSCs express VEGF-Rs and mesenchymal cell marker CD90. (A) Flow cytometric analysis of HemSCs, BM-MSCs, NHDFs, and HDMECs. Each cell type was grown in the EBM-2/20% FBS and assayed at passage 6. Gray histograms show cells labeled with FITC- or PE-conjugated antibodies. Black lines show isotype-matched control FITC- or PE-conjugated antibodies. Incubation with anti-CD31 and anti–VEGF-R2 was carried out following saponin permeabilization of HemSCs, BM-MSCs, and NHDFs. (B) RT-PCR analysis of Oct-4 and AML1 in 2 different HemSCs, Hem 106 and 109, with BM-MSCs and NHDFs shown for comparison. Ribosomal S9 (rS9) served as a control.
RT-PCR analyses showed that HemSCs expressed the embryonic SC transcription factor Oct-4 (Figure 1B), which is known to play a pivotal role in SC specification (19). HemSCs were also positive for AML1 (Figure 1B), a transcription factor required for definitive hematopoiesis (20) and expressed in hemogenic endothelium (21). Expression of Oct-4 and AML1 in HemSCs indicates that HemSCs are multipotent.
We next compared the growth parameters of HemSCs with normal mesenchymal cells and ECs. HemSCs grew to 3–4 times greater density than normal control endothelial and mesenchymal cells, which included HDMECs, cbEPCs, NHDFs, and BM-MSCs (Figure 2A). The proliferative response to serum, basic bFGF, and VEGF-A was determined after cells were serum and growth factor starved for 6 hours (Figure 2B). HemSCs and NHDFs proliferated in response to serum and bFGF, although the response of HemSCs was stronger, as was the basal proliferation. HemSCs, BM-MSCs, and NHDFs did not show increased proliferation in response to VEGF-A, a well-studied endothelial growth and survival factor, while cbEPCs and HDMECs proliferated, as expected.
In vitro growth and multilineage differentiation of HemSCs. (A) Proliferation of HemSCs in EBM-2/20% FBS over 10 days compared with normal endothelial and mesenchymal cells. (B) Proliferation in response to VEGF-A, bFGF, or 5% FBS for 24 hours in serum-free, growth factor–free EBM medium. *P < 0.05 compared with cells in serum-free, growth factor–free medium. (C–E) Clonal HemSCs differentiated into endothelial (C), neuroglial (D), and mesenchymal (E) cells. Scale bars: 50 μm. Insets in C show CD31 and VE-cadherin immunostaining of cells induced in the absence of VEGF-B. All experiments were carried out with cells at passages 6–9.
HemSCs display in vitro clonogenic and multilineage differentiation ability. To study cellular differentiation, we prepared clonal populations from the HemSCs from 3 different proliferating phase IH specimens. This was done by diluting a single-cell suspension of HemSC to 3 cells/ml and plating 100 μl in each well of a 96-well plate; thereafter, wells were inspected to verify that only 1 colony was present in the well. Clonally expanded cells were necessary for multilineage assays of differentiation and later for in vivo studies to exclude the possibility that mixed populations of unipotential cells were responsible for the phenotypes of cells observed following the induction. HemSCs showed in vitro clonal expansion, with efficiency ranging from 6%–30% (6% for Hem95, 23% for Hem97, and 30% for Hem106). BM-MSCs and NHDFs failed to produce clonal populations under identical conditions. To induce cell differentiation toward specific lineages, culture conditions reported in the literature were employed. For endothelial differentiation, HemSCs were seeded at high density on fibronectin-coated plates in serum-free medium with VEGF-B (22, 23). Clonal HemSCs differentiated into ECs, shown by expression of the EC markers CD31 (also known as PECAM-1) and VE-cadherin (Figure 2C). Treatment with nerve growth factor and trans retinoic acid (24, 25) induced morphological changes, expression of the glial cell marker glial fibrillar acidic protein (GFAP), and the neuronal marker β-tubulin III (Figure 2D). Finally, mesenchymal differentiation into adipocytes, osteocytes, and chondrocytes was also seen (Figure 2E) when subjected to well-established protocols (26). BM-MSCs differentiated into fat, bone, and cartilage as expected, whereas NHDFs failed to differentiate into any other cell type in the same inductive medium (data not shown). Interestingly, HemSC-derived ECs exhibited an intracellular localization of CD31 and VE-cadherin (Figure 2C), reminiscent of an earlier study of hemangioma-derived and fetal ECs (10). These findings indicate that other signals or factors may be required to fully differentiate the HemSCs into mature ECs with the expected localization of adhesion molecules at the cell surface. Some IH blood vessels coexpress myeloid markers (27), further illustrating the unique phenotype of IH endothelium. As hematopoietic cells and ECs arise from a common progenitor during development (28), we tested to determine whether HemSCs would produce hematopoietic colonies in vitro. Incubation of clonally expanded HemSCs in MethoCult medium did not yield hematopoietic colonies (data not shown).
HemSCs form hemangioma-like Glut-1+ blood vessels in immunodeficient mice. We tested to determine whether HemSCs would form hemangiomas in immunodeficient nu/nu mice. Clonally expanded HemSCs or normal control cells were resuspended in Matrigel, a solubilized basement membrane preparation that has been shown to support angiogenesis and vasculogenesis (18, 29, 30). Cells were injected s.c. into 6-week-old mice (1.5 × 106 cells/200 μl Matrigel/animal). H&E staining of sections showed numerous rbc-filled vascular channels 7 and 14 days after implantation. After 28 and 56 days in vivo, vessels were still present, but large lipid-filled cells became prominent (Figure 3A). Similar results were obtained with nonclonally expanded HemSCs from 4 different hemangioma specimens (data not shown). Blood vessels were not detected when HemECs (3), HemEPCs (13), hemangioma-derived mesenchymal SCs (HemMSCs) (31), BM-MSCs, cbEPCs (18), or NHDFs were implanted as single-cell types into mice under identical conditions (Supplemental Figure 2).
HemSCs form CD31+GLUT-1+ blood vessels in vivo. (A) Clonal HemSCs were suspended in Matrigel and injected s.c. into nude mice. H&E sections of explants at 7, 14, 28, and 56 days with insets showing explants at the corresponding time points. Scale bar: 50 μm. Passage 9 clonal HemSCs were used. (B) Immunofluorescent staining of day 7 explants. Human CD31 (red) is shown on the left, followed by GLUT-1 (green), a merged image, and a phase contrast image. In the merged image, arrows point to cells along the lumen of the blood vessel that are double-labeled for human CD31 and GLUT-1. rbc in the lumen are also positive for GLUT-1 (green). In the phase image, arrows point to endothelial nuclei. The bottom row shows higher magnification images. Scale bars: 50 μm (top row); 10 μm (bottom row). (C) Flow cytometry of cells reisolated from Matrigel explants after 7 and 14 days in vivo. Cells were labeled with anti-human CD31 without permeabilization to detect surface-localized CD31. HemSCs before implantation were negative for CD31 (left panels). HDMECs and mouse ECs are shown as positive and negative control cells. (D) Retrieved CD31+ cells formed blood vessels in secondary recipient mice. Matrigel implants were removed after 14 days, sectioned, stained with H&E (top panel), and immunostained with anti-human CD31 (bottom panel). Vessel density was 78 ± 35 rbc-filled lumens/mm2. Scale bars: 50 μM.
To verify that the newly formed blood vessels were composed of human ECs, we performed indirect immunofluorescence using an antibody specific for human CD31. Immunostained sections were examined by confocal microscopy. Human CD31 was detected in blood vessels formed from clonal HemSCs, indicating endothelial differentiation and vasculogenesis (Figure 3B). The specificity of the anti-human CD31 was confirmed by lack of staining in mouse tissues (data not shown) and has been demonstrated in other studies (18, 32). We next determined whether the human CD31-positive vessels expressed the GLUT-1–positive immunophenotype, which has been shown to be a specific signature of IH (33). The blood vessels formed from HemSCs in Matrigel were found to coexpress CD31 and GLUT-1 (Figure 3B). High magnification images with DAPI-stained nuclei are shown in Figure 3B to highlight the coexpression of CD31 and GLUT-1. A second marker of the hemangioma phenotype, merosin (34), was also found expressed in the hemangioma lesions formed in mice from HemSCs (Supplemental Figure 3).
To further examine human CD31 expression in the Matrigel model, cells were reisolated from the explants by digestion with a collagenase/dispase solution. Flow cytometry was performed without permeabilization of the cells with anti-human CD31 (Figure 3C). After 7 and 14 days in vivo, 23%–28% of the retrieved cells were found to express human CD31 on the surface. This is consistent with functional endothelial differentiation, demonstrated by blood vessel formation (Figure 3, A and B), and provides additional evidence for the differentiation of HemSCs into ECs.
To further test the stem/progenitor nature and vasculogenic potential of these cells, the CD31-positive cells retrieved from the Matrigel were expanded in vitro in EBM-2/20% FBS, verified as human cells by immunostaining for a human nuclear antigen (Supplemental Figure 4), and injected into secondary recipient nude mice. After 14 days, Matrigel explants were removed, sectioned, and analyzed by H&E to quantify rbc-filled lumens and immunostain for human CD31 (Figure 3D). Vessel density was 78 ± 35 vessels/mm2, which was greater than that observed in primary recipients. The ability to retrieve HemSCs that had undergone endothelial differentiation in vivo and to obtain hemangioma-like blood vessel formation in secondary recipients demonstrates the robust blood vessel–forming potential of the HemSCs.
GFP-labeled HemSCs form blood vessels. To track the cellular fate of the HemSCs in our in vivo model, HemSCs were infected with a lentiviral construct encoding turbo GFP. GFP-positive cells were shown by flow cytometry to be greater than 90% positive for GFP expression (Supplemental Figure 5). The cells were further expanded in vitro for 1 passage and implanted into nude mice under conditions identical to those used in the previous experiments (Figure 3). Matrigel implants were harvested at 2 weeks and analyzed for GFP, human CD31, and GLUT-1. GFP and CD31 were colocalized in lumenal structures indicative of blood vessels (Figure 4A). GFP-positive cells lining vessels were also positive for the hemangioma vessel marker GLUT-1 (Figure 4B). Controls to rule on nonspecific staining with the anti-GFP antibody are shown in Supplemental Figure 6. These data combined with that shown in Figure 3B demonstrate that vessels formed when HemSCs were implanted into nude mice were composed of human ECs; the vessels were not the result of infiltration by murine ECs.
GFP-labeled HemSCs form hemangioma blood vessels in vivo. (A) TurboGFP-expressing HemSCs were injected in Matrigel into mice, explanted at day 14, and localized by staining for TurboGFP (green), CD31 (red), and DAPI (blue). (B) Sections were also stained for TurboGFP (green), GLUT-1 (red), and DAPI (blue). Asterisks mark the lumen of the blood vessels in panels A and B. Scale bars: 50 μM.
In vivo differentiation of HemSCs to adipocytes. Our next objective was to determine whether HemSCs recapitulated the involuting and involuted phases of IH. After 28 days in vivo, HemSC-formed lesions had diminished microvessel density (MVD), and adipocytes emerged between 28 and 56 days (Figure 5A and Figure 3A). Morphometric quantification showed 25 vessels/mm2 at days 7, 14, and 28, which decreased to 15 vessels/mm2 at 56 days (Figure 5A), consistent with the reduced number of vascular channels seen during the evolution of IH. It has been reported that Matrigel supplemented with bFGF induces adipogenesis in vivo (35–37). However, no exogenous bFGF is included in our assay. We tested to determine whether the adipocytes in the Matrigel were host derived (murine) or human and had differentiated from the clonal HemSCs. To do this, GFP-HemSCs were implanted, as described in Figure 4, and removed after 2 months in vivo for analysis of GFP and perilipin A (38). Perilipin A encircled the lipid storage droplets in adipocytes, as expected (Figure 5B). GFP and perilipin A colocalization was seen in the scant cytoplasm of the lipid-filled adipocytes (Figure 5B). Controls to rule out nonspecific staining with the anti-GFP and anti–perilipin A antibodies are shown in Supplemental Figure 6. As an alternative approach to follow HemSC adipogenesis in vivo, sections were immunostained with an anti-human nuclear antigen antibody (Figure 5C). The human nuclear antigen was detected in the nuclei of adipocytes in the HemSC Matrigel implants. Mouse heart tissue sections served as a negative control, and human hemangioma tissue sections served as a positive control (Figure 5C). Some adipocytes negative for the human nuclear antigen were evident, suggesting that recruitment of murine cells into the Matrigel microenvironment may have occurred. These results with tagged, GFP-labeled HemSCs and the anti-human nuclear antigen antibody indicate that HemSCs form adipocytes at time points subsequent to vessel formation. The signals that determine whether and when a HemSC becomes an EC or an adipocyte are unknown.
HemSCs form adipocytes in vivo. (A) Clonal HemSCs were suspended in Matrigel and injected s.c. into nude mice for 7, 14, 28, and 56 days, as shown in Figure 3A. Blood vessels, identified in H&E sections as luminal structures containing rbc, were quantified at the 4 time points. *P < 0.05 compared with other time points. (B) GFP-labeled HemSCs were implanted as described in Figure 4 and analyzed after 56 days for expression of GFP and the adipocyte marker perilipin A. Right panel shows the merged image, middle panel shows GFP (green), and right panel shows perilipin A (red); all panels were stained with DAPI. (C) Sections from the 28-day time point were immunostained with an anti-human nuclear antigen antibody using an HRP-conjugated secondary antibody and a diaminobenzidine substrate. Mouse heart tissue sections showed no immunostaining while nuclei in human hemangioma tissue were uniformly positive, confirming the specificity of anti-human nuclear antigen. Scale bars: 50 μM.
For what we believe is the first time, we have shown that CD133-selected HemSCs recapitulate human IH in a murine in vivo model. Clonal HemSCs produced human GLUT-1–positive microvessels at 7–14 days; after 28 days in vivo, human adipocytes were also detected. HemSCs that had differentiated into endothelium in vivo, as determined by cell-surface CD31 expression, could be retrieved and implanted into secondary recipients, where the cells formed blood vessels once more. This indicates the robust vasculogenic potential of these hemangioma-derived cells. GFP-labeled HemSCs were shown to form vessels and adipocytes at 14- and 56-day time points, respectively, confirming that the vessels and adipocytes were not host derived. Using in vitro cellular activity assays, we showed that HemSCs are highly proliferative and able to differentiate into multiple cell types, including adipocytes. These results demonstrate that HemSCs are the cellular precursors of IH.
CD133 has been used to identify tumor-initiating cells from medulloblastomas, glioblastomas, and prostate and colon carcinomas (16, 17, 39–42). Xenografting CD133+ tumor cells in immune-compromised mice has also been shown to generate a primary tumor (16, 17). In a similar fashion, we demonstrated that the CD133-selected cells from IH formed GLUT-1–positive blood vessels and, at later time points, differentiated into adipocytes, the prominent cellular compartments of proliferating and involuting phase hemangioma, respectively. For the secondary transplantation experiment (Figure 3, C and D), we reimplanted human CD31-selected cells because (a) these cells had shown vasculogenic activity in the primary implant and (b) the level of CD133 expression in the CD31-positive and CD31-negative populations recovered from the primary implant was very low, which would have made reselecting CD133-positive cells difficult. The human CD31-selected cells showed a robust degree of blood vessel formation (78 vessels/mm2) upon secondary transplantation, a finding that supports the vasculogenic potential of these cells.
To date, there are no animal models that truly reflect the human hemangioma phenotype and life cycle. Although murine hemangioendothelioma cells have been studied (43), these do not phenocopy IH or provide a hemangioma model because the tumors do not regress and are fatal within 3 months. In addition, polyoma middle T antigen–transformed cells used in animal models result in "cavernous hemangioma" lesions, more akin to malignant vascular tumors, which are also fatal within 3–10 months (44). Hemangioma tissue fragments xenografted into nude mice have been shown to undergo an initial period of necrosis followed by approximately 2-fold enlargement and then subsequent regression (45), which may provide a model for testing new therapies for IH. Our in vivo model is unique in that we are able to produce IH lesions with a small number of purified primary cells suspended in Matrigel but without exogenous growth factors or supplements. These findings implicate a preset genetic or epigenetic program in the HemSCs that directs the cells to recapitulate the IH tumor, including endothelial and adipocytic differentiation. An important future experiment will be to implant hemangioma-derived CD133+ cells that have not undergone in vitro expansion to determine whether HemSCs lose or gain hemangioma-forming potential during the in vitro culture. It is still not understood what triggers the adipogenesis that characterizes the involuting and involuted phases of IH. It is likely that changes in the microenvironment, such as levels of circulating EPCs, immune cells, and growth factors, initiate the regression of the tumor.
It is well established that hemangioma endothelium expresses GLUT-1, merosin, Lewis Y antigen (CD174), and Fcγ receptor II (CD32) in all 3 phases of the life cycle (34). These markers are also expressed by placental microvessels. This immunohistochemical similarity suggested the hypothesis that IH is formed from embolic placental ECs (34). Microarray analyses have supported this link between placenta and hemangioma (46). It is also possible that the progenitor cells reside in fetal and neonatal tissues or come from the BM and express a transcriptional program similar to that of placenta. To date, placenta-derived cells with in vitro clonogenic and differentiation abilities similar to HemSCs have not been described.
In conclusion, we have identified the hemangioma-initiating cell. We show that a single multipotential cell (HemSC) produces GLUT-1–positive microvessels and that this lesion undergoes an involutive process by differentiation into adipocytes. This could be a result of a somatic mutation within a stem or progenitor cell and subsequent expansion/differentiation. It is also plausible that a normal SC in the context of the postnatal infantile microenvironment becomes disrupted or delayed in its differentiative program, resulting in an accumulation of early progenitor cells. Such a clonal population could then undergo ill-timed or abnormal differentiation, which subsequently resolves. Understanding the cellular origin of IH provides an avenue for deciphering the mechanisms of EC differentiation and delineating targets for adjuvant therapy. Our in vitro and in vivo models of IH offer powerful tools to evaluate potential therapeutic modalities and insight into the biology of de novo formation of blood vessels.
Cell isolation and culture. Specimens of proliferating IH were obtained from the Department of Plastic Surgery at Children's Hospital Boston under a human subject protocol that was approved by the Committee on Clinical Investigation. The clinical diagnosis was confirmed in the Department of Pathology at Children's Hospital Boston. Informed consent was provided for the specimens, according to the Declaration of Helsinki. Single-cell suspensions were prepared from the proliferating phase specimens as described previously (13). HemSCs were selected using anti-CD133–coated magnetic beads (Miltenyi Biotec). CD133-selected cells were cultured on fibronectin-coated (1 μg/cm2) plates with EBM-2 (CC-3156; Cambrex). EBM-2 was supplemented with 20% FBS, endothelial growth media-2 SingleQuot (CC-4176; Cambrex), and 1× PSF (100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin; Invitrogen). Hereafter this supplemented medium is called EBM-2/20% FBS.
Neonatal foreskin and umbilical cord blood were obtained with institutional research board approval from Brigham and Women's Hospital, Boston, Massachusetts, USA. HDMECs were isolated as described (13). Human umbilical cbEPCs were isolated with anti-CD133–coated magnetic beads (Miltenyi Biotec). In addition, BM-MSCs and NHDFs were obtained commercially (Cambrex) and cultured on fibronectin-coated plates in EBM-2/20% FBS under conditions identical to those used for the HemSCs. For in vivo experiments and in vitro differentiation studies, individually expanded clones of HemSCs were prepared by a limiting dilution technique in which cells were diluted in EBM-2/20% FBS media to 3 cells/ml and dispensed at 100 μl per well in 96-well dishes. After plating and until colonies were harvested, wells were inspected to insure that only 1 colony per well was present.
To obtain GFP-expressing cells, 1.6 × 104 HemSCs were plated into wells of a 96-well plate. After 24 hours, fresh medium containing 8 μg/ml hexadimethrine bromide (Sigma-Aldrich) was added and cells were then infected with 10 μl lentiviral-based particles containing TurboGFP encoding vector (Mission TurboGFP Control Vector; Sigma-Aldrich). To obtain stable GFP expression, cells were maintained in 2 μg/ml puromycin medium (Sigma-Aldrich).
Flow cytometry. Cells were labeled with FITC-conjugated mouse anti-human CD31 (Ancell), PE-conjugated mouse anti-human CD34 (Miltenyi Biotec), FITC-mouse anti-human CD146 (Millipore), PE-mouse anti-human CD90 (Millipore), and PE-mouse anti-human neuropilin-1 (NRP-1; Miltenyi Biotec). Cells were also incubated with rabbit anti-human VEGF-R1 (Santa Cruz Biotechnology Inc.) and mouse anti-human VEGF-R2 (Clone KDR1; Sigma-Aldrich) antibody followed by FITC-conjugated secondary antibody (Vector Laboratories). VEGF-R1, VEGF-R2, and CD34 labeling were carried out after permeabilization of the cells with 0.5% saponin (Sigma-Aldrich). Flow cytometry was performed on a BD FACScan. Data were analyzed using FlowJo software.
RT-PCR. Total RNA was extracted from cultured cells using RNeasy Mini Kit (QIAGEN). Purity of the RNA samples was assessed by determining the optical density at 260:280 nm. Three micrograms of total RNA was used for cDNA synthesis using iScript cDNA Synthesis Kit (Bio-Rad). Primers for Oct-4 (47), AML1 (48), and ribosomal S9 (13) were used. All reactions were performed for 30 cycles with the following temperature profiles: 95°C for 3 minutes (initiation; 30 s/cycle thereafter); 55°C for 45 seconds (annealing); and 72°C for 45 seconds (extension). Size of the PCR products was confirmed by running 10 μl of the reaction product on 3% agarose gel.
In vivo murine model of IH. All experiments were carried out with 1.5 × 106 cells per animal. Cells were trypsinized, counted, and resuspended in phenol red-free Matrigel (BD Biosciences). The cell/Matrigel mixture (200 μl/animal) was injected s.c. into the backs of 6-week-old male athymic nu/nu mice (n = 4/group; Massachusetts General Hospital, Boston, Massachusetts, USA). The animals were monitored for weight gain and Matrigel plug size at 7-day intervals throughout the period of the study. Animals were sacrificed and Matrigel plugs harvested at 7, 14, 28, and 56 days. The Matrigel plugs were then either fixed in 10% neutral buffered formalin for immunohistochemistry (described below) or used immediately for cell-retrieval experiments. Cell retrieval was performed by incubating the Matrigel explants in DMEM medium supplemented with 2% FBS and 4× PSF for 1 hour. The explants were then digested with 0.2% collagenase A and 2.5 U/ml dispase (BD Biosciences). rbc were lysed by incubating the cell suspension with chilled NH4Cl (StemCell Technologies). The cells were labeled with FITC-mouse anti-human CD31 antibody as described above. Mouse dermal ECs (kindly provided by Michael Klagsbrun, Children's Hospital Boston) were used to determine the specificity of the CD31 antibody.
Immunohistochemistry. Immunohistochemistry was performed on formalin-fixed/paraffin-embedded Matrigel plugs. The sections were deparaffinized in xylene and hydrated through sequential ethanol gradient. H&E images were captured with Axiophot II fluorescence microscope (Zeiss) equipped with AxioCam MRc5 (Zeiss). Antigen retrieval was carried out by heating the sections in citrate-EDTA buffer (10 mM citric acid, 2 mM EDTA, 0.05% Tween-20, pH 6.2) or Tris-EDTA buffer (10 mM Trizma-base, 1 mM EDTA, 0.05% Tween-20, pH 9). The sections were blocked for 30 minutes in 5% serum and incubated with rabbit anti-human GLUT-1 antibody (1:50; A3536, Dako) or mouse anti-human GLUT-1 antibody (1:25; ab40084, Abcam), followed by FITC or Texas red–labeled secondary antibody (1:200; Vector Laboratories). The sections were washed and blocked again for 30 minutes. Human-specific CD31 monoclonal antibody (1:50; M0823 clone JC/70A, Dako) followed by Texas red anti-mouse secondary antibody (1:200; Vector Laboratories) was used for detection of human-specific microvessels. Sections were also stained for merosin (1:50; MAB1922 clone 5H2, Millipore), human-specific nuclear antigen (HuNuc, 1:25; MAB 1281 clone 235.1, Millipore), and rabbit anti-human perilipin A (perA, 1:100; P1998, Sigma-Aldrich) or goat anti-human perilipin A (1:200; ab61682, Abcam). The human nuclear antigen staining was performed using an HRP-conjugated secondary antibody followed by incubation with diaminobenzidine solution (Vector Laboratories). GFP expression was detected with anti-TurboGFP antibody (1:2000; AB511, Evrogen). The sections were counterstained with DAPI (Vector Laboratories). All fluorescent images were taken with a Leica TCS SP2 Acousto-Optical Beam Splitter confocal system equipped with DMIRE2 inverted microscope (diode 405 nm, argon 488 nm, HeNe 594 nm; Leica Microsystems).
MVD. For the assessment of MVD, 4 fields from mid-Matrigel H&E sections of each of the 4 animals in the group were used. Microvessels were quantified by counting luminal structures containing rbc. MVD was calculated as vessels/mm2.
Assays for in vitro cellular activity. HemSCs were subjected to cellular activity assays that measured cell growth, proliferation in response to growth factors, and differentiation. Growth parameters were assessed by seeding the cells at 5000 cells/cm2 on fibronectin-coated 48-well plates (4000 cells; growth surface, 0.8 cm2) and culturing in EBM-2/20% FBS. The cells were counted every day using a hemocytometer, and the medium was changed every 2 days. For proliferation experiments, cells were plated as described above. Following attachment (24 hours), plating efficiency was determined, medium was removed, and cells were serum starved for 6 hours. Proliferation was assessed after 24 hours in cells exposed to serum-free medium, 10 ng/ml VEGF-A (R&D Systems), 10 ng/ml VEGF-B (R&D Systems), 5 ng/ml bFGF (Roche Applied Science), or 5% FBS.
To study multilineage differentiation, cells were treated with induction media for 14 days. Mesenchymal differentiation of the cells was carried out with adipogenic media (DMEM/10% FBS, 5 μg/ml insulin, 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine, 60 μM indomethacin, and 1× PSF), osteogenic media (DMEM/10% FBS, 1 μM dexamethasone, 10 mM β-glycerophosphate, 60 μM ascorbic acid–2–phosphate, and 1× PSF), and chondrogenic media (DMEM high glucose, 1× insulin-transferrin-selenium, 1:5000 linoleic acid–albumin, 1 μM dexamethasone, 100 μM ascorbic acid-2-phosphate, 10 ng/ml TGF-β1, and 1× PSF). EC differentiation was carried out in serum-free medium containing VEGF-B (EBM-2, 1× insulin-transferrin-selenium, 1:5000 linoleic acid–albumin, 1 μM dexamethasone, 60 μM ascorbic acid–2–phosphate, 10 ng/ml VEGF-B). For neurogenic differentiation, cells were exposed to initiating medium (DMEM/10% FBS, 1 mM β-mercaptoethanol) for 24 hours. Neurogenic induction medium (DMEM, 0.1 μM dexamethasone, 0.25 μg/ml insulin, 5 μM cAMP, 0.5 mM isobutylmethylxanthine, 5 μM trans-retinoic acid, 0.25 mM l-glutamine, and 25 ng/ml nerve growth factor) was then used for 14 days. Hematopoietic activity was assessed in MethoCult medium (StemCell Technologies) at 1 × 103, 1 × 104, 1 × 105, and 1 × 106 cells per 35-mm dish. Mesenchymal differentiation was assessed by oil red O (lipid droplets), alkaline phosphatase induction (substrate-based activity) and Von Kossa (mineralization), and collagen type 2 staining. Immunofluorescent analysis of cells was carried out using goat anti-human CD31 (Santa Cruz Biotechnology Inc.), mouse anti-human VE-cadherin (Immunotech), mouse anti-human tubulin β-tubilin III (Millipore), and goat anti-human GFAP (Dako) antibodies. FITC- and PE-conjugated secondary antibodies (Vector Laboratories) were used for antigen detection. Images were taken with a Nikon Eclipse TE300 (Nikon) using SPOT Advanced 3.5.9 software (Diagnostic Instruments Inc.) and a ×20/0.45 objective lens.
Statistics. The data were expressed as means ± SEM and analyzed by ANOVA followed by Student's two-tailed t test where appropriate. Differences were considered significant at P < 0.05.
This work was supported by an NIH grant (P01 AR048564 to J. Bischoff). We thank Jill Wylie-Sears for her technical assistance, Elke Pravda for confocal microscopic imaging, and Kristin Johnson for the preparation of the figures.
Nonstandard abbreviations used: BM-MSC, BM mesenchymal stromal cell; cbEPC, cord blood EPC; EBM-2, endothelial basal medium 2; EPC, endothelial progenitor cell; GFAP, glial fibrillar acidic protein; HDMEC, human dermal microvascular EC; HemEC, hemangioma EC; HemEPC, hemangioma EPC; HemSC, hemangioma-derived stem cell; IH, infantile hemangioma; MVD, microvessel density; NHDF, normal human dermal fibroblast; SC, stem cell.
Reference information: J. Clin. Invest.118:2592–2599 (2008). doi:10.1172/JCI33493
Zia A. Khan and Elisa Boscolo are co–first authors.
Mulliken, J.B., Fishman, S.J., Burrows, P.E. 2000. Vascular anomalies. Curr. Probl. Surg. 37:517-584.
View this article via: CrossRef PubMed Google Scholar
Bischoff, J. 2002. Monoclonal expansion of endothelial cells in hemangioma: an intrinsic defect with extrinsic consequences? Trends Cardiovasc. Med. 12:220-224.
Boye, E., et al. 2001. Clonality and altered behavior of endothelial cells from hemangiomas. J. Clin. Invest. 107:745-752.
View this article via: JCI CrossRef PubMed Google Scholar
Bielenberg, D.R., et al. 1999. Progressive growth of infantile cutaneous hemangiomas is directly correlated with hyperplasia and angiogenesis of adjacent epidermis and inversely correlated with expression of the endogenous angiogenesis inhibitor, IFN-beta. Int. J. Oncol. 14:401-408.
Berard, M., et al. 1997. Vascular endothelial growth factor confers a growth advantage in vitro and in vivo to stromal cells cultured from neonatal hemangiomas. Am. J. Pathol. 150:1315-1326.
Virchow, R. 1863.Die Krankhaften Geschwulste. August Hirschwald. Berlin, Germany. 306 pp.
Pack, G., Miller, T.R. 1950. Hemangiomas; classification, diagnosis, and treatment. Angiology. 1:405-426.
Malan, E. 1974. Vascular malformations (angiodysplasias). InCarlo Erba Foundation. Milan, Italy. 4.
Smoller, B.R., Apfelberg, D.B. 1993. Infantile (juvenile) capillary hemangioma: a tumor of heterogeneous cellular elements. J. Cutan. Pathol. 20:330-336.
Dosanjh, A., et al. 2000. In vitro characteristics of neonatal hemangioma endothelial cells: similarities and differences between normal neonatal and fetal endothelial cells. J. Cutan. Pathol. 27:441-450.
Shmelkov, S.V., St. Clair, R., Lyden, D., Rafii, S. 2005. AC133/CD133/Prominin-1. Int. J. Biochem. Cell Biol. 37:715-719.
Yu, Y., Flint, A.F., Mulliken, J.B., Wu, J.K., Bischoff, J. 2004. Endothelial progenitor cells in infantile hemangioma. Blood. 103:1373-1375.
Khan, Z.A., et al. 2006. Endothelial progenitor cells from infantile hemangioma and umbilical cord blood display unique cellular responses to endostatin. Blood. 108:915-921.
Bauland, C.G., et al. 2006. The pathogenesis of hemangiomas: a review. Plast. Reconstr. Surg. 117:29e-35e.
Wu, X., et al. 2004. Tissue-engineered microvessels on three-dimensional biodegradable scaffolds using human endothelial progenitor cells. Am. J. Physiol. Heart Circ. Physiol. 287:H480-H487.
O'Brien, C.A., Pollett, A., Gallinger, S., Dick, J.E. 2007. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 445:106-110.
Ricci-Vitiani, L., et al. 2007. Identification and expansion of human colon-cancer-initiating cells. Nature. 445:111-115.
Melero-Martin, J.M., et al. 2007. In vivo vasculogenic potential of human blood-derived endothelial progenitor cells. Blood. 109:4761-4768.
Baal, N., et al. 2004. Expression of transcription factor Oct-4 and other embryonic genes in CD133 positive cells from human umbilical cord blood. Thromb. Haemost. 92:767-775.
Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G., Downing, J.R. 1996. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 84:321-330.
Li, Z., Chen, M.J., Stacy, T., Speck, N.A. 2006. Runx1 function in hematopoiesis is required in cells that express Tek. Blood. 107:106-110.
Reyes, M., et al. 2002. Origin of endothelial progenitors in human postnatal bone marrow. J. Clin. Invest. 109:337-346.
View this article via: JCI PubMed Google Scholar
Jiang, Y., et al. 2002. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 418:41-49.
Egusa, H., Schweizer, F.E., Wang, C.C., Matsuka, Y., Nishimura, I. 2005. Neuronal differentiation of bone marrow-derived stromal stem cells involves suppression of discordant phenotypes through gene silencing. J. Biol. Chem. 280:23691-23697.
Tondreau, T., et al. 2004. Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation. Differentiation. 72:319-326.
Pittenger, M.F., et al. 1999. Multilineage potential of adult human mesenchymal stem cells. Science. 284:143-147.
Ritter, M.R., Reinisch, J., Friedlander, S.F., Friedlander, M. 2006. Myeloid cells in infantile hemangioma. Am. J. Pathol. 168:621-628.
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J.C., Keller, G. 1998. A common precursor for hematopoietic and endothelial cells. Development. 125:725-732.
Ito, Y., Iwamoto, Y., Tanaka, K., Okuyama, K., Sugioka, Y. 1996. A quantitative assay using basement membrane extracts to study tumor angiogenesis in vivo. Int. J. Cancer. 67:148-152.
Hoang, M.V., Senger, D.R. 2005. In vivo and in vitro models of Mammalian angiogenesis. Methods Mol. Biol. 294:269-285.
Yu, Y., et al. 2006. Mesenchymal stem cells and adipogenesis in hemangioma involution. Stem Cells. 24:1605-1612.
Parums, D.V., et al. 1990. JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J. Clin. Pathol. 43:752-757.
North, P.E., Waner, M., Mizeracki, A., Mihm (Jr.), M.C. 2000. GLUT1: a newly discovered immunohistochemical marker for juvenile hemangiomas. Hum. Pathol. 31:11-22.
North, P.E., et al. 2001. A unique microvascular phenotype shared by juvenile hemangiomas and human placenta. Arch. Dermatol. 137:559-570.
Kawaguchi, N., et al. 1998. De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor. Proc. Natl. Acad. Sci. U. S. A. 95:1062-1066.
Kimura, Y., Ozeki, M., Inamoto, T., Tabata, Y. 2002. Time course of de novo adipogenesis in matrigel by gelatin microspheres incorporating basic fibroblast growth factor. Tissue Eng. 8:603-613.
Tabata, Y., et al. 2000. De novo formation of adipose tissue by controlled release of basic fibroblast growth factor. Tissue Eng. 6:279-289.
Greenberg, A.S., et al. 1991. Perilipin, a major hormonally regulated adipocyte-specific phosphoprotein associated with the periphery of lipid storage droplets. J. Biol. Chem. 266:11341-11346.
Singh, S.K., et al. 2003. Identification of a cancer stem cell in human brain tumors. Cancer Res. 63:5821-5828.
Salmaggi, A., et al. 2006. Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype. Glia. 54:850-860.
Collins, A.T., Berry, P.A., Hyde, C., Stower, M.J., Maitland, N.J. 2005. Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res. 65:10946-10951.
Richardson, G.D., et al. 2004. CD133, a novel marker for human prostatic epithelial stem cells. J. Cell Sci. 117:3539-3545.
Obeso, J., Weber, J., Auerbach, R. 1990. A hemangioendothelioma-derived cell line: its use as a model for the study of endothelial cell biology. Lab. Invest. 63:259-269.
Bautch, V.L., Toda, S., Hassell, J.A., Hanahan, D. 1987. Endothelial cell tumors develop in transgenic mice carrying polyoma virus middle T oncogene. Cell. 51:529-537.
Tang, Y., et al. 2007. A novel in vivo model of human hemangioma: xenograft of human hemangioma tissue on nude mice. Plast. Reconstr. Surg. 120:869-878.
Barnes, C.M., et al. 2005. Evidence by molecular profiling for a placental origin of infantile hemangioma. Proc. Natl. Acad. Sci. U. S. A. 102:19097-19102.
Tai, M.H., et al. 2005. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis. 26:495-502.
Choi, S.J., et al. 2003. AML-1A and AML-1B regulation of MIP-1alpha expression in multiple myeloma. Blood. 101:3778-3783.
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There is a possible misconception which people might have about how Bitcoin Wallets work, which I would say, because I had this misconception myself until only very recently. Many popular Bitcoin Wallets present themselves to the user as having one balance at one time.
Each time we receive funds, most of the time, we will want to receive those funds to a separately-created Address. And the difference between the Address and the Public Key, is mainly one in formatting of the binary data.
What many Wallets do not show the user, is that each receiving Address continues to hold its own balance. This means that when we tell such a Wallet to Send an amount of Bitcoins to another recipient, which is larger than the balance we hold on any one of our own Addresses, what our Wallet tends to try to do, is to make numerous transactions, one from each Address, all to the same recipient, until the total amount of funds has reached the amount we asked to Send.
Each of those Addresses, being a Public Key, has its own Private Key, which we must unlock across-the-board, at least until the transaction is complete. And each Private Key, therefore, only has a limited amount of Funds it can Send. Balances are not allowed to go negative.
And so what can happen to many users, is that our Wallets become fragmented, with only a small balance associated with each of our Addresses, many of which might have started out as having a larger, received balance. And sometimes, the P2P network can be reluctant to process such a transaction, because it represents a load on the network.
And so an operation that some Wallets recommend, is that if we have many small balances, eventually to transfer a larger sum, out of those balances, to a new, single receiving Address we have in our own, same Bitcoin Wallet. This incurs the transaction fee normally associated with such an operation, but the only benefit to us, is that we will have a single, larger balance afterward.
Other types of Wallets do not show us, how much of our balance is associated with each Address.
What this also means, in connection with what I wrote before, about being able to Sweep one Address, is that this ability is usually not so sweeping in practice. Since a wallet with 10 Addresses that hold balances, can also have 10 Private Keys, the program from which we might want to do the Sweeping, will need each of the Private Keys, in order to empty out a whole Wallet, for example.
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Because it is a crypto-currency, public lists of transactions are not meaningful by default, beyond revealing the Addresses used. They cannot typically be associated back to specific people.


text:
NHI hearings to start in Gauteng tomorrow
Committee chairperson Dr Sibongiseni Dhlomo says while they have attended several public hearings, a decision will be made to benefit of the country.
FILE: Chris Hani Baragwanath Academic Hospital. Picture: Sethembiso Zulu/EWN.
NHI Bill
NHI Hearings
NHI Public Hearings
Jason Felix | 347 days ago
CAPE TOWN – Parliament's health committee chairperson Dr Sibongiseni Dhlomo says there's no need for the hysteria around the national health insurance (NHI) Bill.
He says while they have attended several public hearings, a decision will be made to benefit of the country.
The NHI has been a hotly contested matter with many questioning its viability.
According to Health Minister Zweli Mkhize NHI could be fully implemented by 2026.
But this plan has not come easy for government as it has been questioned by several stakeholders.
Even in some communities, people are questioning this plan.
Parliament's health portfolio hosted public hearings on the NHI in Beaufort West.
Dhlomo adds they would recommend the best decision for Parliament to debate and at the end be implemented.
"There is a group of other South Africans who say, while we welcome this Bill we have a number of concerns that we don't see good staffing in our hospitals and find there is a shortage of medicine in some areas."
The committee says it's confident the public hearings into NHI in Gauteng will be welcomed.
The hearings will kick off in Gauteng on Monday.
The committee has been travelling across the country since the NHI Bill was passed in parliament last year.
Dhlomo said, "One must appreciate that the process has been extremely democratic and fair going to other areas.
We are going to Gauteng now, that's the last province. The committee had its presence in Beaufort West. If we had 200 people we would be happy, and if we had 900 people in the metro it would be okay."
The Bill will enable government to meet its goal of universal health coverage, similar to that offered by the United Kingdom's National Health Service.
WC ANC calls on Winde to remove MEC Bredell over ethics breach


Copies of this magazine shaped Passion Pod, "Delayed Gratification",are always tricky to prise from the clutches of friends and family; a pretty mighty sign that these guys are on to a winner. Set up by journalists Rob Orchard and Marcus Webb, this publication is all about taking its time. Produced quarterly it looks (and smells!) ridiculously beautiful. It pootles its way back through the bits and bobs that have been going on over the last few months and with the benefit of hindsight, prides itself on "being the last to breaking news". V clever and massively refreshing.
But the truly bestest thing about this week's Passion Pod is that after a year and half of DG being up and running, the day that we chatted to Rob was his first one working full time on it. If that doesn't encourage your Passion Pod juices, what on earth will?


text:
Q: Does gravity act in the centre of the Earth? If we we dig a hole from north pole to the south pole of the Earth and we throw a ball in this hole where would it stop? Will it come back on the surface or it will stop at the centre?

A: if u drill such a hole and drop a ball then the result will be that it will harmonically oscillate about the centre of earth with the radius as amplitude
however if ball is directly released at the centre. it will stay still since the net gravitational force is 0.
edit:- as has been pointed by the first commentor , thank you.
the answer is for a perfectly uniformly dense spherical ball and that too when the hole u drill is sufficiently small to not alter the mass of earth in any considerable way


Growth Groups are small groups meeting during the week to study God's Word, pray together, and to support and care for one another. These smaller groups provide us with the opportunity to dig deeper, ask questions, help each other grasp God's truth and its implications for our lives, and pray together, in ways that aren't always possible in our larger Sunday gatherings.
We have growth groups meeting during the day and most evenings as well as a Mum's group with a crèche. We welcome anyone to join a Growth Group, and would be glad to help you find a group that will suit you.
For more information about times and locations, contact our office by emailing [email protected] or by calling (02) 4647 2195.