text:
Industry Organization
Vietnam's oil and gas industry is currently the country's biggest foreign currency earner and a major procurer of imported technology, services and equipment. In 2009, Vietnam produced 24.3 million tons of oil equivalent, and earned $14.24 billion in revenue, accounting for about 16 percent of GDP; and exported 16.3 million tons of crude oil, worth $7.82 billion, accounting for 14 percent of total national exports. Oil and gas is one of the top priority sectors for development by the Government of Vietnam since it is viewed as central to national economic growth and energy security. The oil and gas industry in Vietnam is under the principal jurisdiction and management of the Ministry of Industry and Trade (MOIT). PetroVietnam, the national oil and gas group reports directly to the Prime Minister and holds a monopoly in the upstream, mid-stream and virtually all key downstream areas of the industry. In 2009, PetroVietnam maintained the No. 1 rank in Vietnam's Top 500 biggest companies. In 2009 alone, the taxes paid by PetroVietnam accounted for 24 percent of Vietnam's state budget.
Upstream and Midstream
Vietnam is currently ranked fourth in Southeast Asia after Indonesia, Malaysia, and Brunei for oil and gas production and oil reserves. Vietnam's continental shelf is about one million km2, comprising major tertiary basins and groups of basins: Song Hong, Phu Khanh, Cuu Long, Nam Con Son, Malay-Tho Chu, Tu Chinh-Vung May, Hoang Sa (the Paracel Islands) and Truong Sa Group (the Spratly Islands). Of these, the Cuu Long and Nam Con Son basins have shown the most hydrocarbon potential.
To date, about 100 hydrocarbon-bearing prospects have been found in almost 50 fields, with estimated reserves of approximately 600 million tons of crude oil and 644 billion cubic meters (bcum) of natural gas (23 trillion cubic feet -Tcf). Among the 50 structures with oil and gas discoveries, there are 20 commercial fields.
From 1988 to 2008, PetroVietnam signed 60 oil and gas exploration and production contracts with foreign companies in the form of Product Share Contracts (PSC), Business Cooperation Contracts (BCC), Joint Ventures (JV) and Joint Operation Companies (JOC). Of these, 35 contracts are currently in effect. In 2008, PetroVietnam entered into agreements for 16 projects in foreign countries, consisting of 6 projects in Southeast Asia, 6 projects in the Middle East and Africa, and 4 projects in South America. In 2009, PetroVietnam signed 13 contracts with local partners, 2 contracts with foreign ones, and 7 cooperation agreements with the national oil and gas companies of Nicaragua, Bolivia, Argentina, Kazakhstan, Mozambique, Angola and Sudan.
In 2009, the country produced 24.3 million tons of oil equivalent, comprising 16.3 million tons of crude oil and condensate and 8 billion cubic meters of gas, an increase of 8 percent compared to the company's 2008 production. In the medium term, oil production is expected to decline gradually due to the deteriorating performance of the White Tiger field while other new discoveries will not offset this loss in production. Gas production, however, will rise significantly since several gas fields will be put in production in the near future. At present, about 85 percent of the natural gas produced in Vietnam is used for power generation, 10 percent for fertilizer and the remaining 5 percent for industries and households. Gas is transported via a network of gas pipelines from offshore gas fields to onshore processing facilities and power complexes
In 2009, three new oil wells were put into production including Bunga Orkid, Nam Rong-Doi Moi and Dong Rong. In 2010, Petrovietnam plans to start drilling six new oil wells. However, PetroVietnam has also invested $600 million in 21 overseas projects. Along with three gas pipelines Bach Ho, Nam Con Son and Ca Mau, PetroVietnam is building the fourth pipeline from the Thai frontier to Can Tho Province's O Mon and also the second Nam Con Son pipeline to drill gas in West Ocean and Nam Con Son basin. Cite the Chevron deal here. Vietnam's domestic demand for crude oil and gas in the future is expected to increase, especially as the country expands refinery capacity.
After an eight-year delay, in November 2005, PetroVietnam started construction of its first oil refinery at a cost of $3 billion with a capacity of 6.5 million tons per year in Dung Quat, Quang Ngai Province (central Vietnam). Operational as of February 22, 2009, the Dung Quat refinery uses local feedstocks from the Bach Ho field and imports from the Middle East. Dung Quat refinery achieved 100 percent capacity in January 2010. Petro Vietnam plans to raise the capacity of Dung Quat Refinery from 6.5 million tons to 10 million tons.
In April 2008, Vietnam began the construction of the $8 billion Nghi Son Refinery and Petrochemical complex in Nghi Son, Thanh Hoa Province in the North. The complex is under construction and will be able to process 8.4 million tons of crude oil per year when it is scheduled to come on-stream in 2013.
The third refinery, scheduled to be constructed in 2010 and operational in 2014, is the Long Son Refinery Ba Ria-Vung Tau Province in the South. Long Son Refinery needs investment capital of $5 billion and will be able to refine 10 million tons of crude oil per year.
Best Prospects/Services
Under its WTO commitments, the Vietnamese government has opened its oil and gas sector to foreign companies, which it hopes will bring in capital, expertise and technology to help achieve the country's major industry goals. Over the last few years, the government has made visible efforts to make the sector more attractive to foreign companies, by enacting the amended Petroleum Law in 2008, instituting management reforms at PetroVietnam, and cracking-down on business corruption. Foreign oil and gas companies active and successful in Vietnam include ConocoPhillips, British Petroleum (BP), Chevron, Talisman, KNOC (Korea), ONGC Vadesh (India), Idemitsu (Japan), Zarubezneft (Russia), and Petronas Carigalli (Malaysia).
In December 2009, the Vietnamese Government issued Decree 115, essentially supplementing the 2008 Petroleum Law and the 2001 Petroleum Bidding Regulation. The decree outlines the kinds of entities that are allowed to conduct business in the petroleum sector, and confirms that only PetroVietnam and its subsidiaries may conduct exploration activities as primary contractor. The decree outlines licensing processes, tax issues and bidding regulations. A number of decrees delineate surcharges on profit oil and other regulations for this sector, and U.S. entities in this field are encouraged to engage competent legal counsel as they develop their market-entry strategies.
According to Vietnam's Oil and Gas Master Plan toward 2015 and vision to 2025, the industry will require an investment of $203 billion to achieve the goals set forth by the government for the 2006-2025 period, in which PetroVietnam's share will be $81.54 billion (accounting for 40 percent).
These policies, as well as the recent positive developments in the oil and gas sector, have generated a steadily increasing demand for equipment and services that will continue in the years to come.
American technologies, expertise and experience are well respected in the oil and gas industry in Vietnam. U.S. companies are highly competitive in supplying sophisticated equipment, advanced technologies and professional services to both new and existing projects.
Significant business opportunities for U.S. companies exist in the upstream, midstream and downstream segments of the oil and gas industry. More details on investment opportunities and a list of specific upcoming projects in the oil and gas sector will be provided to U.S. companies upon request. Potential buyers include PetroVietnam and its subsidiaries, joint ventures and affiliates, as well as foreign oil and gas companies operating in Vietnam, which normally prefer sourcing from the U.S., Europe and Japan.
Read the full market research report


text:
Pair charged over shop blaze
Police are investigating two separate fires in Bundaberg last night.
Two men have been charged with arson over a fire at a shop about 6:30pm (AEST).
The 18 year old and 21 year old are due to face the Bundaberg Magistrates Court today.
Meanwhile, an investigation is underway into a house fire at Branyan in the city's south-west.
Fire crews were called to the house about 7:30pm last night.
No-one was injured but the building was destroyed.
Topics: courts-and-trials, fires, industrial-fires, residential, arson, bundaberg-4670

subset: commoncrawl


text: To get from Rieti to Udine by train you can catch one of the 10 trains that everyday travel between these two cities. The first train to Udine leaves from Rieti at 06:10 and the last train departs at 21:22. If you choose a local train the trip lasts on average 07:24 hours. For your trip from Rieti to Udine by train you can choose to leave from one of Rieti main stations: Rieti On Wanderio you can compare all schedules, fares and railway companies to Udine and book the best itinerary starting from Rieti.


text:
Project Lifting Device for Dismantling Nuclear Power Plant
Our current project "Lifting Device for Dismantling Nuclear Power Plant" includes developing a technical solution for handling radioactive leavings from nucelar fuel rods and other contaminated reactor components.
The project is co- financed by the european EFRE found.
Turn over machine for plates
Machine for 180 degree turning of heavy plates up to 30t
Vacuumchambers for Electrobeam Welding Machines
Vacuumchamber, equipped with transport devices and auxiliary gear
Steelworks and rolling mill technology
Finishing linesTransport devicesManipulators
Mechanical auxiliaries
Dressing and straightening lines
Tilting devices
Vacuum chamber for Hahn-Meitner Institute (HMI) Berlin
Equipment for Electrobeam Welding Machines
Vacuum chambers for large scale Electrobeam Welding MachinesHandling equipment, appliancesAuxiliary equipment


text:
Our reputation as a law firm comes from the hard work and dedication put forth by both our staff and commitment of Top Anaheim criminal attorney Spencer Seyb. Our ratings by previous clients speak for themselves and we want to help you. We have experience in all types of criminal cases in Anaheim and in Orange County. Our staff is ready to help you 24 hrs.
Our Top Anaheim criminal defense attorneys are easy to reach day or night and have handled many DUI and criminal cases involving very serious crimes as well as misdemeanor crimes in Orange County.
Our Anaheim DUI lawyers are available 24 hours a day to help you defend your freedom from a DUI charge or conviction in Anaheim or anywhere in Orange County. If you have been arrested or charged with a DUI crime in California, it is important you consult with an attorney and more importantly kindly decline discussing any details with the arresting Police officer in Anaheim.
We offer a flat fee representation, payment plan and a free consultation to all potential clients.
Our Anaheim criminal defense attorneys can represent you for any type of felony or misdemeanor charge in Orange County. We have defended many clients with success and have been able to get charges dropped, dismissed, and reduced. In any criminal defense case, details play a key role in a successful defense. Call us 24 hours if you have been charged with a crime.
When it comes to quality representation for our clients, our top criminal lawyers are affordable and aggressive, detail oriented and will provide each client with the dedication that they deserve. You will have someone on your side to defend your freedom.
Anaheim Criminal Lawyers provide criminal defense representation for individuals who have been charged with a crime whether it be a felony or misdemeanor crime in Anaheim and the entire Orange County Area. Our goal is to work diligently for our clients and do our absolute best to obtain a case dismissal, or earn a lesser plea resulting in reduced charges on behalf of our clients. When you retain our Anaheim criminal attorneys, you are hiring a legal team that cares about their clients, and the one on one attention your case deserves. If you are in a situation where you are at risk of going to jail or have been charged with serious crimes including sex crimes, assault, weapons or guns charges, and / or homicide, our team of Anaheim criminal defense lawyers can be of assistance to you. Your freedom matters, call our law office 24 hrs a day at 949-565-2929.


text:
Article Preview
Mother Hysterical at Boy's Bier
More than 300 persons filed past the body of James Powell yesterday afternoon in Harlem.
July 19, 1964
Thank you for visiting The New York Times archive.
New York Times subscribers* enjoy full access to TimesMachine—view 129 years of New York Times journalism, as it originally appeared. 99¢ for your first 4 weeks.
Or, purchase this article individually for $3.95 and download a high-resolution PDF.
Buy Article
* Does not include NYT Now or Premium Crosswords subscribers.
Already a subscriber? Log in to view this article »


text:
Season 2, Episode 1 There's Got to Be a Morning After
First Aired: September 1, 1994
Max and Kyle (Erika Alexander, T.C. Carson) are mortified that they spent the night together and wish they could turn back the clock. Overton: John Henton. Khadijah: Queen Latifah. Terrence: Cress Williams. Regine: Kim Fields.
Guest Cast
Cress Williams Terrence
Rolando Molina Waiter
Cast & Details See all »
- Premiered: August 1, 1993
- Rating: TV-PG
- Premise: The lives and loves of a magazine editor and her roommates living in a Brooklyn brownstone, with plotlines revolving around their ever-changing relationships with neighbors, coworkers and boyfriends. Winner of two NAACP Image Awards. (more)
Trending TonightSee all »
Doubt8:00 pm | CBSI'll Watch111 Watching
Orphan Black10:00 pm | BBCI'll Watch84 Watching
Still Star-Crossed10:00 pm | ABCI'll Watch72 Watching
Living Single: Season 1
By WarnerBrothersBuy New: $11.99
TV Female Foursomes and Their Fans: Featuring the Golden Girls, Designing Women, Living Single, Sex and the City, Girlfriends, Cashmere Mafia, and...
By McFarlandBuy New: $20.05
The Concise Bachelor's Manual: The Gentleman's Guide to Separation, Divorce and Living Single: Including Handy Checklists
By CreateSpace Independent Publishing PlatformBuy New: $9.95
Popular Shows
Popular Movies
- 1. War Dogs
- 2. Pete's Dragon
- 3. Sausage Party
- 4. Suicide Squad
- 5. Star Trek Beyond
- 6. Jason Bourne
- 7. Captain America: Civil War
Popular Celebrities
- 1. Rami Malek
- 2. Taylor Schilling
- 3. Constance Zimmer
- 4. Emilia Clarke
- 5. Tom Hiddleston
- 6. Kit Harington
- 7. Gina Rodriguez


text:
A car DVD player will add hours of entertainment, not only for your kids and passengers, but also for the many people that are following behind you! A cell phone video clip system might also put resale price for your automobile, so there are several advantages of getting a auto electronic digital movie disc competitor. Needless to say, the absolute primary benefit needs to be the quiet calm atmosphere that surrounds your vehicle after the movie starts out along with the children rest with the mouths sealed. Silence is great. Cellular Digital video disc gamers are great for lengthy excursions throughout the united states or simple versions only a few municipalities more than. These competitors provide anything to do for people individuals vacationing along with you. auto radio
- The DIN just covers the height and the width of the car DVD, however. The best tactic.
- There are many different variety cell phone DVD.
- car roof mount monitor.
There are numerous assortment mobile phone DVD gamers out there now. Brand names such as Coby, Sony and Boss and Toshiba are one of the ideal athletes out there. These players have many choices such as over head dome lamps, AV inputs, and wifi potential for wireless network headsets. If your main reason for purchasing an overhead car DVD player is for the children, then you will definitely want to get them wireless headphones. Very quickly, in any way you will certainly be so sick and tired of ability to hear the exact same dvd enjoying repeatedly, therefore the earbuds will prevent you from tugging out your frizzy hair.
Prevent you
Most of the time, an auto Digital video disc participant could have high-quality display screens that array in proportion from 9 inches to in excess of 11 in .. Many participants will play CD's and also DVD's, so paying attention to your selected music is achievable. Besides the DVD person, a motor vehicle encompass program will probably be wonderful match to the complete mobile phone online video technique. An auto music system and a motor vehicle video clip system will give you the very best of both media worlds. A process like this might be good when taking a crack on long journeys. You can just draw into the remainder place, place your seating rear, start up a motion picture, and feel as though you are in a cinema when you are relaxing.
Up a motion picture and feel
A lot of expense portable athletes include components for example a tiny remote and an IPOD docking program. It is recommended that you employ a specialized to set up your automobile Digital video disc gamers. You can also try to install it yourself if you have the skill and know how. An overhead DVD player is not the only kind of player available for your vehicle. That's an interesting point to make. There are some which do not must be mounted, the truth is, they can fit across the entrance face and seats the rear passengers. Battery packs function this style of automobile DVD person and many even include an adaptor plug to use in your tobacco cigarette less heavy. This type is the cheaper of the two options, as you would expect. You are able to quickly acquire one for less than $100. Selling prices for the overhead automobile Digital video disc forms will cost you a lot more and definately will change according to the brand name you pay for. These rates vary from $150 to over $500. car rear view camera
Navigation systems DVD vehicle gamer car
Regardless of whether this DVD participant suits your motor vehicle is determined by a couple of things, the DIN proportions of the opening in your dash board and also the degree of your own dashboard.
By a couple of things
DIN is just the German abbreviation for Deutsche Industrie Usual. This is the German Field Normal for all motor vehicle parts and lots of other electrical and mechanical factors. The one that has an effect on you is 1 DIN and 2 DIN and it also talks about the height and width of all the car Digital video disc players and car stereos. LCD Car Monitor
Unit DVD player GPS
If it is long and narrow then it is a 1 DIN hole if there is a larger squarish hole in your dash then you will have a 2 DIN hole. Except if you wish to do some remodeling work with your dash it can be a great idea to find the proper sizing DIN.
Your dash
However, the DIN just covers the height and the width of the car DVD. The ideal approach is to examine the vehicle owner guide to the sizes of your motor vehicle dash and after that pile that facing the dimensions of the car DVD using this itemizing or through the manufacturer's web site. If it is too long then it might not be the best model to choose. Car Electronics
Equipment cameras for cars back view
- Regardless of whether this Digital video disc gamer suits your.
- However, the DIN just covers the height and the width of the car DVD. The best method would.


text:
2 Min Read
DETROIT (Billboard) - Motley Crue's Tommy Lee is not exactly Dr. Feelgood these days. The drummer will be off his kit for several Crue Fest 2 shows after burning his left hand while playing with a set of sparklers.
Sevendust's Morgan Rose "came charging in like a knight in shining armor" to fill in for Lee, according to a note on bassist Nikki Sixx's MySpace site.
Lee, his left thumb and forefinger wrapped in bandages since the accident last week, is still taking part in the shows, coming on first to explain his absence -- "First of all, I suck. I was having fun and I burned myself," he told Saturday's crowd at the DTE Energy Music Theater near Detroit -- then to hand a bottle of Jagermeister to the crowd and, during the encores, play piano on "Home Sweet Home" and thump on a drum with his right hand for "Girls, Girls, Girls."
There's no official word about when Lee will be able to play drums again. Crue Fest 2 runs through September 2.
In his MySpace posting, Sixx noted that "accidents do happen and, yes, frustration does happen to everybody, including Tommy, who would rather play drums (even with a damaged hand) than just about anything in the whole world. But following doctor's orders for a few shows is what we have to (do) right now (or cancel shows). We all wanna say we're sorry Tommy has to sit out a couple shows, but we hope to have him back up on the drums ASAP."
Editing by SheriLinden at Reuters


text:
Disruption in the Payments World
AbstractBoston, MA, USA May 7, 2008
The consumer payments world is in transition. The current euphoric era for many issuers will be disrupted and quite possibly eliminated over the next three years.
Celent expects some major shake-ups to occur in the payments industry in the near future. A new report, , examines the storms brewing that could have dire consequences for many issuers and offers insight into strategies that could help them weather these storms.
First, Celent expects that interchange rates will begin to decline. The evidence is mounting against the issuing business, and lawsuits will continue to add pressure to act. Free rewards programs will be the first real casualty as issuers are forced to reevaluate their card portfolios and justify high-cost programs. In the long run, the card industry will be forced to return to a fee-based system that aligns the cost to the beneficiary. This will likely persuade consumers to seek their free lunch elsewhere, and the door to other payment methods will be opened even more.
Second, Celent expects that a large payments company will throw its hat into the ring and launch an alternative network that will rival MasterCard and Visa. Lastly, credit-only or credit-centric issuers are expected to diversify their payments mix (a necessity during this economic time and consumer trend to debit) and focus on building their debit payment options by way of decoupled debit programs.
As a result of these events, issuers will have many things to consider over the next five years. To hedge against these changing times, Celent recommends the following strategies:
- Diversify the payments mix. Regardless of the actual impact, one thing is quite apparent: banks will have to view their payments package as a whole and formulate a long-term strategy that not only includes multiple payment products but is flexible to work with the changes that lie ahead.
- Scrutinize rewards funding. The way issuers fund their rewards programs should be heavily scrutinized. They need to consider a Plan B if they are funded largely by high interchange rates.
- Rethink the debit card and DDA relationship. Those with weak debit card programs need to consider customer stickiness attributes and the tactics they can use to either make their debit card more attractive or entice their customers to continue to use their cards.
"Without question, the consumer payments world is in transition," notes Ariana-Michele Wittlake, senior analyst with Celent's banking group and author of the report. "Not only will issuers have to revisit their lending strategies, but they will have to grow more innovative in attracting and retaining cardholders as their revenue sources become compromised by competitive threats, lower interchange, and the general consumer trend to consolidate payments to low-cost channels."
In this report, Celent discusses the likely impact interchange, alternative payments and decoupled debit will have on payments, particularly on credit card payments. In addition to this analysis, Celent offers insight into how banks can benefit from these events and how others can hedge their risk from them.
The 48-page report contains 13 figures and two tables. A table of contents is available online.
Members of Celent's Retail Banking research service can download the report electronically by clicking on the icon to the left. Non-members should contact firstname.lastname@example.com for more information.
Celent is a research and advisory firm dedicated to helping financial institutions formulate comprehensive business and technology strategies. Celent publishes reports identifying trends and best practices in financial services technology and conducts consulting engagements for financial institutions looking to use technology to enhance existing business processes or launch new business strategies. With a team of internationally based analysts, Celent is uniquely positioned to offer strategic advice and market insights on a global basis. Celent is a member of the Oliver Wyman Group, which is a wholly-owned operating unit of Marsh & McLennan Companies [NYSE: MMC].
Media Contacts
North America
Michele Pace
Tel: +1 212 345 1366
Europe (London)
Chris Williams
Tel: +44 (0)782 448 3336
Asia (Tokyo)
Yumi Nagaoka
Tel.: +81 3 3500 3023
Table of ContentsBoston, MA, USA May 7, 2008
|Executive Summary
|The Tornado: Interchange
|Rewards -- The Last Straw
|The Aftermath
|Ability to Surcharge
|Right to Discriminate
|Price Caps
|The Likely Outcome
|The Rising Threat of Alternative Players
|Criteria for Success
|Rifts in the Payments Barrier
|Nontraditional Competitors Entering the Fray
|Traditional Incumbents
|Alternative Takes on the ACH
|Online Payments and the ACH
|Potential Impact of Alternative Payments
|Decoupled Debit
|Decoupled Benefits
|Decoupled Challenges
|Current Initiatives


text:
i always seem to catch this guy looking at me from across the room and I don't know what to do so I pretend I don't see him because I don't wanna send the wrong signal. I like him tho. what should I do?
Most Helpful Guy
i agree with what TheIncredibleD and the Anonymous girl said. it might be awkward for you at first but keep eye contact and smile. and try to go talk to him.00|00|0


text:
In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells
- First Online:
- Received:
- Accepted:
- 527 Downloads
The neogenesis of islets from cultured human adult pancreatic tissue has been reported. The islet progenitors have been thought to be ductal cells. Since previous experiments have been 'contaminated' by a number of pre-existing islet cells, we examined their involvement in islet cell neogenesis.
Fresh human pancreatic cells with different purities of islet cells were grown in monolayer culture and labelled with bromodeoxyuridine. Transitional cells were analysed by double immunofluorescence staining. For purified ductal cell culture, pre-existing islets were eliminated on a magnetic cell separation system.
We confirmed that less than 1% of the endocrine cells proliferated, mainly during the first 48 h of culture. However, a 10-fold larger proportion of the cells acquired a transitional phenotype by starting to coexpress the ductal marker cytokeratin 19 (CK19). These cells represented more than 10% of all endocrine cells after 1 day in culture, and 6% at 5 days of culture. Using magnetic cell sorting, we eliminated cells expressing neural cell adhesion molecule (N-CAM), after which we obtained 99.7% pure non-endocrine CK19-rich cell populations. These cell populations could be expanded in vitro. However, their endocrine differentiation capacity was severely reduced as compared with the original mixed cell cultures.
These results suggest that islet neogenesis in this culture system at least partly represents the de-differentiation of islet cells into a duct-cell-like phenotype, with further re-differentiation in appropriate conditions. The plasticity of differentiated human pancreatic cell types may thus be an important mechanism of human pancreas regeneration.
KeywordsDe-differentiation Islet Neogenesis Pancreas Plasticity Re-differentiation
- BrdU
cultivated human islet bud
cytokeratin 19
- Chro A
chromogranin A
hepatocyte growth factor
magnetic cell sorting
neural cell adhesion molecule
tetrarhodamine isothiocyanate
Islet-cell transplantation in diabetic patients would greatly benefit from methods that increase the numbers of transplanted beta cells either in vitro prior to engraftment or through increased beta cell replication or neogenesis within the graft. New beta cells might be generated in two ways: by replication of differentiated islet cells; and by differentiation from stem/progenitor cells. Based on studies in mice, it has recently been proposed that replication of pre-existing beta cells is the predominant mechanism operating in adult animals [1]. However, the replication of human beta cells is extremely low, both during fetal development [2] and in adult islets [3]. Several studies suggest that neogenesis of islets could occur in the adult pancreas. However, the exact nature and identity of islet-cell precursors remains a puzzle. Experimental models in rodents have shown that beta cells can regenerate after tissue injury, apparently through the differentiation of precursor cells located within pancreatic ducts [4, 5]. Morphological observations also suggest the appearance of neogenic islet cells with ductal origin in the human pancreas [6]. There is direct evidence of islet cell differentiation from experimentally transplanted human ductal cells taken from young donors [7]. Furthermore, a recent analysis of the cellular composition of clinical islet grafts shows that long-term graft function correlates with the number of transplanted ductal cells [8]. In addition to this evidence of duct cells as the islet precursors, a number of studies suggest that a more primitive stem cell could reside within the pancreas or islets [9, 10].
We and others have shown the in vitro generation of endocrine cells from adult pancreatic ductal cell culture [11, 12, 13]. One problem of experiments performed with human pancreatic cells is the presence of pre-existing islet cells at the onset of culture. It has been proposed that bilateral transdifferentiation between human pancreatic exocrine and endocrine cells could occur in long-term culture [14]. Several other studies also demonstrate that differentiated islet cells retain the plasticity to de-differentiate when cultured either as a monolayer [15] or in three-dimensional structures [16]. Recently it has been shown that human adult islets can de-differentiate through epithelial-to-mesenchymal transition to generate proliferative vimentin-positive cells. By serum deprivation these precursor cells can re-differentiate into hormone-expressing islet-like clusters [17]. In the present study, using the previously reported culture model [13], we studied the replication and de-differentiation of pre-existing islet cells in monolayer culture, and applied magnetic cell sorting to estimate the contribution of these cells to in vitro islet neogenesis.
Subjects and methods
Tissue preparation and in vitro cultures
Human islets were isolated according to previously described methods [18, 19] in Uppsala, Sweden. After Ficoll gradient purification, the fractions with different purities of islet cells were collected and shipped on ice to Helsinki for subsequent cultures. Pancreatic tissues from 10 donors were used for these studies (mean age 53±14 years, range 30–67 years). All procedures were approved by institutional ethical committees in Sweden and Finland. The donors or their next of kin gave written informed consent to the use of their pancreas tissues.
As previously described, but with a minor modification [13], the fresh pancreatic cell clumps were first expanded into monolayers in six-well culture plates (Corning, Corning, NY, USA) in CMRL-1066 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (PromoCell, Heidelberg, Germany). After 5–7 days of expansion, the media were changed for serum-free DMEM/F12 (National Public Health Institute, Helsinki, Finland) supplemented with ITS (5 mg/l insulin+5 mg/l transferrin+5 μg/l sodium selenite; Sigma, St. Louis, MO, USA), 2 g/l bovine serum albumin, 8 mmol/l glucose and 10 mmol/l nicotinamide (Sigma). The cells were then overlaid with Matrigel, a basement membrane preparation from Engelbreth–Holm–Swarm mouse tumour cells (BD Biosciences, Bedford, MA, USA), according to the manufacturer's instructions with the exception of dilution (1:10) and overnight gelling at 37°C. Within 2–3 weeks after gel overlay, three-dimensional structures, cysts and cultivated human islet buds (CHIBs), which protruded from monolayers, were hand-picked for analysis. The remaining monolayer cells were collected using a solution of 0.05% trypsin and 0.02% EDTA (Invitrogen).
Bromodeoxyuridine labelling and immunocytochemical staining
To determine endocrine cell proliferation during the monolayer expansion period, the cultured cells were labelled with 10 μg/ml bromodeoxyuridine (BrdU; ZYMED, San Francisco, CA, USA) for 24 h at different time-points before fixation in 4% paraformaldehyde (PFA). The cells that were positive for endocrine marker chromogranin A and proliferation marker BrdU were expressed as a percentage of endocrine cells.
Cytospins were prepared from dissociated (0.05% trypsin and 0.02% EDTA) cell suspensions, which were spun to microscope slides (SuperFrostPlus, Menzel, Braunschweig, Germany) by centrifugation at 55 g for 8 min. Cytospin slides were fixed in 4% PFA for 15 min and rinsed in PBS before staining. Harvested cysts/CHIBs were fixed for 1 h in 4% PFA, rinsed with PBS, suspended in a 2% agarose–PBS solution and centrifuged at 220 g for 1 min to form compact pellets, which were then embedded in paraffin for sectioning.
Immunostaining was performed in the above preparations to identify various cell types using the following primary antibodies: guinea-pig anti-porcine insulin 1:100, rabbit anti-human glucagon 1:500, rabbit anti-human somatostatin 1:500, rabbit anti-human pancreatic polypeptide 1:500, rabbit anti-human chromogranin A (Chro A) 1:500, mouse anti-human cytokeratin 19 1:50, mouse anti-human vimentin 1:200, and mouse anti-BrdU 1:100 (all from DAKO, Carpinteria, CA, USA); mouse anti-synaptophysin 1:5 (Boehringer Mannheim, Mannheim, Germany); rabbit anti-neural cell adhesion molecule (N-CAM) 1:50 (kindly provided by V. Cirulli, The Whittier Institute for Diabetes, La Jolla, CA, USA). Non-specific binding was blocked by preincubation in 3% normal serum (ZYMED) from the species in which the secondary antibody was raised, followed by incubation of primary antibodies for 1 h at room temperature. Biotinylated goat anti-rabbit and biotinylated rabbit anti-mouse IgGs (ZYMED, 1:200) were used as secondary antibodies. Peroxidase-conjugated streptavidin (ZYMED, 1:200) was used by developing the substrate of 3-amino-9-ethylcarbazole. Light counterstaining was performed with haematoxylin. Microwave treatment in citrate buffer was necessary to retrieve the antigenicity of BrdU, whereas 0.1% pepsin–0.1 mol/l HCl was optimal for cytokeratin 19 (CK19) and hormone retrieving in paraffin sections. For double staining of BrdU and Chro A, the Vectastain ABC-kit (Vector, Burlingame, CA, USA) was used. Double immunofluorescent staining was performed to check for co-localisation of endocrine markers, N-CAM, and CK19, using conjugated secondary antibodies as follows: fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit, FITC-conjugated donkey anti-guinea-pig, tetrarhodamine isothiocyanate (TRITC)-conjugated donkey anti-mouse, and TRITC-conjugated donkey anti-guinea-pig IgGs (DAKO, 1:50).
Insulin and DNA measurement
For the analysis of insulin and DNA content, the cells in one well of six-well plates were collected and washed twice in PBS, resuspended in 300 μl distilled cold water, and homogenised by sonication on ice. Aliquots of the homogenates in duplicate were either analysed fluorometrically for their DNA content [20] or extracted with acid ethanol overnight and measured for insulin content using a solid-phase RIA kit (DPC, Los Angeles, CA, USA).
Cell separation
The initial cell clusters were dissociated with 0.16 mg/ml trypsin and 0.1 mmol/l EDTA [21] and filtered through a 30-μm nylon mesh (Miltenyi Biotec, Bergisch Gladbach, Germany) to remove residual cell clumps. The single cell suspension was incubated for 15 min at 6 °C with microbeads conjugated to a monoclonal antibody against N-CAM (Miltenyi Biotec) diluted 1:5. Cell separation was carried out on a MiniMACS magnetic cell separation system according to the manufacturer's instructions (Miltenyi Biotec). The N-CAM-negative cells were purified by passage through two consecutive MS columns while N-CAM-positive cells were retained in the columns. The separated cells were plated on gelatin-coated 12-well plates and cultured in CMRL-1066 medium (Invitrogen) supplemented with 10% fetal calf serum (PromoCell). In some experiments, 15 ng/ml hepatocyte growth factor (HGF) was used to promote maximal expansion of the monolayers. Differentiation of the expanded cells was induced by Matrigel overlay and serum-free DMEM/F12 differentiation medium as before. In addition, several other protocols were applied to induce differentiation in these cells, including transfer of the cells after the expansion phase into high-density culture either in conical culture wells or on Matrigel in the serum-free medium.
In the first part of this study, we used islet isolates with variable purity from five donors to study proliferation of endocrine cells and discover evidence of de-differentiation of pre-existing islet cells during monolayer culture.
Endocrine differentiation
Cell sorting experiments
Immunocytochemical analysis of dissociated human pancreatic cells before and after MACS with N-CAM antibody-coated beads
Percent of total cells
Chromogranin A
Cytokeratin 19
Before separation
In this study, we again show that human islet-cell neogenesis can be induced during the culture of mixed human pancreatic cells originating from islet isolation fractions of low purity. We also show that a similar neogenic process is induced from very pure islet fractions. These experiments are characterised by an initial phase of proliferation, associated with a decrease in endocrine cell number, and a later differentiation phase, which results in a net increase of endocrine islet cells. In our previous study [13] we found that the neogenic islet cells were derived from CK19-positive ductal epithelium-like cells which proliferated during the initial days of culture. This time we focused on the role of the pre-existing differentiated islet cells, which are present at the onset of culture. Our results show that a significant proportion of islet cells start to express CK19 soon after plating, thus attaining a transitional phenotype. Although it is theoretically possible that these transitional cells could represent pre-existing ductal cells that start to express endocrine proteins, this is unlikely because these cells are encountered at an early stage of culture, characterised by decreasing numbers of endocrine cells, and a decrease in the cellular insulin content by 50% [13]. The transitional cells were only observed for a limited time, suggesting that they stopped expressing markers of fully differentiated islet cells (such as Chro A). Significant epithelial cell proliferation was only observed in CK19-positive cells, but not in Chro A-positive cells. Consequently, it is possible that the de-differentiated islet cells started to proliferate after losing their endocrine phenotype, and may have acted as precursors of newly differentiated islet cells during the differentiation phase. We tested this hypothesis by eliminating the pre-existing islet cells before starting the cultures.
The role of beta cell proliferation has been emphasised based on cell lineage experiments performed in mice [1]. However, many studies show that there are obvious differences between species in the capacity of beta cells to replicate. Proliferation of human beta cells in vivo appears to be extremely low at all ages [2, 6, 22], and this is true also for isolated islets even after transplantation to nude mice [3, 7, 23]. However, when placed in suitable culture conditions, human beta cells may transiently proliferate but this is invariably associated with loss of differentiated function, at least in monolayer cultures [15, 24]. In this study, we also observed a low-level transient stimulation of beta cell replication at the time when transitional cell types emerged, consistent with the association of proliferation and de-differentiation.
All experimental studies published so far on human islet cell neogenesis have been hampered by 'contamination' through pre-existing islet cells in the starting material [7, 12, 13, 25]. This is a difficult problem because it appears to be impossible to identify any fractions from dispersed human pancreatic cells processed for islet isolation that would be completely devoid of islet endocrine cells. We developed a method for the elimination of these endocrine cells based on their expression of N-CAM [21]. Magnetic microbeads coated with N-CAM antibody effectively bound to the islet cells, allowing us to generate 99.7% endocrine-cell-free human pancreatic cells. When plated in culture, most of these cells expressed CK19 and proliferated. However, the differentiation phase was dramatically altered. The formation of three-dimensional cystic structures with small islet buds, which we have regularly seen in 50 consecutive experiments, did not occur when the Matrigel overlay and serum-free culture medium were applied for the expanded N-CAM-negative cell population. This was not the result of poor viability of the cells, because their rate of proliferation was not clearly different from that of the unseparated cell population. However, because of the relatively low numbers of cells that could be processed with the MACS system, and the inevitable loss of cells during separation, we were not able to achieve equally large confluent monolayers as with the unseparated cell mass. Using a smaller scale, we applied various alternative protocols to induce differentiation. The only positive results were observed when the expanded cells were transferred in relatively high density to culture wells coated with Matrigel. During 2–3 weeks in serum-free differentiation medium, these cultures formed a number of cysts resembling the original 'CHIBs' (Fig. 6f). However, there were practically no dense cell buds growing out of these cysts, and although a few Chro A-positive cells could be detected by immunocytochemistry, there were very few insulin-positive cells and also the insulin content of the extracted cultures remained low.
The specific expression of N-CAM in adult pancreatic islets of rodents has been reported [21, 26], but in the human pancreas, N-CAM may be found also in ducts [27, 28]. We performed the MACS experiments to deplete the pre-existing islets. However, our results show that a cell population coexpressing N-CAM and CK19 was also depleted from the N-CAM-negative fraction. These cells were also detected by double immunocytochemistry in the uncultured fresh material. It is possible that the N-CAM/CK19 double-positive cells could represent a pool of islet cell progenitors or partially de-differentiated islet cells. This interesting pool of cells needs to be characterised in more detail in further studies.
The plasticity of differentiated pancreatic cells may be an important mechanism behind islet-cell expansion, as has been demonstrated by several studies in rodents. Transdifferentiation of acinar cells to ductal cells and further differentiation of these cells into islets has recently been demonstrated in vivo [29] and in vitro [30]. There are fewer observations in human pancreatic cells but clear evidence exists, particularly for the islet/ductal transitional cells both in vivo [6] and in vitro [14, 15, 31]. However, only a recent report [31] and our study have observed a relatively large number of transitional cells within all four types of endocrine cells and presented the quantitative results while only occasional co-staining of insulin and ductal markers is found by others. Our demonstration of endocrine–ductal transitional cells and the fact that depletion of endocrine cells from the starting material severely reduces the endocrine differentiation of the expanded CK19-positive cells promotes the hypothesis that this particular form of plasticity could be important for human islet expansion. This would imply that instead of direct replication, the human islet cells could de-differentiate into a duct-like phenotype that could then re-differentiate into islet cells after a phase of proliferation. The data presented here suggest that the endocrine precursors within the ductal cell compartment might actually belong to this pool of committed cells circulating between the endocrine and ductal phenotypes. More direct evidence for this might be obtained if the freshly isolated endocrine and ductal cells could be separately labelled prior to the in vitro culture experiments.
Recently, the process of de-differentiation and re-differentiation in cultured human islets cells has also been reported by Gershengorn et al. [17]. However, in contrast to the islet–ductal transition found in our study, they showed a reversible epithelial-to-mesenchymal transition. The transitional vimentin-positive cells were positive for proinsulin mRNA until passage 10, suggesting that de-differentiated beta cells were proliferative. The majority of proliferating vimentin-positive cells were also positive for nestin, which makes it possible that these cells were identical to the populations reported by Zulewski et al. [9]. However, the re-differentiated epithelial cells had a very low insulin expression level of less than 0.02% of that seen in human islets. In comparison, the CHIBs were much more differentiated, with 5% of the insulin level found in pure human islets. However, it is clear that the expansion of the de-differentiated epithelial cells in our study was very limited in comparison with the long-term proliferation reported for the mesenchymal cells [17].
Taken together, we can conclude that the islet cell neogenesis in our culture system at least partly represents the de-differentiation of non-proliferative islet cells into a proliferative duct-cell-like phenotype with further re-differentiation in appropriate conditions. Further studies are needed to establish whether all ductal cells, and also acinar cells, participate in the pool of endocrine precursors, or whether only a pool of cells committed for endocrine differentiation circulate between endocrine and ductal phenotypes. This concept is also compatible with the notion strongly suggested by mouse experiments [1] that self-duplication rather than stem cell differentiation is responsible for the expansion of the adult beta cell mass.
This work was supported by grants from the Juvenile Diabetes Research Foundation, the Sigrid Juselius Foundation, the Finnish Diabetes Research Foundation, the Academy of Finland and the Helsinki University Hospital Research Funds (EVO). The authors thank Dr K. Salmela and the Nordic Network for Clinical Islet Transplantation for the procurement of human pancreatic cells. Ms P. Narinen is thanked for her skilled technical assistance.


text:
The FFIEC's Internet site now contains additional information on the Community Reinvestment Act. The expanded CRA web site now includes Interagency CRA Interpretive Letters FFIEC (with the identity of the addressees and proprietary information suppressed unless government sponsored), Examination Procedures, the CRA Regulation and Questions and Answers, as well as links to the CRA home pages of each of the agencies.
The new FFIEC CRA web pages are located at www.ffiec.gov/cra/dcca or at the FFIEC home page (www.ffiec.gov) under "Regulations, Ratings, Schedules and Documents" under "Community Reinvestment Act" within the section on "Information Services."
If you have any questions, please contact either Cathy Gates (202) 452-2099 or Anna Rotenberg (202) 736-1921 in DCCA at the Board.
The Practice of Microenterprise in the U.S.: Strategies, Costs, and Effectiveness, published by The Aspen Institute's Self-Employment Learning Project. This report provides a detailed picture of seven microenterprise lending programs and offers an analysis of their progress and challenges that lie ahead for the field of microenterprise programs. To obtain copies, contact The Aspen Institute, P.O. Box 222, Queenstown, Md., 21658, or call (410) 820-5326.
Keep up with what's new and noteworthy at the St. Louis Fed. Sign up now to have this free monthly e-newsletter emailed to you.
FedCommunities.org is a portal to community development resources from all 12 Federal Reserve Banks and the Federal Reserve Board of Governors.


text:
TRISHA YEARWOOD has a place in Oscar history! Before Dazed and Confused, new Oscar winner MATTHEW McCONAUGHEY got his big break as the male eye candy in 1992's "Walk Away Joe" music video.
While the two have never connected, Trisha feels somewhat responsible for discovering Matthew before he made it big in Hollywood.
She says, "Honestly, I'm so happy to see him recognized for his work in Dallas Buyers Club. And if he ever wants to be in another Trisha Yearwood video, I won't make him audition."


text: I am resending an email from a couple of years back, which looked fine previously but now the on iPhone the animated gif is slightly cropped by about 20-30px on the right hand side. I haven't changed the code and the @media is the same. Has anyone had this same issue? It's actually a Christmas card so the animation is the content.


text:
I am smart, strong, and strange
Like action movies, but no real favorites. I do like the older classics from the 80's and 90's, like Short Circuit, Howard the Duck, Zapped, Weird Science,The Last Dragon, Last Starfighter, WarGames. Some of the newer stuff, Matrix, Ultraviolent, Transformers, Underworld series. Pretty much anything staring Bruce Lee and the old Kung-Fu theater movies. Funny movies are also good.
Shows are kinda hit an miss cause of my schedule, but do like Mythbusters, Outrageous acts of Science, Aerial America, How its Made, DarkMatter, Law&Order, Avatar, Bones. Also most anime and cartoons, always a plus. Classic like the Miyazaki films, many of the Gundam series, G-force, Voltorn and news stuff like Gate, One Punch ect. Also old school, He-man, GI Joe, Transformers, Centurions (bonus for anyone that actually remembers this).
I'll listens to almost all types of music from Rap, Rock,Hip-Hop, R&B, Soul, Old School, Funk, Blues Metal, J an K-pop and others. I also like classical music like opera an traditional music from different cultures, the only thing i won't do is country, can't stand Country.
Partial to Asian style cuisine, but I like all kinds of food, even the more bizarre types of food, though not Andrew Zimmerman's level, I am willing to try almost anything once. My family was military so I traveled a bit and got to try all kinds of different foods from different parts of the world.