Xinjiang Party Secretary Chen Quanguo made a name for himself with his repressive style of rule as party secretary of Tibet from 2011 to 2016. Since his transfer to the Uyghur region in August this year, his tactics have become only more brutal.
Since taking the reins in Xinjiang, Quanguo has launched new polices targeting the religious freedom and cultural identity of the Uyghurs and intensified existing policies like the Communist Party's "strike hard" campaign against Uyghur.
Chen Quanguo effectively considers all Uyghurs and other Muslims in Xinjiang as political threats and "undependable" because of their distinct religious and ethnic identities. The political re-education camps that have popped up in almost every district of Xinjiang that currently detain tens of thousands of Uyghurs are the brainchild of 62-year-old Quanguo. His strike even harder approach in Xinjiang may have helped secure his current seat on the CCP's 19th Politburo, further cementing his influence in the regime.
In March, the Xinjiang CCP issued the ?????????????? (Regulation on Counterextremism in Xinjiang Uyghur Autonomous Region). The regulation requires a massive campaign against politically "untrustworthy" and "incorrect" people plagued by "ideological illness" because of their religious and national identities. The "re-education" camp that detain people from almost every Uyghur city are part of Quanguo's effort to cure Uyghurs of that "ideological illness" that he sees as their religious and ethnic identity so that they may be more easily assimilated and controlled.
The Chinese government often embellishes its brutal campaigns against Uyghurs with seemingly benign labels. The party has used various politically correct names for the re-education camps, including ???????? (Education and Transformation Centers), Career Development Centers, Professional Education Schools, Socialism Training Schools, and Counter-extremism Training Schools.
An official explained the frequent change in labeling, saying "obviously, the reason or changing the name is to avoid giving others a bad impression".
Going to a re-education and transformation center is a free opportunity for the ideologically infected to receive treatment and cure their disease. Young friends, how are you? I am very pleased to share my views on this topic. Following the recent strengthening of the strike hard campaign, some of the people in Xinjiang – mainly younger people – have been sent to re-education and transformation centers. But their parents, relatives, and even the majority of Uyghurs don't know about the re-education and transformation centers and worry about it. Today, we'll answer the questions you've been wondering about.
The people sent to the re-education and transformation center are actually ideologically diseased people infected with ideas of religious extremism and violent terrorism – so they have to be treated.
In order to cure ideologically diseased people in time and guarantee the effectiveness of the treatment, the CCP of Xinjiang decided to set up re-education and transformation camps in every prefecture, city and county all over Xinjiang. The government has organized special officials to teach the people the laws and regulations of the country and the autonomous region, the ethnic and religious policies, various beneficial party policies, and the state language – Chinese.
First, the government will clarify what is wrong and what is right, what they should do and what they should not do. The government assures the people that it will exterminate any impact of religious extremism and violent ideas. It will ensure that those who are ideologically infected will regain their ideological health and return to their homes to reunite with their family members after their minds have been cleaned from harmful ideas.
"I only had a one-time experience of listening to or watching a religious preaching or violent extremism audio or video. I recognized my wrong and won't listen to or watch it again. I have so many important things to do, so can I not go to the re-education and transformation center?".
Some may say the stay at the re-education and transformation center is too long, can't it be shorter? The answer to those questions are NO. An infection of religious and violent extremism is serious. If anyone escapes from treatment, he will throw himself and society in danger.
Re-education and transformation centers are special hospitals to cure ideological illnesses. The hospital is free, and food and housing is provided. The government built new re-education centers all over Xinjiang to provide the conditions to educate and change the people. The government has appointed a lot of key officials as doctors to these hospitals to treat ideological illness.
Family members of re-educated people, don't worry. Nobody will be left hungry at the re-education centers. There is no cold, no labor, but there is rare and free opportunity for people to be trained so that they may change. The government's guarantees to re-educate the people and after that they will live normal lives like others.
The audio is available through various government propaganda offices in Xinjiang.
"Five kinds of suspicious people have been detained and sent to re-education camps – people who throw away their mobile phone's SIM card or do not use their mobile phone after registering it; former prisoners; blacklisted people; suspicious people who have some fundamental religious sentiment; and the people who have relatives abroad," a female police officer from far western Xinjiang told RFA.
Chinese auhtorities have formed an official grading standard using those abstract notions to determine their targets. Uyghurs are scored according to their religious background, political views, and other factors. Those who get a score under 60 are considered dangerous and are sent to the camps.
The majority of those targeted are Uyghurs. Other Turkic minorities have also been targeted, but no Han Chinese have been the subject of China's "re-education" efforts in Xinjiang. Earlier this month, local officials in Xinjiang told RFA that thousands of Uyghurs and other Muslim ethnic minorities – including Kyrgyz and Kazakhs – are being held in re-education camps without contact with their families under a policy designed to counter extremism in the region.
Sources believe there are virtually no ethnic Han Chinese held in the Xinjiang re-education camps, despite Han Chinese making up the majority of Xinjiang's population. Those sources also indicate that the number of detainees in southern Xinjiang – where there are higher concentrations of Uyghurs – significantly exceed that of the northern part of the region.
"The Chinese authorities are holding people at these 'political education' centers not because they have committed any crimes, but because they deem them politically unreliable".
Since April, Uyghurs who have traveled abroad have been accused of harboring "extremist" and "politically incorrect" have been detained in re-education camps throughout Xinjiang until they admit that they committed a "wrong" by leaving the country.
"I learned through my work that among the detainees [from my district] 13 people were held for traveling abroad with a tourist company, " said ___. "One person had gone on hajj to Mecca two years ago, and two others had studied in Turkey for a short time before returning home".
Anyone with a religious background may be targeted, including religious teachers, imams, and youth who learn to practice their religion at home..
A family of four Uyghurs –including two children – were taken to a political education facility in western Xinjiang in April for traveling abroad for business and for the Hajj, according to a Human Rights Watch report.
Xinjiang authorities also imprisoned an Uyghur man after accusing him of "religious extremism" for scolding his son for drinking alcohol.
Those detained in the camps are treated like criminals and are not free to see their family members. They are interrogated by police and face imprisonment if they do not comply with the rules and regulations of the camp. And they have no legal protections.
Those who have actual physical illnesses are kept inside the camps instead of being sent to hospitals. The local authorities keep them busy with labor and aren't concerned about the needs of the people, aside from the very basic necessities like food and sleep. Some elderly Uyghurs and children have died because they were left without the care of their loved ones who were sent to the camps.
Those at the camp are forced to learn CCP ideology. The government has set up classrooms in addition to interrogation rooms and barracks inside the camps. The instructors and other staff, like the "students" at these "re-education" camps who are essentially prisoners, live inside the camp and share the same courtyard with the detainees. The centers' main gates are guarded 24 hours a day and instructors are required to obtain permission before leaving the facility.
Those who fail to actively learn the political ideology force fed to them at the camp face imprisonment. It's these kinds of extreme measures that suggest the true aim of the camps are not to educate as much as it is to strip the detainees of their Uyghur identity and force them to accept a Chinese identity.
While the number of those detained cannot be adequately confirmed, almost all sources indicate mass detentions and extrajudicial imprisonment are becoming the norm in Xinjiang. RFA has reported that nearly half of the Uyghurs in Hotan have been targeted for re-education camps.
The camps in Ghulja county , Ili Kazakh prefecture and Korla City hold at least 3600 detainees each, local officials told RFA's Uyghur Service. Those camps are run under the label of "career development centers" to mask their true nature.
According to one of the teachers in a re-education camp in Ghulja City, there are five camps in a just one of Ghulja's countries – Turpanyuzi.
Sources in Bayin'gholin Mongol Autonomous Prefecture's Korla city, where Uyghurs have previously protested house raids during China's "strike hard" anti-terrorism campaigns, told RFA that the municipality has three re-education camps with at least 2,100 detainees. A "socialism institute" in the municipality detains more than 40 religious figures.
"A few people were released after two to three months. But most detainees sent to the camps remain indefinitely."? ?
Most observers believe that The Chinese government's aim is to streamline ideology in an area it perceives to be troubled by radical violence and Uyghur nationalist rights movements and perhaps even erase the western region's connections with outside world.
Frances Eve, researcher at Chinese Human Rights Defenders, the ramped up suppression in Xinjiang will likely continue past the Party Congress's because it appears Chen was brought to Xinjiang to replicate the heavy-handed tactics he used in Tibet.
The lack of a response from the international community is somewhat surprising in the face of mounting evidence of the re-education camps.
The New York-based advocacy group Human Rights Watch called on the Chinese government to free the thousands of Xinjiang people placed in the camps since April 2017 and close them down.
The Xinjiang political education detention centers — where inmates who have not broken any laws are detained extrajudicially, indefinitely and without the knowledge of their families – run contrary to China's constitution and violate international human rights law, Human Rights Watch noted.


Hurconotes Page 23 : Gold And Silver Table Lamps. Modern Floor Lamps Images. Red Drum Lamp Shade. Tripod Ikea. Antique Brass Pharmacy Lamp.
By Adorlee Ferreira. Lamps. Published at Thursday, January 03rd, 2019 - 18:51:12 PM.
By Nichol Brunet. Lamps. Published at Tuesday, January 01st, 2019 - 10:24:38 AM.
By Gaetana Leroux. Lamps. Published at Tuesday, December 25th, 2018 - 05:52:34 AM.
By Aceline Pelletier. Lamps. Published at Tuesday, December 11th, 2018 - 12:16:24 PM.
By Aubrey Hamon. Lamps. Published at Saturday, December 08th, 2018 - 05:36:13 AM.
By Pascale Buisson. Lamps. Published at Monday, December 03rd, 2018 - 11:12:42 AM.
By Galla Gaillard. Lamps. Published at Saturday, November 17th, 2018 - 23:04:35 PM.
By Lavern Nguyen. Lamps. Published at Sunday, November 11th, 2018 - 16:27:09 PM.
By Adorlee Ferreira. Lamps. Published at Thursday, November 08th, 2018 - 21:46:17 PM.
By Tempeste Boyer. Lamps. Published at Monday, November 05th, 2018 - 14:34:55 PM.


text:
NIOC to Start Selling Oil on Stock Market by October 28
Sunday، 21 October 2018 |
TEHRAN (Shana) -- The CEO of the National Iranian Oil Company (NIOC) said his company would start selling oil on the domestic stock market by October 28.
Addressing the opening ceremony of the 4th Iranian Petroleum and Energy Club Congress & Exhibition in Tehran on Tuesday, Ali Kardor said necessary arrangements for the supply of crude oil in the stock exchange had been provided and the condition to participate in the transactions, was paying an equivalent of 10% of the price of oil in Rials by buyers.
The base price will be set at $79.15 for the oil but the final price would be determined based on the international trading prices of the National Iranian Oil Company, the official said.
He said talks for striking deals under the newly-devised model of oil contracts were ongoing, adding, "We are in talking for three fields; moreover, with Total's exit from the South Pars Phase 11 project, the engineering data, maps, etc. regarding the projects have been made available to the CNPCI."
Kardor further said that the National Iranian South Oil Company (NISOC) had signed a memorandum of understanding with Pergas Consortium for developing Keranj Oilfield in Iran which would soon turn into a contract.
About PEDC
Pasargad Energy Development Co. (PEDC) is an independent energy company based in Iran. PEDC is an integrated energy company whose activities span the entire value chain including exploration, production, refining, petrochemicals marketing, power generation & renewables as well as new high-tech venture capitalizing. Our headquarter is located in Tehran. PEDC is present across the Iranian continental shelf and international market through its subsidiaries. We create value for our shareholders through active investment, high-level monitoring of our projects, building world-class partnership and managing our risks effectively and making balanced portfolio. Meantime, we contribute to the sustainable development of the energy sector and communities in our country. PEDC's competitiveness relies upon our values-based performance culture, with a strong commitment to transparency, cooperation and continuous operational improvement. As a wholly owned subsidiary of the Pasargad Financial Group, with Pasargad Bank being a distinctive shareholder, we owe our success to our experience, management knowledge and strong local/international partnerships since 2008. PEDC is committed to maintaining sustainability and being recognized as the strategic partner of choice in Iran's energy and petrochemical sector.
Oil Market company National Iranian NIOC CEO said domestic on 28 would The By of selling start his stock October
Iran Firms Wielding 70% Share in Oil Production Enhancement, Maintenance Projects: CEO

redpajama_set_name: RedPajamaCommonCrawl


A major fire erupted at the boundary of the Sanjay Gandhi National Park near Arun Kumar Vaidya Marg in Goregaon on December 3 around 6:30 pm in an open plot near an IT Park Mumbai.
As soon as the firefighting department received a call from the spot they immediately rushed towards the spot to douse the fire. 10 fire tenders were present on the spot to fight the flames.
According to the sources, the blaze was of Level 3 and no casualties or injuries were reported in this mishap. The cause of the fire was yet to be ascertained. This massive fire was visible form a long distance which created panic among the residents. It started spreading towards residential areas such as New Mhada Colony near the New Dindoshi Green Hill Society in Goregaon.
Due to lack of access in the forested area of the Aarey milk colony, the firefighter officials along with the fire vehicles were finding it very difficult to reach the spot.
Sources revealed that green tree branches which do not catch fire easily were used to bring the flames in control. After so many attempts the fire was doused after 12 hours on December 4.
Note: The land where the fire has erupted belongs to F E Dinshaw trust property and has been taken over by Raheja builders. It lies in the Dindoshi Hills between SGNP and Aarey Colony.


GetHuman-sandkey's next move: go to the Groupon help center.
Groupon doesn't have a customer phone number that is answered by an agent. Instead, GetHuman-sandkey will want to follow the link below to head to their help center. We will check on GetHuman-sandkey later to make sure it's all set or try to help escalate.
GetHuman-sandkey did not yet indicate what Groupon should do to make this right.
We are waiting for GetHuman-sandkey to fix the problem and share the solution with the rest of us customers.
See how GetHuman-sandkey fixes it.


text:
Q: add delay to this jquery on hover I'm using this code:
var timeout    = 500;
var closetimer = 0;
var ddmenuitem = 0;

function navBar_open()
{  navBar_canceltimer();
   navBar_close();
   ddmenuitem = $(this).find('ul').css('visibility', 'visible');}

function navBar_close()
{  if(ddmenuitem) ddmenuitem.css('visibility', 'hidden');}

function navBar_timer()
{  closetimer = window.setTimeout(navBar_close, timeout);}

function navBar_canceltimer()
{  if(closetimer)
   {  window.clearTimeout(closetimer);
      closetimer = null;}}

$(document).ready(function()
{  $('#navBar > li').bind('mouseover', navBar_open) //mouseover
   $('#navBar > li').bind('mouseout',  navBar_timer)}); //mouseout

document.onclick = navBar_close;

which works fine
what i'd like to do is add a delay to the mouseover event
i'll be honest, I found this code on another site and don't completely understand how it works.
I get that when the user mouses out, the navBar_timer function is called, which adds some kind of delay before the dropdown menu is hidden again, but i don't quite see how to implement a hover on the mouseover.
any guidance would be appreciated
thanks

A: What version of Jquery are you using? If you're using the new one (1.4), you should be able to leverage the new $.delay() function. Then all you'd have to change is one line within navBar_open() to:
ddmenuitem = $(this).find('ul').delay(timeout).css('visibility', 'visible');

A: Try to change this:
$(document).ready(function()
{  $('#navBar > li').bind('mouseover', navBar_open) //mouseover
   $('#navBar > li').bind('mouseout',  navBar_timer)}); //mouseout

To this:
$(document).ready(function() {
    $('#navBar > li').hover(function() {
        closeHoverTimer = window.setTimeout(navBar_open, 500); //500ms timeout);
    }, function() {
        navBar_timer();
        window.clearTimeout(closeHoverTimer);
    });
});

A: this is what you are looking for... click http://cherne.net/brian/resources/jquery.hoverIntent.html

A: Something like this will do the trick with jquert 1.4 and above. No need for hoverIntent plugin:
$("#yourdiv").hover(function(){
    $(this).stop(true).delay(400).animate({"height":"300px"},800, "easeOutBounce");
},function(){
    $(this).stop(true).animate({"height":"25px"}, 300, "easeOutBack");
});

just add the delay function after the stop element. If you hover over the element it waits 400ms before expanding the menu. If you move the mouse out of the element before the 400ms timeframe the menu will not open.

A: I second Reigel's recommendation to use the hoverIntent plugin.
To delay other jquery functions, i tend to use this function:
  var delay = (function(){
    var timer = 0;
    return function(callback, ms){
      clearTimeout (timer);
      timer = setTimeout(callback, ms);
    };
  })();

And call it:
delay (function () {
    // add whatever function you want delayed by 2 secs
}, 2000);

A: This should work as well:
    $('#navBar > li').hover(
        function() {
            var newthis = $(this);
            timer = setInterval(function() {showTip(newthis)}, delay);
        },
        function() {
            clearInterval(timer);
            $(this).find('ul:visible').fadeOut(speed);
        },
        showTip = function(newthis) {
            clearInterval(timer);
            newthis.find('ul:hidden').fadeIn(speed);
        }
    );

redpajama_set_name: RedPajamaStackExchange


0%68°42°Sunny with a high of 68 °F (20.0 °C). Winds ESE.Night - Clear. Winds variable at 3 to 6 mph (4.8 to 9.7 kph). The overnight low will be 45 °F (7.2 °C).
50%71°57°Rain today with a high of 71 °F (21.7 °C) and a low of 57 °F (13.9 °C). There is a 50% chance of precipitation.
Today - Sunny with a high of 68 °F (20.0 °C). Winds ESE.
Tonight - Clear. Winds variable at 3 to 6 mph (4.8 to 9.7 kph). The overnight low will be 45 °F (7.2 °C).


Beautiful kitchen in 500,000 sq. ft. multi tenant building, perfect for catering, restaurant / quick casual startup, or cooking classes. Please contact for more information, pictures, and a tour.
Come by and take a tour of this massive kitchen and move in right away!


text:
Lac du Pasteur är en sjö i Kanada.   Den ligger i regionen Mauricie och provinsen Québec, i den sydöstra delen av landet,  km nordost om huvudstaden Ottawa. Lac du Pasteur ligger  meter över havet. Arean är  kvadratkilometer. Den högsta punkten i närheten är  meter över havet,  km söder om Lac du Pasteur. Den sträcker sig 2,6 kilometer i nord-sydlig riktning, och 1,4 kilometer i öst-västlig riktning.

I övrigt finns följande vid Lac du Pasteur:

 Lac Boucane (en sjö)
 Lac Wabush (en sjö)

I omgivningarna runt Lac du Pasteur växer i huvudsak blandskog. Trakten runt Lac du Pasteur är nära nog obefolkad, med mindre än två invånare per kvadratkilometer.  Trakten ingår i den hemiboreala klimatzonen. Årsmedeltemperaturen i trakten är  °C. Den varmaste månaden är juli, då medeltemperaturen är  °C, och den kallaste är januari, med  °C. Genomsnittlig årsnederbörd är  millimeter. Den regnigaste månaden är september, med i genomsnitt  mm nederbörd, och den torraste är mars, med  mm nederbörd.

Kommentarer

Källor

Insjöar i Mauricie
Insjöar i Kanada större än 1 kvadratkilometer

redpajama_set_name: RedPajamaWikipedia


To view this site you must be of legal drinking age in your country of residence.
This Cuvée has zesty citrus flavours and yeasty complexity with a crisp clean finish.
Bursting with zesty citrus flavours and yeast complexity that produces a clean crisp finish.


text:
About Jackson
Help us Find a Cure
401 Project Events
Our hope is to find a cure for MECP2 Duplication Syndrome before it is too late for Jackson.
There is Hope!
In 2012, The 401 Project was started by families who's children are/were affected with this disorder.
We are dedicated to
CURE MECP2 DUPLICATION SYNDROME!
"Given the experience with models of Rett syndrome we believe the odds are excellent that several symptoms of the duplication will be reversed."
— Dr. Huda Zoghbi Feb 2013
To cure MECP2 Duplication Syndrome.
In 2012, a concerted parent-driven effort was established to raise funds for the initial MECP2 reversal experiments conducted at the Baylor College of Medicine. Each MECP2 family pledged to raise $401.00 in funds to cover the research program cost. The initiative was a success, and the 401 Project was born. We've maintained the 401 Project name as it is fundamental to our philosophy: the collection of individual efforts produces high impact results.
The 401 Project was established by parents of children affected by MECP2 Duplication Syndrome. Today, the 401 Project is maintained by a group of parent facilitators who guide fundraising programs, scientist interactions, research finding translations, and social media activities. There are very minimal direct costs associated with running the 401 Project, and these costs are covered by the parent facilitators. Every penny donated to the 401 Project goes to research.
OUR RELATIONSHIP WITH THE RETT SYNDROME RESEARCH TRUST
All funds raised through the 401 Project initiative are directly deposited into the Rett Syndrome Research Trust MECP2 Duplication Syndrome Fund. The Rett Syndrome Research Trust (RSRT) is a registered non-profit organization, and all donations are fully tax deductible.
MECP2 Duplication Syndrome was discovered in 2005 by Dr. Huda Zoghbi during the course of her research on Rett Syndrome. Both Rett Syndrome and MECP2 Duplication Syndrome are caused by mutations in the MECP2 gene. Rett Syndrome-affected individuals do not produce enough MECP2 protein in their brains while MECP2 Duplication-affected individuals produce far too much MECP2 protein. Research conducted on the earlier-discovered Rett Syndrome was easily leveraged to initiate promising research to cure MECP2 Duplication Syndrome.
RSRT was founded in 2008 by Monica Coenraads. Since 1999, Monica has led the surge in research for genetic disorders caused by mutations in the MECP2 gene. The MECP2 Duplication community is beyond fortunate that Monica has championed MECP2 Duplication Syndrome research under the RSRT umbrella. Under Monica's leadership, over $40,000,000 has been raised for research on MECP2-associated genetic disorders. RSRT operates by constantly engaging academic scientists, clinicians, industry, investors, and affected families. These relationships catalyze the development and execution of a research agenda that neither academia nor industry could achieve alone. RSRT identifies, solicits, evaluates, prioritizes, funds and monitors ambitious research projects that have the greatest likelihood of impacting the lives of those afflicted with Rett Syndrome and MECP2 Duplication Syndrome.
* Taken from www.401project.com
To See Research Projects Underway:
www. 401project.com


2. Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, USA.
Pancreas duodenum homeobox 1 (PDX1) is essential for pancreas development and β-cell formation; however more studies are needed to clearly illustrate the precise mechanism regarding spatiotemporal regulation of Pdx1 expression during β-cell formation and development. Here, we demonstrate that SIRT1, FOXA2 and a number of proteins form a protein complex on the promoter of the Pdx1 gene. SIRT1 and PDX1 are expressed in the same set of cells during β-cell differentiation and maturation. Pancreas-specific disruption of SIRT1 diminished PDX1 expression and impaired islet development. Consequently, SIRT1 mutant mice develop progressive hyperglycemia, glucose intolerance, and insulin insufficiency, which directly correlate with the extent of SIRT1 deletion. We further show that SIRT1 interacts with and deacetylates FOXA2 on the promoter of the Pdx1gene, and positively regulates its transcription. These results uncover an essential role of SIRT1 in β-cell formation by maintaining expression of PDX1 and its downstream genes, and identify pancreas-specific SIRT1 mutant mice as a relevant model for studying insulin insufficiency.
Keywords: SIRT1, FOXA2, PDX1, insulin.
Diabetes is a severe metabolic disease worldwide that can be caused by many factors. In human, inactivation of pancreas duodenum homeobox 1 (PDX1) causes early-onset type II diabetes mellitus [50, 51]. Experiments in mice revealed that PDX1 regulates the transcriptional network of pancreatic endocrine progenitor cells and that PDX1 deficiency blocks pancreas formation, suggesting that PDX1 is essential for pancreas development and β-cell formation [26, 27, 41, 42]. Thus, it is important to understand how PDX1 is regulated. Previous studies indicated that PDX1 is highly expressed throughout the entire pancreatic epithelium in the early stages of embryogenesis; however, its expression becomes more restricted to insulin-producing β-cells at later stages of pancreas development [41, 52].
The PDX1 promoter contains several nuclease hypersensitive sequences (HSS) that are regulated by transcriptional factors [16, 61]. This enhancer region, known as area I (bp -2761 to -2457), area II (bp -2153 to -1953, also referred as PH2 domain), area III (bp -1879 to -1600) , and area IV , is bound by transcription factors, including HNF1a, Foxa2, Foxa1, NHF6, Pax6, MafA and Ptf1 to direct Pdx1 expression in pancreatic progenitors/or in adult β-cells [2, 14, 25, 31, 37, 46, 47, 53]. Area II (PH2) is only present in the mammalian PDX1 gene promoter [16, 17]. Recently, GATA4 and GATA6 binding sites have been discovered in area III, which are critical for pancreas organogenesis [5, 65].
Forkhead transcription factor FOXA2 (formally HNF3β) binds to area II through a consensus FOXA binding site and is required for PDX1 expression . Pancreas-specific disruption of FOXA2 diminishes PDX1 transcription and impairs pancreas formation in mice . These data establish an important role for FOXA2 in regulating PDX1 expression and pancreas development. Several proteins, including PAX6, FOXA1, and FOXO1, have been reported to affect expression of PDX1 through their interactions with FOXA2 [14, 28, 45]. However, the underlying mechanism regarding their relationship with FOXA2 in regulation of PDX1 expression deserves further investigation.
Mammalian SIRT1, a homolog of yeast Sir2, is a founding member of a family of 7 members (SIRT1-7) of NAD+ dependent type III histone and protein deacetylases [15, 19-21, 23, 30, 36, 39, 48]. SIRT1 is indispensable for mammalian development as majority of SIRT1 mutant mice die during embryonic development or early postnatal stages [7, 38, 58]. Tissue specific knockout using a Cre-loxP mediated approach to overcome the early lethality revealed important roles of SIRT1 in calorie restriction, energy expenditure, lipid metabolism, and mammary lobuloalveolar development, as well as neuronal differentiation and neurodegeneration [6, 9, 12, 35, 43, 55, 56]. A study of β cell-specific SIRT1-overexpressing transgenic mice showed that increased dosage of SIRT1 in β-cells improves glucose tolerance and enhances insulin secretion in response to glucose . Other investigations also demonstrated that chemicals that activate SIRT1 activity play protective roles on islet insulin secretion [22, 67]. Recently, a study identified an inherited heterozygous mutation in human SIRT1 that leads to an autoimmune-mediated β cell destruction, and diabetes . However, a role for SIRT1 in β cell development and differentiation remains elusive due to the early lethality of SIRT1 null mice.
In this study, we performed a proteomic analysis and found that SIRT1, FOXA2 and a number of other proteins form a protein complex on the promoter of PDX1 in cultured cells. We further demonstrated that SIRT1 deacetylates FOXA2 and plays a critical role in regulating PDX1 expression. Finally, using the Cre-loxp conditional knockout approach, we showed that the absence of SIRT1 in the pancreas results in impaired β cells formation followed by progressively developed insulin insufficiency.
Floxed Sirt1 gene: Forward- 5' CTT CCT TGC CAC AGT CAC TC 3'; Reverse- 5' CAT CTA AAC TTT GTT GGC TGC 3'. Pdx-Cre: Forward- 5' CCT GTT TTG CAC GTT CAC CG 3'; Reverse- 5' ATG CTT CTG TCC GTT TGC CG 3'. All experiments were approved by the Animal Care and Use Committee of the National Institute of Diabetes, Digestive and Kidney Diseases (ACUC, NIDDK).
All the transfections were carried out with LipofectamineTM 2000 (Invitrogen). pUse-Sirt1 is from Upsate. pCruz-SIRT1 and pCruz-Sirt1(HY) were used previously . shRNA against mouse SIRT1(pBSU6-Sirt1) was constructed as described previously . GFP-FOXA2 expression vector was previously described .
The biotin-labeled wild-type (WT: [Biotin 5] ACCAGAAGTGCTAAGCAAACATCCTGGGGTG) or mutant (MT: [Biotin-5] ACCAGAAGTGCTAAGCCCCTATCCTGGGGTG) oligo of FOXA2 binding site on Pdx1 promoter was used for biotin pull down assay with the nuclear lysates from 20 million GFP-FOXA2 transfeced 293HEK cells. The biotin-bound streptavidin beads were extensively washed with 500 mM KCl and loaded onto 4-12% Tris-SDS PAGE. The gel was later stained with Silver Staining (SilerQuestTM Staining Kit, Invitrogen). The bands were excised, distained, and digested with trypsin. The tryptic peptides were analyzed by nanoflow reversed-phase liquid chromatography (nanoRPLC) mass spectrometry using a linear ion-trap (LIT) mass spectrometer (LTQ, ThermoElectron, San Jose, CA). The LTQ was operated in a data-dependent mode in which the seven most abundant peptide molecular ions in every MS scan were sequentially selected for fragmentation and acquisition of MS2 spectra. Tandem mass spectra were searched against the human protein database using SEQUEST software (ThermoFinnigan, San Jose, CA). A similar in-gel digestion approach was also used to analyze the FOX2A gel band following in vitro deacetylation by SIRT1. Acetylation of lysine (+42.0106 Da) and oxidation of methionine (+15.9949 Da) residues were included as dynamic modification when the MS2 spectrum was searched against the human FOXA2A protein sequence. The SEQUEST filtering criteria for initial identification of peptides were cross-correlation (Xcorr) scores of 1.9 for [M + H]1+, 2.2 for [M + 2H]2+, 3.1 for [M + 3H]3+ ions; and a minimum delta correlation (ΔCn) of 0.08. The MS2 spectra of eligible peptides were manually inspected to confirm the identification of correct peptide sequence and modifications.
To validate the Mass Spectrometry data, pulldown with either WT or MT biotinylated oligos was performed and Western blot analysis was carried out with antibodies against individual protein. The following antibodies were used: PARP1 (Millipore), KU70 (Santa Cruz), KU80 (Abcam), and GFP (MP).
Twenty million 293T cells were co-transfected with myc-p300 and GFP-FOXA2 or one of the flag-FOXA2 (wildtype, K226R, K275R or K226R/K275R). The transfectants were then treated with either TSA (5μM) or NIM (20mM) or both for 8 hours before harvest. The cellular lysates were immunoprecipitated with GFP antibody, Ac-K antibody, or flag antibody respectively. Generally, 2-3 mg nuclear lysates were used. For flag-tagged protein, after being immunoprecipitated with flag antibody (Sigma, M2), the beads were eluted with 3Xflag peptide (Sigma) and eluates were loaded onto 10% Tris-glycine SDS PAGE for Western blot with Ac-K antibody. When testing SIRT1 function on FOXA2 acetylation, myc-p300 and GFP-FOXA2 plasmids were co-transfected into 293 HEK cells, together with either GFP or SIRT1 or SIRT1 (HY).
Flag-tagged SIRT1 or flag-tagged SIRT1(HY) was transfected into 293T cells. The flag-tagged FOXA2 was co-transfected with myc-p300. The cells were lysed in buffer (20mM Hepes, pH7.9, 180mM KCl, 0.2mM EGTA, 1.5mM MgCl2, 20% glycerol, 0.1% NP40) with 0.5uM TSA and 10mM Nicotinamide (NIM). The flag-tagged proteins were then immunoprecipitated with anti-Flag M2 agarose beads (Sigma) and eluted with 3X flag peptide. Afterwards, the purified proteins were suspended with 20ul deacetylation buffer (20mM Hepes, pH7.9, 67mM KCl, 5mM MgCl2, 0.2mM EDTA) with or without NAD+ (Sigma), incubated at 30 °C for 2hr, and then reactions were stopped by adding 2X loading buffer. After electrophoresis, the membrane was immunoblotted by anti-Ac-K antibody, followed with M2 antibody; then the gels were stained with Coomassie blue and processed for mass spectrometry analysis.
To mutate the lysine residues that are acetylated in FOXA2, flag-tagged murine FOXA2 plasmid in a modified pCMV vector was used as a parental template. The PCR method (PCR SuperMix high fidelity, Invitrogen) was applied and mutation was confirmed with sequencing. Mutagenesis was carried out by BioInnovatise Inc. (Rockville, MD 20852). The primers are as follows: Foxa2F1: ggtggtggatccatgctgggagccgtgaagatgg; Foxa2R1: GGTGGTAAGCTT TTA GGA TGA GTT CAT AAT AGG; K226F1: cccgctcgccagacaggcctggcaagggctcc; K226R1: GGA GCC CTT GCC AGG CC T GTC TGG CGA GCG GG; K275F1: TGCGGC CAGTAGCGGA GGCA GGAG GA CCGCTCCTGG GTCCCAG; K275R1: CTG GGA CCC AGG AGC GGT CCT CCT GCC TCC GCT ACT GGC CGC A. "Red color" indicates mutant nucleotides.
Mice were maintained under fed condition with regular chow. At different ages, glucose was measured with ContourR glucose meter (Bayer) and ContourR blood glucose test strips (Bayer).
Serum was used for insulin measurements with either a Rat Insulin ELISA Kit (Crystal Chem, Inc.) or an RA method performed by the Metabolic Core Facility of NIDDK.
Mice at different ages were euthanized with CO2. Collagenase Type V from Sigma was perfused into pancreas. The entire pancreas was collected, and digested at 37°C. Islets were purified by Ficoll gradient (Ficoll-paqueTM Plus, GE Healthcare), and hand-picked using a microscope.
Total RNA was isolated with STAT-60TM (Tel-Test, Inc.) from islets or MIN6 cell. cDNA was synthesized with Cells-to-cDNA TMII (Ambion, Inc.). Primers used are shown in the Supplementary (Additional file 1).
Western blot was carried out with Licor (Lincoln, Nebraska) using antibodies against SIRT1 (Upstate), FOXA2 (Santa Cruz), acetyl-lysine (Chemicon and Abcam). Immunoprecipitation was carried out as described previously .
Pdx-PH2/pGL3B was obtained from Addgene. The plasmid was transfected into MIN6 cells or beta-TC6 cells either alone or together with SIRT1, FOXA2, FOXA2 mutant alone or both. The luciferase activity was assessed with the Dual-LuciferaseR Reporter Assay System (Promega).
The entire pancreas was collected, flattened, and fixed in 10% formalin. The fixed pancreas was embedded with paraffin. Five μm sections were cut with MICROM (HM 340E). The sections were stained with H&E, or processed for immunohistochemistry or immunofluorescence. The antibodies used are: anti-Sir2a (Millipore), anti-glucogon and anti-insulin (Sigma), anti-proinsulin (R&D system), anti-somastatin (MP), anti-pancreatic polypeptide (ICN), anti-Glut2 (Chemicon), anti-Pdx-1 (a gift from Christopher Wright, Vanderbilt University), anti-F4/80 (Serotec), anti-MPO (Biocare Medical), anti-NF-κB (Santa Cruz), anti-CD3 (Dako), anti-IL6 (Becton Dickenson), anti-TNFα (R&D), anti-IL1β (R&D). The immunohistochemistry was performed with HistomouseTM SP (Invitrogen). The secondary antibodies for immunofluorescence staining were from Invitrogen and Sigma.
Student's t-test is used for statistical analysis. ±SD is reflected in corresponding panels as bars.
To identify potential factors involved in regulating Pdx1 expression through the interaction with FOXA2, the consensus FOXA2 binding site in the region of PH2 domain of Pdx1 promoter was utilized for a biotin pull-down assay. A GFP-FOXA2 or FLAG-FOXA2 construct was expressed in either HEK 293 cell line that was originally derived from human embryonic kidney or Min6 cell line, a pancreatic beta cell line derived from a transgenic mouse expressing the large T-antigen . The nuclear lysates from the transfectants were used for a pulldown analysis with biotinylated wild-type and mutant oligos for the consensus FOXA2 binding site in the PH2 domain (Fig. 1A). Wild-type oligo precipitated several distinct protein bands, while mutant oligo did not pull down any of these proteins (Fig. 1B). Analysis of the differential bands by mass spectrometry identified several proteins, including PARP1, HLTF1, KU70, KU86, SSBP, and SIRT1 (Fig. 1B). Individual oligo pull-downs and Western blots by antibodies against PARP1, KU70, KU86 and SIRT1 confirmed their interaction with the wild-type oligo in both 293 and Min6 cells (Fig. 1C-D). Once the consensus FOXA2 binding site was mutated, SIRT1, PARP1, KU70, KU86 and FOXA2 disappeared together, indicating that the binding of SIRT1, PARP1, KU70, and KU86 to Pdx1 promoter is mediated by FOXA2 recognition sequence.
Two of the proteins identified as part of the complex, PARP1 , and KU70  were reported to interact with SIRT1 as substrates. A third protein, KU86, could be acetylated as well although the corresponding acetyltransferase was not identified . Therefore, we chose to study SIRT1's relationship with FOXA2 and PDX1 expression.
Using reciprocal immunoprecipitation, we confirmed the interaction between SIRT1 and FOXA2 in both 293T cells (Fig. 1E) and Min6 cells (Fig. 1F), suggesting FOXA2 could be one of the SIRT1's substrates. Next, we tested whether FOXA2 is acetylated and whether SIRT1 could deacetylate FOXA2. 293T cells were transfected with p300 and FLAG-FOXA2. Then FLAG-FOXA2 was pulled down with anti-FLAG M2 antibody and western blotted with anti-acetyl lysine antibody. The reverse way was also performed with the same lysates. As shown in figure 1G, FOXA2 was acetylated in the presence of p300 and the level of acetylation is correlated with the amount of FOXA2. The acetylation of FOXA2 was enhanced in the presence of nicotinomide (NIM) (Fig. 1H), indicating that class III HDACs are involved. Wild-type SIRT1, but not a deacetylase deficient mutant form of SIRT1, was able to deacetylate FOXA2 (Fig. 1H). A similar acetylation pattern was observed when purified FLAG-FOXA2 and SIRT1were added together into a test tube in the presence or absence of NAD+ (Fig. 1I). Only in the presence of NAD+, was wild-type SIRT1 capable of taking off the acetyl group from FOXA2. The deacetylase deficient mutant of SIRT1 (SIRT1-HY) was unable to catalyze the removal of acetyl group from FOXA2 (Fig. 1H-I). Mass spectrometry (MS) analysis identified two potential acetylation sites within FOXA2 (i.e., K226 and K275). K226 resides in FOXA2 DBD domain, and K275 is at the junction between Wing 2 and NLS (Nuclear localization sequence). K226 and K275 are conserved in both human and mouse FOXA2 (Fig. 1J). Site mutagenesis experiments were performed to mutate these two lysine residues (K) to arginine residues (R). Three FOXA2 mutants were generated (FOXA2K226R, FOXA2K275R and FOXA2K226R/K275R=DM). In Min6 cells, mutation of both sites to arginine residues completely abolished acetylation of FOXA2 by p300 (Fig. 1J). Mutating each lysine residue individually does not have an obvious effect on the acetylation status of FOXA2 (Fig. 1J), confirming the identification of only two p300-dependent acetylation sites within FOXA2 by MS under our experimental condition.
Min6 cell line was applied to study the relationship between SIRT1 and FOXA2 further. Acute knockdown of Sirt1 resulted in significant decrease of Pdx1 and a number of its downstream genes, including insulin, glucokinase (Gk) and Glut2 (Fig. 2A). Of note, overexpression of SIRT1 in these cells resulted in only a slight up-regulation of Pdx1 (Additional file 1: Supplementary Fig. 1A, B), suggesting that the amount of SIRT1 is not a rate-limiting factor for the expression of Pdx1 in these Min6 cells.
To investigate whether SIRT1 binds to Pdx1 promoter, we performed a chromatin immunoprecipitation (ChIP) assay and found that SIRT1 occupies the Pdx1 promoter near FOXA2 binding site (PH2) (Fig. 2B) . Using a luciferase reporter for Pdx1 transcription, we found that expression of FOXA2 and wild type SIRT1, but not SIRT1-HY, synergistically induced Pdx1 reporter activity (Fig. 2C), suggesting that deacetylase activity of SIRT1 is required for this action. siRNA-mediated knockdown of Sirt1 also reduced induction of Pdx1 promoter activity by FOXA2 (Fig. 2D), indicating that SIRT1 is required for FOXA2 to further elevate Pdx1 promoter.
Next, we studied whether K226 and K275 in FOXA2 play a role in SIRT1 induction of Pdx1 expression. Expression of FOXA2 K226 and/or K275 mutants in min6 cells were able to induce the Pdx-luc activity close to the level where SIRT1 and wild type FOXA2 combination could get (Fig. 2E). Of note, expression of SIRT1 does not further increase Pdx-luc activity on the top of FOXA2 mutations (Fig. 2E). Since arginine mutation mimics deacetylated form of lysine, these data is consistent with a role of SIRT1 deacetylating FOXA2 on these sites to increase its transcriptional activity on Pdx1 promoter.
With these data, we rationalized that mutation of lysine 226 and 275 to arginine enhances FOXA2 binding to Pdx1 promoter. To test this hypothesis, FOXA2 or its different mutants were co-transfected with SIRT1 into min6 cells, and ChIP experiments were performed with these transfectants (Fig. 2F). In the presence of SIRT1, FOXA2 binds to Pdx1 promoter stronger compared with FOXA2 alone (p<0.001). K226R or K275R single mutation enhanced the binding of FOXA2 to Pdx1 promoter (p<0.01). When both K226 and K275 were mutated to arginine (FOXA2 DM), FOXA2 DM achieved the highest binding to Pdx1 promoter. The binding synergy between SIRT1 and FOXA2 diminished when lysine to arginine mutants were applied. This evidence underscores the importance of SIRT1 and lysine sites K226/K275 in maintaining transcriptional activity of FOXA2 on Pdx1 promoter.
To assess the potential impact of SIRT1 deficiency on β-cell formation and pancreas development, a pancreas-specific disruption of SIRT1 using a Cre-LoxP mediated approach was performed (Additional file 1: Supplementary Fig. 2A). The Sirt1 floxed mice were crossed with transgenic mice carrying a Cre gene under the control of a Pdx1 promoter (Pdx1-Cre)  to achieve pancreas-specific knockout of SIRT1 (Sirt1PKO) (Supplementary Fig. 2B-C).
The Sirt1PKO mice appeared normal during early postnatal developmental stages. At 14 days of age, all Sirt1PKO mice examined had a normal range of glucose. However starting from 3 weeks (P21), when the mice were weaned, 6 out of 41 (15%) of Sirt1PKO mice displayed a significant increase in glucose (Fig. 3A). Over time, the number of mice with hyperglycemia significantly increased. By 20 weeks of age, 60% of Sirt1PKO mice were hyperglycemic. Many of these Sirt1PKO mice died at later stage. This contributes to the high mortality rate of Sirt1PKO mice over 16 weeks of age (Fig. 3B).
Dynamic changes in blood glucose levels of a cohort of 23 Sirt1PKO mice were followed up to 4 months of age. Of these mice, 5 at 3 weeks, 7 at 6 weeks, and 14 at 16 weeks displayed hyperglycemia (blood glucose levels >200 mg/dL) (Fig. 3C, Additional file 1: Supplementary Table 1A). In contrast, the blood glucose level remains almost steady in the wild-type control group (Fig. 3C, Supplementary Table 1B).
To understand the cellular and molecular basis for the observed hyperglycemia, we analyzed Sirt1PKO mice, irrespective of whether or not they were hyperglycemic. Analysis of mice at P21 indicated that hyperglycemic Sirt1PKO animals had an average of 8 islets, with the blood glucose concentration reaching over 600 mg/dL. The average number of islets of normaglycemic Sirt1PKO was 68, about half the number found in wild-type controls (Fig. 3D).
A protein complex formed in the promoter of Pdx1 revealed by oligo pulldown and proteomics. (A) Wild-type and mutant oligo sequences that were used for biotin pulldown. In wild-type oligo, AAAC is the consensus site for FOXA2 binding. In mutant oligo, AAAC is mutated to CCCT. (B) A silver stained picture from GFP-FOXA2 pulldown sample is shown. (C-D) Biotin pulldown validation was performed in 293T (C) and Min 6 (D) cells using nuclear extracts that over expressing FLAG-FOXA2 to confirm the identity. (E-F) Reciprocal immunoprecipitation to show SIRT1 and FOXA2 interact with each other. Input: 10% of total protein. (E) 293T cells with overexpression of Flag-FOXA2. (F) Min6 cells with endogenous protein lysates. (G) FOXA2 is acetylated by p300. 293T cells were transfected with FLAG-FOXA2 and p300, then the lysates were immunoprecipated with anti-FLAG antibody, western blotted with either FOXA2 antibody or Ac-K antibody. (H) Treatment of NIM or SIRT1 mutant (SIRT1(HY)) can enhance the acetylation level on FOXA2. (I) Wild-type SIRT1deacetylates FOXA2 in vitro. Purified FLAG-FOXA2 and SIRT1 or SIRT1(HY) were added together into a test tube with or without NAD+. (J) Mutation of K226 and K275 completely abolished the acetylation on FOXA2 in Min 6 cells.
Regulation of Pdx1 transcription by SIRT1 and FOXA2. (A) shRNA-mediated knockdown of Sirt1 in Min6 cells reduces expression of genes that are involved in pancreas development and insulin production. *p<0.01. (B) Both SIRT1 and FOXA2 bind to the PH2 domain of the Pdx1 promoter revealed by qChIP. Arrows show the positions of primers used in the assay. (C) Wild-type SIRT1 (T1) and FOXA2 (A2) synergistically elevate the luciferase activity driven by the PH2 domain of the Pdx1 promoter. Insert shows expression of SIRT1 and FOXA2. (D) Knockdown of Sirt1 reduces the activity of FOXA2 on Pdx1 promoter. Insert shows level of SIRT1 knockdown. (E) Mutation of two lysine residues in FOXA2 abolished the synergistic effect of FOXA2 and SIRT1 on Pdx1 promoter, but improve the FOXA2 transcriptional activity. All the transfection contained the same amount of DNA, GFP plasmid was used to make up the total DNA amount. (F) Mutation of two lysine residues in FOXA2 enhances the binding ability of FOXA2 to Pdx1 promoter assessed by ChIP.
Sirt1PKO mice display hyperglycemia and glucose intolerance. (A) Percentage of Sirt1PKO mice that contain high blood glucose level (>250 mg/ml) during development. Number of mice is show on the top of each bar. (B) Survival curve of Sirt1PKO mice during 4 months after birth. (C) Summary of blood glucose levels in animals at different ages. Twenty-three Sirt1PKO animals and twelve control mice were followed for their glucose level during period of P14 to 4 months. (D) Islet number and glucose level of P21 animals. Each group contains 4-6 animals. * p<0.001.
We suspected that phenotypic variation might be due to incomplete Cre-mediated recombination, which is always the case based on our experience on Cre-loxP mediated disruption of many other genes in various organs/tissues [34, 57, 62, 64]. Indeed, Western blot against SIRT1 from different genotypes of pancreases demonstrated that hyperglycemic Sirt1PKO mice contain much less SIRT1 compared with normaglycemic Sirt1PKO mice (Additional file 1: Supplementary Fig. 2C). Thus, the severity of SIRT1 mutant phenotype directly correlates with the extent of SIRT1 deletion, highlighting a critical role of SIRT1 in regulating and maintaining normal development of islets. Therefore, our further study focused on the hyperglycemic Sirt1PKO mice.
Histological analysis revealed that Sirt1PKO mice had reduced size and irregular shaped islets (Fig. 4A-C). Studying pancreas at P21, apoptosis (TUNEL assay) was not detected in the islets of either Sirt1PKO mice or wild-type mice. The proliferation status was investigated with Ki67 antibody staining. As shown in figure 4D-F, in wild-type pancreas, Ki67 positive cells can be observed in both acinar and islet, but SIRT1 deficient pancreases contain 6-fold reduction of Ki67 positive cells.
It has been shown that over-expression of SIRT1 protects β-cells against cytokine toxicity by suppressing NF-kB signals . Therefore, we examined whether loss of SIRT1 could trigger an autoimmune response that destroys islets. Monitoring immunohistological markers, such as IL-1β, TNFα, IL-6, CD3, F4/80, MPO, NF-kB, did not detect obvious signs of inflammation in the mutant islets (Additional file 1: Supplementary Fig. 3). These data ruled out the possibility that the impaired formation of islets in Sirt1PKO mice is caused by an autoimmune response triggered by SIRT1 deficiency.
PDX1 is a master regulator for islet formation and expression of insulin. Due to the positive relationship between SIRT1 and PDX1 displayed above (Figs. 1, 2), a gene expression analysis was conducted to ascertain the molecular mechanism underlying the phenotype. Realtime RT-PCR analysis revealed that islets from hyperglycemic Sirt1PKO mice had about 5% of Sirt1 mRNA, markedly reduced transcription levels of Pdx1 and a number of Pdx1 downstream genes such as Glut2 and insulin (Fig. 5A). We followed insulin levels in Sirt1PKO mice for 6 months. Hyperglycemic Sirt1PKO mice consistently exhibited very low levels of insulin (Fig. 5B).
Next, we performed immuno-fluorescent staining to examine the impact of SIRT1 deletion on different cell types in pancreatic islets. Consistent with the real-time PCR data (Fig. 5A), a dramatic reduction of SIRT1 protein in the islet of hyperglycemic Sirt1PKO mice was observed compared with wild- type control mice (Fig. 5C). A graded reduction of cells that are positive for PDX1, GLUT2, insulin, and proinsulin was also observed in the islet of these mutant mice (Fig. 5C), suggesting the specific reduction of β-cells.
Notably, α cells (positive for glucagon) became randomly distributed in the mutant islets (Additional file 1: Supplementary Fig. 4A). They also seemed relatively more abundant in mutant islets compared to wild type islets. However, when serum glucagons was measured, no difference was detected between wild-type and Sirt1PKO mice. Conversely, there was no obvious increase of δ cells (positive for somatostatin) (Supplementary Fig. 4B) or PP cells (positive for pancreatic polypeptide) (Supplementary Fig. 4C).
Our in vitro studies illustrated that SIRT1 positively regulates Pdx1 expression through interacting with and deacetylating FOXA2 (Figs. 1 and 2). Using the Sirt1PKO pancreas as a negative control, we now ask whether SIRT1 and FOXA2 interact with each other in vivo. Whole pancreas lysates from wild-type or Sirt1PKO mice at P14 were used to perform a reciprocal CO-IP between SIRT1 and FOXA2. Indeed, SIRT1 and FOXA2 interact with each other in wild type mice. The interactions between these two proteins were not detected in the mutant samples (Fig. 5D-E). We next examined whether FOXA2 is acetylated in vivo at this stage. Whole pancreatic lysates were precipitated with acetyl-lysine antibody and Western blotted with FOXA2 antibody, and ratio of acetyl lysine vs total FOXA2 was quantified. The results indicated that FOXA2 is acetylated and Sirt1PKO samples contain higher level of acetylation on FOXA2 compared with controls (Fig. 5F). Finally, we investigated whether FOXA2 binds to PDX1 promoter in vivo and whether loss of SIRT1 affects its binding by ChIP assay. Clearly, FOXA2 binds to PH2 domain of PDX1 promoter in wild type pancreas, and deletion of Sirt1 greatly reduced this binding (Fig. 5G). Thus, with Sirt1PKO mice model, we demonstrated that SIRT1 and FOXA2 indeed interact with each other in vivo during pancreas development. SIRT1 deacetylates FOXA2 and enhances FOXA2 transcriptional activity on Pdx1 promoter either directly or by affecting FOXA2's ability to recruit other transcriptional activators/repressors, and this process is critical for pancreatic β-cell formation.
Sirt1PKO mice display abnormal islet development. (A-C) H&E staining of pancreas sections is shown from wild-type (A) and Sirt1PKO (B-C) mice. Sirt1PKO animals exhibit various abnormal morphology of the islet. Arrows indicate islets. (D-F) Ki67 staining of pancreas at P21. The pancreas from Sirt1PKO mice has 6 folds reduction of Ki67 positive cells. Arrows indicate the Ki67 positive cells.
Deletion of SIRT1 in pancreas impaired β-cell formation. (A) Gene expression pattern in islets with different genotypes. Each group contains islets from 4 individual animals. * p<0.01. (B) Serum insulin level from animals. *p<0.001. (C) Immunofluorescent staining of the islet. (D-E) Reciprocal immunoprecipitation with whole pancreases lysates shows that SIRT1 and FOXA2 interact with each other in vivo. (F) FOXA2 is acetylated in vivo. The ratio of acetylated FOXA2 (top lane) vs total FOXA2 (second lane) is presented in number at the bottom. (G) FOXA2 binds to PH2 domain of PDX1 promoter in vivo. ChIP assay was performed with a FOXA2 antibody. For experiments in D-G, pancreases from 9 wild-type mice or 13 Sirt1PKO mice at P14 were pooled together to get enough samples for each experiment.
SIRT1 deacetylates many proteins that are implicated in multiple important biological processes, including DNA damage repair, cell cycle checkpoints, metabolic regulation, animal aging, and cancer [11, 13, 20, 21, 23, 30, 36, 48, 55, 56, 66]. Here, our results demonstrate that SIRT1 positively regulates pancreatic β cell formation and differentiation by interacting with FOXA2 and synergistically regulates PDX1 transcription. Since mutation of K226 and K275 to arginines (which mimic the deacetylated form) in FOXA2 abolished the synergistic induction of FOXA2 and SIRT1 on PDX1-Luc reporter, enhanced the binding of FOXA2 to endogenous PDX1 promoter, and increased FOXA2 transcriptional activity on Pdx1-Luc vector in min6 cells, we conclude that SIRT1 activates FOXA2 through deacetylation on K226 and K275. We further showed loss of SIRT1 impairs expression of PDX1 and blocks β-cell formation, leading to severe hyperglycemia.
A recent study showed that FOXA2 acetylation on K259 promotes FOXA2 transcriptional activity to regulate hepatic lipid oxidation in ob/ob and db/db mice . In our study, SIRT1 deacetylates FOXA2-K226 and K275 in both human HEK-293 cell line and mouse pancreas beta cell derived Min6 line, whereas K259 was not detected. Since acetylation on K259 only takes place when fasting or glucogon treatment was applied, and SIRT1 displays the highest activity in fasting condition , it is possible that K259Ac is not a direct SIRT1 target under normal physiological condition. It is also plausible that SIRT1 deacetylates different residues of FOXA2 in different tissue context. Nonetheless, these data picture a complex mechanism in finely tuning FOXA2 transcriptional activity during development, differentiation and metabolism. This issue deserves further investigation in order to fully understand disease progress in liver and pancreas.
Of note, it has been previously shown that overexpression of SIRT1 in β cells in transgenic mice enhances insulin secretion in response to glucose through inhibiting transcription of the Ucp gene . Other study also showed that a transgenic mouse strain with moderate overexpression of SIRT1 exhibits normal insulin sensitivity but exhibits decreased food intake and locomotor activity, resulting in diminished energy expenditure . In cultured β cell lines, SIRT1 represses transcription of the Ucp2 gene by binding directly to the Ucp2 promoter . Here we demonstrate for the first time that SIRT1 is critical in β cell differentiation and maturation, since SIRT1 deficiency inhibits β-cell formation. We further manifested that SIRT1 deacetylates FOXA2 and forms a protein complex with FOXA2 on the promoter of Pdx1 and serves as a potent regulator for expression of Pdx1 during the early stages of pancreas development and islet formation. Of note, our pulldown using the consensus FOXA2 binding site in the PH2 domain revealed a protein complex including SIRT1, FOXA2, PARP1, HLTF1, KU70, KU86, and SSBP on the Pdx1 promoter, it is likely that these proteins form a complex in the promoter of Pdx1. The spatial relationship of each component in the complex might be an interesting topic to be addressed in future. Furthermore, SIRT1 is also a histone deacetylase, it is possible that SIRT1 might affect this protein complex formation through epigenetic modification on histones on the promoter of Pdx promoter. All these issues deserve further studies. Nonetheless, because PDX1 acts as a master factors in the pancreas and regulates expression of many genes that are essential for insulin production and secretion [26, 27, 41], we conclude that the impact of SIRT1 on β-cell formation is primarily through its regulation of Pdx1 expression (Fig. 6).
There is no doubt that Sirt1 transcription is subjected to spatial and temporal regulation. Our previous study showed that the tumor suppressor BRCA1 could positively regulate Sirt1 expression during tumorigenesis . Given the important roles of SIRT1 in numerous biological processes [11, 13, 21, 23, 30, 36, 48], the underlying mechanism for SIRT1 regulation should be an important focus for future investigation. The regulation and activation of SIRT1 in β-cells should serve as a model for bridging islet development, insulin secretion, and glucose metabolism.
An integrated model displays a critical role of SIRT1 in maintaining PDX1 expression and β-cell formation. (A) SIRT1 interacts with, and deacetylates FOXA2 on the promoter of PDX1 and serves as a potent regulator for expression of PDX1 and its downstream genes during the early stages of pancreatic development and β-cell formation. Concurrently, expression of SIRT1 is also subjected to spatial and temporal regulation. (B) Targeted disruption of SIRT1 increases the acetylated form of FOXA2, impairs PDX1 expression, and blocks β-cell formation, leading to hyperglycemia and insulin insufficiency.
We thank Dr. Douglas Melton for PDX1-Cre mice, and Dr. Kenneth S. Zaret for GFP-FOXA2 expression vector. We thank Drs. Oksana Gavrilova, Tyler J. Lahusen, Cuiling Li, and Cuiying Xiao for technical assistance. We also thank the members of the Deng lab for critical review of the manuscript. This research was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA.
Corresponding author: Tel: (301) 402-7225; Fax: (301) 480-1135 Email: chuxiadniddk.nih.gov.


text:
Kidney patients sometimes miss out on their sessions: Kumar
Kelly Vacala
Patients have often missed out on their kidney dialysis treatment due to its exorbitant cost.
Consumer Council Chief Executive, Premila Kumar says before the 2018/2019 budget announcement, a group of these patients visited them raising their concerns on the issue.
Kumar says that this was then part of their submission to the budget and they've commended government for the $3.5million subsidy for kidney dialysis from the new budget.
She says the cost of dialysis has always been a struggle for kidney patients and their families.
"For years they have been suffering and been forking out large sums of money. And sometimes when they don't have enough money than they have to miss a session and just do two sessions per week. I mean for them to get $250 every session and in total of $750 every week, is a lot of money. Most of us don't even earn $750 a week."
Economy Minister, Aiyaz Sayed-Khaiyum had announced that people with household income over $30,000 will be offered dialysis for $150 a treatment and families below the $30,000 threshold will be able to attain the same service for $75.
The Consumer Council says this will greatly ease the cost of dialysis on patients and their families.


Conviva's solutions optimize live and on-demand video streams, providing a personalized HD viewing experience for each and every viewer. Preemptive and automated dynamic bit-rate adjustment and source switching across multiple content delivery networks empowers content owners to maximize the performance of their video while maintaining and protecting viewer, brand, and business relationships.
Online audiences want to watch content as it was meant to be seen... without buffering and at the highest definition possible. When streaming quality is high, viewers watch more than twice as long, but unfortunately this does not happen all the time causing publishers a significant loss in revenues and audience viewing time. Even 1% of buffering results in a loss of almost 4 minutes of viewing time, while higher definition streams show 50% longer engagement.
Conviva is all about improving viewing experience automatically while providing publishers with real-time data to constantly tune all aspects of their video business for optimum quality and profitability.
Imagine knowing what every single viewer is experiencing each second they watch your content. Now imagine a solution that preemptively adjusts each stream to BOTH raise the picture quality AND prevent interruptions like buffering and choppiness. These are the core capabilities of Conviva; Stream optimization (Precision Video) and real-time viewer Analytics (Insights).
Protect each and every viewer. By focusing on each individual viewer's experience and taking unique action to protect the quality of every stream, online video can be even more profitable.
Maximize your digital media investment. Conviva provides content owners actionable data to make the most profitable business decisions and takes preemptive action to provide a high-quality, buffer-free experience that makes a profitable digital media strategy.
Our integration process adds the Conviva libraries to the customer's player in order to capture metrics and improve playback.
Pricing depends on a variety of factors. Please reach out to our sales team.


So I've finally got the revised version of The Uncanny X-Men Chapter 4 up. However, that took longer than I expected and as such, everything else has been delayed somewhat. The next chapter of The Amazing Spider-Man won't be up until tomorrow.
Second Revised Chapter of Uncanny X-Men is Up!
The First Chapter of Amazing Spider-Man is up!
Goddamn, two fanfic chapters in one day? I've never been that productive.
Come and check it out! The Amazing Spider-Man!
Revised First Chapter of The Uncanny X-Men Is Up!
Short post today. I'm getting set up on AO3, and as such I'm going to be re-writing the entries in The Uncanny X-Men. Nothing major, but there's ideas and concepts that didn't get into the previous versions I did and I want to add them. Also there are some characterization mistakes that I made that need to be corrected.
As the entries are re-written, I will be re-uploading them to both Tumblr and Fanfiction.net. So keep an eye out!
Hi guys! So I've got some good news: the next chapter of The Uncanny X-Men should be out within the next couple of weeks! Three at the most.
In the meantime, however, I thought I would share some of my thoughts about the upcoming Black Panther movie.
Thing is, I've never read a Black Panther comic and only one, maybe two others with him in it. My exposure to the character has been pretty much limited to Avengers: Earth's Mightiest Heroes and Captain America: Civil War. So this is about as close to the point of view of the general audience who have never heard of Black Panther before as you're going to get.
Tomorrow, My Thoughts On Black Panther!
So I'm still working on my analyses of Universal Horror films. Given how my work on Dracula (1931) turned out, however, I think that those analyses need something of re-think. At least in how I do them.