Is the Pope about to bestow John Lennon a sainthood!?

A short reflection on the last day of the Church Year
Sunday, 23 November 2008

Reuters report that the Vatican propaganda paper, Osservatore Romano, has stated that the Vatican (aka the Pope) has forgiven John Lennon his 1966 declaration that “the Beatles now are more famous than Jesus Christ”.

I wonder what John Lennon would have said about this resurrection from the high prelate in Rome!

I suspect at least he would not have liked the Vatican’s motivation: "The remark by John Lennon, which triggered deep indignation ..., after many years sounds only like a 'boast' by a young working-class Englishman faced with unexpected success”.

Or is it a ploy by the Vatican when celebrating the last Sunday of the Church Year, the End of the World?

ZenMaster

Pure Insulin-producing Cells Produced from ESCs

Cells effective in treating diabetes in lab model
Friday, 21 November 2008

Singapore researchers have developed an unlimited number of pure insulin-producing cells from mouse embryonic stem cells (ESCs).

These pure insulin-producing cells, which according to electron microscopy studies, have the same sub-cellular structures as the insulin-producing cells naturally found in the pancreas, were highly effective in treating diabetes in the mouse model.

The transplants of pure insulin-producing cells reduced the blood glucose levels of diabetic mice with high blood glucose levels.

The experiments also showed that the subsequent removal of the transplanted cells from the diabetic mice restored the blood glucose to its original high level.

None of the diabetic mice involved in the transplant experiments developed teratoma, which are a type of tumour often associated with ESCs and which could complicate their use in human therapeutic treatment.

Furthermore, the pure insulin-producing cells managed to retain their insulin-production and glucose-sensing capacity over time.

The Singapore researchers' achievement provides proof of principle that this strategy could be applied to human ESCs to obtain similar pure insulin-producing cells.

These research findings were published in two separate papers in the July and August 2008 online versions of the journal Stem Cell Research.

Conducting the research were scientists at the Institute of Medical Biology (IMB), which is under Singapore's Agency for Science, Technology and Research (A*STAR), and the Yong Loo Lin School of Medicine (YLLSoM) at the National University of Singapore (NUS).

The team of researchers was co-led by Dr. Lim Sai Kiang, an IMB principal investigator and a research associate professor at the YLLSoM Department of Surgery, and Dr. Li Guodong, a research associate professor at National University Medical Institutes, YLLSoM, NUS.

Commenting about these findings, Dr. Gordon Weir, Director of the Clinical Islet Transplantation Program at Harvard Medical School, who also holds appointments at the Harvard Stem Cell Institute and Joslin Diabetes Centre, said:

"The amount of careful work done by this group of researchers is impressive. We need something to put into diabetic patients to treat their condition, and these findings tell us interesting things about the development of beta cells."

The strategic approach by the group offers avenues for further research in the treatment for diabetes.

"Our ability to isolate and then multiply insulin-producing cells from differentiating ESCs provides an unlimited supply of pure insulin-producing cells to study in unprecedented detail many aspects of these cells," said Dr. Lim.

"Besides providing a tool to facilitate basic research in test tubes and animals, these insulin-producing cells may be also used to replace the isolated native pancreatic cells that are hard to obtain in a large amount, for pharmacological tests," added Dr Li.

The research was supported primarily by grants from A*STAR's Biomedical Research Council, Juvenile Diabetes Research Foundation International, and National Medical Research Council of Singapore.

References:
Generating mESC-derived insulin-producing cell lines through an intermediate lineage-restricted progenitor line
Li GD, Luo R, Zhang J, Yeo KS, Lian Q, Xie F, Tan EKY, Caille D, Kon OL, Salto-Tellez M, Meda P, and Lim SK
Stem Cell Research 2008, in press and available online 8 August 2008.

Derivation of functional insulin-producing cell lines from primary mouse embryo culture
Li GD, Luo R, Zhang J, Yeo KS, Xie F, Tan EKY , Caille D, Que J, Kon OL, Salto-Tellez M, Meda P, and Lim SK
Stem Cell Research 2008, in press and available online 31 July 2008.
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Tissue Engineering for Transplanting from Own Stem Cells II

Use of several types of adult stem cells grow new trachea
Thursday, 20 November 2008

The first tissue-engineered trachea (windpipe), utilising the patient's own stem cells, has been successfully transplanted into a young woman with a failing airway. The bioengineered trachea immediately provided the patient with a normally functioning airway, thereby saving her life.

These remarkable results provide crucial new evidence that adult stem cells, combined with biologically compatible materials, can offer genuine solutions to other serious illnesses.

In particular, the successful outcome shows it is possible to produce a tissue-engineered airway with mechanical properties that permit normal breathing and which is free from the risks of rejection seen with conventional transplanted organs. The patient has not developed antibodies to her graft, despite not taking any immunosuppressive drugs. Lung function tests performed two months after the operation were all at the better end of the normal range for a young woman.

The pan-European team from the universities of Barcelona, Bristol, Padua and Milan report on this pioneering work in an article published early online and in an upcoming edition of The Lancet.

The loss of a normal airway is devastating, but previous attempts to replace large airways have met serious problems. The 30-year-old mother of two, suffering from collapsed airways following a severe case of TB, was hospitalised in March 2008 with acute shortness of breath rendering her unable to carry out simple domestic duties or care for her children. The only conventional option remaining was a major operation to remove her left lung, which carries a risk of complications and a high mortality rate.

Based on successful laboratory work previously performed by the team, and given the urgency of the situation, it was proposed that the lower trachea and the tube to the patient's left lung (bronchus) should be replaced with a bioengineered airway based on the scaffold of a human trachea.

A seven-centimetre tracheal segment was donated by a 51-year-old transplant donor who had died of cerebral haemorrhage. Spain has a policy of assumed consent for organ donation. Using a new technique developed in Padua University, the trachea was de-cellularised over a six-week period so that no donor cells remained.

Stem cells were obtained from the recipient's own bone marrow, grown into a large population in Professor Martin Birchall's lab at the University of Bristol, and matured into cartilage cells (chondrocytes) using an adapted method originally devised for treating osteoarthritis by Professor Anthony Hollander at the University of Bristol.

The donor trachea was then seeded with chondrocytes on the outside, using a novel bioreactor which incubates cells, developed at the Politecnico di Milano, Italy, allowing them to migrate into the tissue under conditions ideal for each individual cell type. In order to replicate the lining of the trachea, epithelial cells were seeded onto the inside of the trachea using the same bioreactor.

Four days after seeding, the graft was used to replace the patient's left main bronchus. Professor Paolo Macchiarini of the University of Barcelona performed the operation in June 2008 at the Hospital Clínic, Barcelona.

Professor Macchiarini, lead author on the paper, said:

"We are terribly excited by these results. Just four days after transplantation the graft was almost indistinguishable from adjacent normal bronchi. After one month, a biopsy elicited local bleeding, indicating that the blood vessels had already grown back successfully".

Martin Birchall, Professor of Surgery at the University of Bristol, added:

"Surgeons can now start to see and understand the very real potential for adult stem cells and tissue engineering to radically improve their ability to treat patients with serious diseases. We believe this success has proved that we are on the verge of a new age in surgical care".

Anthony Hollander, Arthritis Research Campaign Professor of Rheumatology and Tissue Engineering at the University of Bristol, concurred:

"This successful treatment manifestly demonstrates the potential of adult stem cells to save lives".

The patient, Claudia Castillo, a young woman from Colombia but now living in Spain, had no complications from the operation and was discharged from hospital on the tenth post-operative day. She has remained well since and has a normal quality of life. She is able to care for her children, walk up two flights of stairs and occasionally go out dancing in the evenings.

She said:

"Above all I would like to thank Dr. Macchiarini and his medical team who did the research, for the time and dedication they devoted to my case to make sure that everything turned out alright."

Reference:
Clinical transplantation of a tissue-engineered airway
Paolo Macchiarini, Philipp Jungebluth, Tetsuhiko Go, M Adelaide Asnaghi, Louisa E Rees, Tristan A Cogan, Amanda Dodson, Jaume Martorell, Silvia Bellini, Pier Paolo Parnigotto, Sally C Dickinson, Anthony P Hollander, Sara Mantero, Maria Teresa Conconi, Martin A Birchall
The Lancet, Early Online Publication, 19 November 2008, doi:10.1016/S0140-6736(08)61598-6

See also:
Tissue Engineering for Transplanting from Own Stem Cells I
CellNEWS - Thursday, 20 November 2008
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Tissue Engineering for Transplanting from Own Stem Cells I

First tracheal transplant without immunosuppression
Thursday, 20 November 2008

Summary:

• Tissue engineering has made possible this doubly innovative operation – the first trachea transplant and the first tissue transplant to be performed without the need for immunosuppression.
• Professor Paolo Macchiarini, Head of the thoracic surgery department of Hospital Clínic of Barcelona, has led the basic research and the international team formed by the universities of Bristol, Padua and Milan, who contributed to this success.
• The transplanted tissue is a hybrid from a donor that was re-populated with stem and epithelial cells from the recipient. Five months later, Claudia Castillo, who required the operation to save a lung following tuberculosis, is in perfect health.

After 4 years of going from consultation to consultation, Claudia Castillo finally found a solution to her respiratory problems. The young Colombian woman suffered from a cough that took a long time to be diagnosed as tuberculosis. She arrived at Hospital Clínic of Barcelona with complications and there, she met Professor Paolo Macchiarini, Head of the Thoracic Surgery Department, who led the international team that made possible the first trachea transplant and the first tissue transplant without immunosuppression. She underwent an operation on the upper part of the trachea but nothing could be done to repair the blockage in the left lung. The infection had led to a severe collapse just before the branch of the trachea and this obstruction prevented air from reaching the lung. The only treatment option at the time involved removing the affected lung. As the young mother of two children, removing the lung would have considerably reduced quality of life for Claudia Castillo. In March 2008, her situation worsened to the point where she was unable to carry out domestic chores or look after her children, so intervention became urgent. In June, after obtaining authorization from the ethics committee of Hospital Clínic of Barcelona and from the Catalan Transplant Organization (OCATT), the first trachea transplant and the first tissue transplant of any kind without immunosuppression took place.

The study, published online on Wednesday by the journal The Lancet, with Professor Paolo Macchiarini as the principal author, together with his colleagues Dr. Philip Jungebluth, Dr. Tetsuhiko Go and Dr. Jaume Martorell, presents the details of this transplant – the first treatment alternative for treating the collapsed trachea that the patient was suffering from. The technique consists of depleting the trachea to be transplanted of the donor's cells and repopulating it with cells from the recipient before the operation. Thus, thanks to tissue bioengineering, the donor trachea becomes a hybrid that the recipient's body identifies as its own, thereby making immunosuppression unnecessary. The transplant and most of the processes involved were carried out at Hospital Clínic of Barcelona, but this would have been impossible without the collaboration of the University of Bristol (UK), the University of Padua (Italy) and the University of Milan (Italy). Professor Paolo Macchiarini led the prior basic research.

The process of preparing the trachea requires many cycles of washing to eliminate all the donor cells – many more than those suggested by the basic research. The tissue was a 7-cm segment of trachea from a 51-year-old donor who had died from brain haemorrhage. The team of Dr. Maria T. Conconi at the University of Padua (Italy) confirmed that, after 25 washing cycles, the trachea treated at Hospital Clínic was free from donor antigens – the molecules that would cause the tissue to be rejected by the recipient. Meanwhile, at the University of Bristol, the teams of Professor Martin Birchall and Professor Anthony Hollander cultivated the recipient's cells that would later be introduced into the trachea. These cells were epithelial cells taken from the trachea and cartilage cells (chondrocytes), differentiated from stem cells taken from the patient's bone marrow. This technique was initially designed to treat cases of osteoarthritis. Back at Hospital Clínic, the team of Professor Paolo Macchiarini introduced these cells into the trachea using a bioreactor designed by the team of Dr. Sandra Mantero at the University of Milan. The epithelial cells were inserted into the inner surface of the trachea and the chondrocytes covered the outer surface. The donor tissue thus became a hybrid very similar to new tissue from the patient herself.

The operation was performed 4 days later at Hospital Clínic, where the thoracic surgery team extracted the damaged section of trachea and replaced it with the new trachea. This pioneering operation was not without question marks but if anything had gone wrong, it would have been changed to a lung-resection operation – the classical treatment choice. Thanks to the skill of the surgeons and the huge international effort, the operation was a success. Five months later, the lung that had been so long out of use was providing normal respiration.

This innovation in biomedicine and surgery may become an alternative for diseases of the upper airways, such as congenital deformities or primary tumours, which cannot currently be treated using conventional surgical techniques. The clinical application of stem cell cultures and the prevention of the problems deriving from immunosuppression are a milestone in the history of transplantation. There are already some cases being studied that may benefit from the new technique and research continues into improving the process. If all goes well, Claudia Castillo will be just the first patient to benefit from a new advance led by researchers from Hospital Clínic of Barcelona.

Reference:
Clinical transplantation of a tissue-engineered airway
Paolo Macchiarini, Philipp Jungebluth, Tetsuhiko Go, M Adelaide Asnaghi, Louisa E Rees, Tristan A Cogan, Amanda Dodson, Jaume Martorell, Silvia Bellini, Pier Paolo Parnigotto, Sally C Dickinson, Anthony P Hollander, Sara Mantero, Maria Teresa Conconi, Martin A Birchall
The Lancet, Early Online Publication, 19 November 2008, doi:10.1016/S0140-6736(08)61598-6

See also:
Tissue Engineering for Transplanting from Own Stem Cells II
CellNEWS - Thursday, 20 November 2008
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Extinct Woolly-mammoth Genome Sequenced

Close to that of the modern-day African elephant's genome
Thursday, 20 November 2008

Scientists at Penn State are leaders of a team that is the first to report the genome-wide sequence of an extinct animal, according to Webb Miller, professor of biology and of computer science and engineering and one of the project's two leaders. The scientists sequenced the genome of the woolly mammoth, an extinct species of elephant that was adapted to living in the cold environment of the northern hemisphere. They sequenced four billion DNA bases using next-generation DNA-sequencing instruments and a novel approach that reads ancient DNA highly efficiently.

"Previous studies on extinct organisms have generated only small amounts of data," said Stephan C. Schuster, Penn State professor of biochemistry and molecular biology and the project's other leader.

"Our dataset is 100 times more extensive than any other published dataset for an extinct species, demonstrating that ancient DNA studies can be brought up to the same level as modern genome projects."

The researchers suspect that the full woolly-mammoth genome is over four-billion DNA bases, which they believe is the size of the modern-day African elephant's genome. Although their dataset consists of more than four-billion DNA bases, only 3.3 billion of them — a little over the size of the human genome — currently can be assigned to the mammoth genome. Some of the remaining DNA bases may belong to the mammoth, but others could belong to other organisms, like bacteria and fungi, from the surrounding environment that had contaminated the sample. The team used a draft version of the African elephant's genome, which currently is being generated by scientists at the Broad Institute of MIT and Harvard, to distinguish those sequences that truly belong to the mammoth from possible contaminants.

"Only after the genome of the African elephant has been completed will we be able to make a final assessment about how much of the full woolly-mammoth genome we have sequenced," said Miller. The team plans to finish sequencing the woolly mammoth's genome when the project receives additional funding.

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Ball of permafrost-preserved mammoth hair containing thick outer-coat and thin under-coat hairs. Credit: Stephan Schuster lab, Penn State University.

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The team sequenced the mammoth's nuclear genome using DNA extracted from the hairs of a mammoth mummy that had been buried in the Siberian permafrost for 20,000 years and a second mammoth mummy that is at least 60,000-years-old. By using hair, the scientists avoided problems that have bedevilled the sequencing of ancient DNA from bones because DNA from bacteria and fungi, which always are associated with ancient DNA, can more easily be removed from hair than from bones. Another advantage of using hair is that less damage occurs to ancient DNA in hair because the hair shaft encases the remnant DNA like a biological plastic, thus protecting it from degradation and exposure to the elements.

The researchers previously had sequenced the woolly mammoth's entire mitochondrial genome, which codes for only 13 of the mammoth's roughly 20,000 genes but is relatively easy to sequence because each of the mammoth's cells has many copies. In their most recent project, the team sequenced the mammoth's nuclear genome, which codes for all the genetic factors that are responsible for the appearance of an organism. The two methods combined have yielded information about the evolution of the three known elephant species: the modern-day African and Indian elephants and the woolly mammoth. The team found that woolly mammoths separated into two groups around two million years ago, and that these groups eventually became genetically distinct sub-populations. They also found that one of these sub-populations went extinct approximately 45,000 years ago, while another lived until after the last ice age, about 10,000 years ago. In addition, the team showed that woolly mammoths are more closely related to modern-day elephants than previously was believed.

"Our data suggest that mammoths and modern-day elephants separated around six-million years ago, about the same time that humans and chimpanzees separated," said Miller.

"However, unlike humans and chimpanzees, which relatively rapidly evolved into two distinct species, mammoths and elephants evolved at a more gradual pace," added Schuster, who believes that the data will help to shed light on the rate at which mammalian genomes, in general, can evolve.

The team's new data also provide additional evidence that woolly mammoths had low genetic diversity.

"We discovered that individual woolly mammoths were so genetically similar to one another that they may have been especially susceptible to being wiped out by a disease, by a change in the climate, or by humans," said Schuster.

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Drawing of a woolly mammoth.

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Members of the team previously ruled out humans as a cause of extinction for at least one of the Siberian sub-populations — the group appears to have gone extinct at least 45,000 years ago at a time when there were no humans living in Siberia. However, much debate still remains regarding the causes of extinction for the other group and for those populations that lived in other places, such as North America.

Currently, the team is searching the mammoth's genome for clues about its extinction.

"For example," said Miller, "most animal genomes contain integrated viral sequences and, though these are not directly associated with disease, evidence of multiple recent integration events could indicate a perturbation of virus-host interaction that might be responsible for disease. Alternatively, it might turn out that long generation times and limited out-breeding result in accumulation of deleterious genetic mutations. We are considering a number of possible causes of extinction."

The new data are allowing the Penn State team to begin looking for genetic causes of some of the mammoth's unique characteristics, such as their adaptation to extremely cold environments. For instance, the team already has identified a number of cases in which all previously sequenced mammals, except mammoths, have the same protein segment.

"One has to wonder whether a particular protein that has remained the same in animals for several billion years of combined evolution and then became different in mammoths could result in a mammoth-specific trait," said Miller.

Investigating the unique characteristics of woolly mammoths, and why they went extinct, are just some of the many tasks that the research team plans to pursue now that they have access to such a large quantity of sequence data.

"This really is the first time that we have been able to study an extinct animal in the same detail as the ones living in our own time," said Schuster.

Another significant aspect of the study is that it was completed by a small group of scientists at a relatively low cost and over a short period of time, whereas previous reports of modern mammalian genome sequences — including human sequences — have taken millions of dollars and several years of analysis by large groups of scientists to complete. Miller hopes that after he completes a few additional genome projects he can produce computer software that will enable others to perform low-cost mammalian genome analysis, and Schuster already is preparing to decode extinct genomes at an even faster pace.

Schuster hopes that lessons learned from the mammoth genome about why some animals go extinct while others do not will be useful in protecting other species from extinction, such as the Tasmanian devil, whose survival is threatened by a deadly facial cancer.

"In addition," added Schuster, "by deciphering this genome we could, in theory, generate data that one day may help other researchers to bring the woolly mammoth back to life by inserting the uniquely mammoth DNA sequences into the genome of the modern-day elephant. This would allow scientists to retrieve the genetic information that was believed to have been lost when the mammoth died out, as well as to bring back an extinct species that modern humans have missed meeting by only a few thousand years."

About the project:
In addition to being members of the faculty of Penn State's Eberly College of Science, Miller and Schuster are researchers associated with Penn State's Center for Comparative Genomics and Bioinformatics. The study also involved researchers from the Severtsov Institute of Ecology and Evolution and the Zoological Institute in Russia, the University of California, the Broad Institute, the Roche Diagnostics Corporation, and the Sperling Foundation in the United States. Penn State, Roche Applied Sciences, a private sponsor, the National Human Genome Research Institute, and the Pennsylvania Department of Health funded this research.

More information about this project is on the Web at the Mammoth Genome Project.

Reference:
Sequencing the nuclear genome of the extinct woolly mammoth
Webb Miller, Daniela I. Drautz, Aakrosh Ratan, Barbara Pusey, Ji Qi, Arthur M. Lesk, Lynn P. Tomsho, Michael D. Packard, Fangqing Zhao, Andrei Sher, Alexei Tikhonov, Brian Raney, Nick Patterson, Kerstin Lindblad-Toh, Eric S. Lander, James R. Knight, Gerard P. Irzyk, Karin M. Fredrikson, Timothy T. Harkins, Sharon Sheridan, Tom Pringle & Stephan C. Schuster
Nature 456, 387-390, 20 November 2008, doi:10.1038/nature07446
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Kangaroo Genome Mapped

Kangaroo Genome Mapped
Tuesday, 18 November 2008

Australian researchers will today launch the world first detailed map of the kangaroo genome, completing the first phase of the kangaroo genomics project.

Researchers at the ARC Centre of Excellence for Kangaroo Genomics (KanGO), including University of Melbourne, ANU, WEHI, University of Sydney, University of NSW and the Australian Genome Research Foundation (AGRF) have built a framework to assemble the genome of a model kangaroo, the tammar wallaby.

"A good map is crucial for finding our way around a new genome," said KanGO Director Prof. Jenny Graves, who divides her time between ANU and University of Melbourne.

"It enables us to explore how the genome of mammals - including humans - is organized, how it functions, and how it evolved."

"Now the world can use information on kangaroo genes and sequences to explore how mammals develop and function," she said.

DNA sequence obtained by the Australian Genome Research Facility (AGRF) with funding from the Victorian government will be arranged using the genome map.

Researchers say the international race to sequence the genomes of significant species is driven by the power of genome comparisons – particularly of species that are distantly related – to reveal secrets of the genome in humans, as well as other mammals.

"Australia's weird and wonderful animals are making crucial contributions," Professor Graves said.

"The kangaroo has helped to consolidate Australia's reputation in this important genomics era," she said.

Graves says genomic information is extremely powerful. She says KanGO researchers used the kangaroo genome map to solve fundamental genetic puzzles, for instance discovering the gene that controls the sex of a baby, and overturning theories of the origin of our blood proteins.

The map and sequence will open up new areas of research into how genes are turned on and off during development of all mammals.

"Kangaroos are a marvellous model for studying human development and reproduction because they are born very early and complete much of their development in the pouch - rather than the womb," said Laureate Professor Marilyn Renfree of the University of Melbourne's Zoology Department, who takes over as KanGO Director today.

"This makes them a powerful tool for studying the genes and hormones involved in mammalian reproduction and development."

Professor Graves says that access to the next generation sequencing technologies will mean that the wealth of genetic information in Australia's native flora and fauna can now be tapped into.

"This will provide a depth of understanding never thought possible until recently and lead to new and exciting applications in the field of biotechnology."
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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‘Orphan’ Genes Importance in Evolution

‘Orphan’ Genes Importance in Evolution
Tuesday, 18 November 2008

Closely related animal species share most of their genes and look almost identical. However, minor morphological differences allow us to tell them apart. What is the genetic basis for these differences?

Often, the explanation is provided by minor changes in spatial and temporal activity of transcription factors - "regulator" genes that are conserved throughout the animal kingdom. However, every group of animals also possesses a small proportion of genes, which are, in contrary, extremely variable among closely related species or even unique. For example, a gene may be present in one species or animal group, but not in any other. Such genes are referred to as "novel," "orphan" or "taxonomically restricted". Their function and origin are often obscure. What are these genes needed for?

A new paper, published in this week's issue of the online open access journal, PLoS Biology, explores this question in the freshwater polyp Hydra, which belongs to the same branch of the evolutionary tree as jelly fish. These animals are small (several mm long), predatory creatures, with a tube like body-form that ends in a mouth surrounded by mobile tentacles. They are of particular interest to scientists for their regenerative properties, and because they appear to be biologically immortal; not undergoing the aging process that affects all other known animals.

In this paper, a team of scientists from the Christian-Albrechts-Universität zu Kiel in Germany, used transgenic polyps to uncover the role of "orphan" genes in these morphologically-simple animals. The work, led by Thomas Bosch reports that a family of "novel" genes is responsible for morphological differences between two closely related species of fresh water polyps called Hydra. Their most remarkable finding is that a secreted protein, encoded by "novel" gene Hym301, controls the pattern in which the tentacles in Hydra develop.

"We knew that these genes were important, but it was in no sense simple to demonstrate that," says Konstantin Khalturin, first author of the PLoS Biology paper.

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A freshwater polyp Hydra and its tentacles during bud formation.

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In one species, Hydra oligactis, emergence of its tentacles during bud formation is not synchronised; in Hydra vulgaris all five tentacles develop simultaneously and symmetrically; in Hydra vulgaris polyps genetically altered to produce large amounts of protein from the “orphan gene” Hym301, tentacles are formed in an irregular and asymmetric pattern.

The data indicate that "novel" genes are involved in generation of novel morphological features that characterise different species, thus pointing the way to a new, more complete understanding of how evolution works at the level of a particular group of animals. Emergence of "novel" genes may reflect evolutionary processes, which allow animals to adapt in the best way to changing environmental conditions and new habitats.

Reference:
A novel gene family controls species-specific morphological traits in Hydra
Konstantin Khalturin, Friederike Anton-Erxleben, Sylvia Sassmann, Jörg Wittlieb, Georg Hemmrich, Thomas C. G. Bosch
PLoS Biol 6(11)(2008): e278 doi:10.1371/journal.pbio.0060278
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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US Scientists Self-censor During Bush Administration

US Scientists Self-censor During Bush Administration
Tuesday, 18 November 2008

A survey of scientists whose studies became the focus of a public debate about NIH grant funding has found that many of them engaged in self-censorship as a result of the controversy. The study, published in the open access journal PLoS Medicine, found that following the criticism of their research, scientists removed politically sensitive language from grant applications and stopped studying certain topics. These self-censorship tactics were employed despite the fact that all of the criticised studies — most of which investigated sexual behaviour, drug-use, and other HIV-related questions — were defended in an NIH internal review and retained their funding.

Joanna Kempner of Rutgers University in New Jersey surveyed the researchers who were the subject of a debate in the US in July 2003, which began when a Congressional Representative proposed an amendment to rescind five NIH grants after publicly criticising the studies as "less worthy of taxpayer funding" than research into "devastating diseases." The amendment failed to pass but the controversy resulted in the internal review of more than 250 grants by the NIH, which concluded that each study was scientifically sound.

After conducting in-depth interviews with thirty researchers whose funding was reviewed, Kempner surveyed the principal investigators of all the studies. Only a third of the 82 respondents felt they were less likely to receive funding from the NIH in the future, but a majority reported undertaking strategies designed to disguise the most controversial aspects of their research. Half (51%) said they removed potential "red flag" words from the titles and abstracts of their grant submissions, including the words gay, lesbian, homophobia, anal sex, needle-exchange, and AIDS. Kemper reports that one interviewee said "I do not study sex workers, I study 'women at risk.'"

Almost a quarter of the researchers had either reframed their studies to avoid research on marginalized or stigmatized populations or had chosen to drop studies that were thought to be political sensitive, such as those on sexual orientation, abortion, childhood sexual abuse, and condom use. The survey also found that four of the principal investigators had made career changes and left academia as a result of the controversy.

Joanna Kempner stresses that the controversy also galvanized sections of the research community with 10% of respondents reporting a strengthened commitment to see their research completed, including those who had reported self-censorship practices. She says that the findings are a powerful example of how the political environment can shape what scientists chose not to study.

Reference:
The chilling effect: How do researchers react to controversy?
Kempner J
PLoS Med 5(11): e222. doi:10.1371/journal.pmed.0050222
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Cellular Damage in Huntington's Disease

New clues emerge from study
Monday, 17 November 2008

"Huntington's disease presents an ideal vantage point to study neurodegenerative disease, because we know the misfolded protein that's responsible," says Martin Duennwald, formerly a postdoctoral researcher in the lab of Whitehead Institute for Biomedical Research member Susan Lindquist.

"But we don't understand how this protein causes cellular damage and death for the neurons that are affected."

In a study published in Genes & Development online on November 17, however, Duennwald and Lindquist report the discovery of a mechanism driven by the misfolded proteins that could be one early trigger for cell death.

In the U.S., about 1 in 20,000 people suffers from Huntington's. Better understanding of the cellular toxicity may allow new therapies for this fatal and incurable disorder.

"This is a diabolical disease, because the misfolded protein interacts with and probably traps many other proteins in the cell," notes Lindquist, who is also a Howard Hughes Medical Institute investigator and a professor of biology at Massachusetts Institute of Technology.

Scientists have long known that a single mutated gene that creates proteins with abnormally long repeats of the amino acid glutamine (“Q”) drives Huntington’s. In certain neurons, these "polyQ-expanded" proteins misfold and clump together, damaging and eventually killing the cells.

But the steps that kick off the process of cell damage and death have remained a mystery, remarks Duennwald, now a principal scientist at Boston Biomedical Research Institute in Watertown, Mass.

In the study, Duennwald first examined what makes polyQ-expanded proteins toxic in yeast. He then performed similar experiments in two kinds of mammalian cells — rat cells that model neurons and mouse striated cells (from the part of the brain most afflicted in Huntington's).

He found that cells generated with polyQ-expanded fragments quickly showed problems with proteins that had been marked for degradation in the endoplasmic reticulum (ER, a cell component that folds and finalizes proteins). Such proteins were not expelled for tagging and degradation in the cytosol, the intracellular fluid, outside the ER.

"With no garbage disposal, all of a sudden the ER is flooded with proteins that need to be degraded," he says. This breakdown in protein quality control may lead toward cell damage and death.

"We were quite surprised because the ER didn't seem to have any connection with the misfolded proteins in the cytosol," Duennwald adds.

"This study tells us to investigate cellular pathways beyond the usual suspects."

He went on to uncover the basis for this breakdown: The polyQ-expanded fragments glom onto the key VCP/Npl4/Ufd1 protein complex that aids in the transport and degradation of the proteins that flunk quality control in the ER. When Duennwald genetically modified cells to over-express two crucial proteins in the protein complex, the toxic effect dropped.

Additionally, his experiments showed that polyQ-expanded proteins avoid a main method by which cells deal with misfolded proteins. Generally, a class of proteins called "chaperone" or "heat shock" proteins move in and either help the misfolded proteins assume their normal shape or help to get rid of them.

"Amazingly, polyQ-expanded proteins don't elicit the heat shock response, and that might contribute to their toxicity," Duennwald says.

Such findings may help in eventually treating the disease. The research suggests that activating the cell's protein quality control mechanisms may provide novel and effective strategies for combating Huntington's and other illnesses driven by polyQ-expanded proteins.

Reference:
Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity
Martin L. Duennwald and Susan Lindquist
Genes & Development, December 2008
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How Cilia Make Us Asymmetric

FoxJ1 helps cilia beat a path to asymmetry
Monday, 17 November 2008

New work at the Salk Institute for Biological Studies reveals how a genetic switch, known as FoxJ1, helps developing embryos tell their left from their right. While at first glance the right and left sides of our bodies are identical to each other, this symmetry is only skin-deep. Below the surface, some of our internal organs are shifted sideways — heart and stomach to the left, liver and appendix to the right.

Creating this left-right asymmetry is a key step in early embryonic development, and requires hundreds of tiny hair-like structures called nodal cilia to beat in unison. Like microscopic conductors, cilia orchestrate a flow of embryonic fluid from right to left that allows the growing tissues to orient themselves. The current study provides new insight into the crucial role FoxJ1 plays in directing the development of these cilia.

"This one transcription factor regulates a whole suite of genes needed to coordinate the formation of nodal cilia," says Christopher R. Kintner, Ph.D., a professor in the Molecular Neurobiology Laboratory, who led the study. Strikingly, FoxJ1 can induce cilia to form on the surface of cells that do not usually have them, the Salk researchers report in this week's early online edition of Nature Genetics. Their findings may one day lead to a cure for ciliopathies, diseases that result from malfunctioning or damaged cilia.

Cilia — tiny hair-like protrusions found on certain cell types — come in three flavours. Motile cilia crowd the surface of specialized cells and move in harmony to generate liquid flow. They are used to sweep mucus and dirt out of our lungs and in females to propel the egg from the ovary through the Fallopian tube into the uterus. Unlike motile cilia, sensory cilia usually number just one per cell and are used to relay information back to the cell about its surroundings.

A third and less characterized subtype are nodal cilia. Nodal cilia share certain features with both their sensory and motile counterparts; they exist one per cell yet function to generate the fluid movement during embryo development that is crucial to forming the left-right asymmetry.

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In the developing embryo, nodal cilia (shown in green) orchestrate a flow of embryonic fluid from right to left that allows the growing tissues to orientate themselves. Credit: Courtesy of Jennifer Stubbs, Salk Institute for Biological Studies.

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"We were interested in the developmental cues that drive the formation of these different cilia subtypes," says Kintner. Clues from previous work in mice persuaded Kintner and his team to take a closer look at cilia in the South African clawed frog Xenopus, a model popular with developmental biologists, and zebrafish.

In mice, FoxJ1 is needed to drive the formation of motile but not sensory cilia. The Salk researchers depleted FoxJ1 in both Xenopus and zebrafish by injecting embryos with morpholinos, synthetic DNA-like structures that bind to nucleic acids and work like dimmer switches to turn down gene expression. When FoxJ1 was turned down, nodal cilia development was disrupted, causing organ displacement and defects in the left-right asymmetry.

The real surprise came when the scientists increased the levels of FoxJ1.

"We started seeing cilia popping up all over the place," says Kintner, "and they were not random subtypes; they looked just like the nodal cilia that form on the cells to generate the embryonic left-right flow."

"These ectopic cilia were really interesting," adds Jennifer Stubbs, first author of the study and a graduate student in the Kintner lab, "and no one had been able to show them in any other system."

These findings call into question current theories as to how FoxJ1 regulates motile cilia. Motile cilia are anchored to the cell surface at sites called basal bodies, and FoxJ1's role in their development was thought to act primarily by regulating this docking process. Since activating FoxJ1 was sufficient to drive the formation of cilia in usually cilia-less cells, however, Kintner and colleagues reasoned that FoxJ1 must play a broader role in promoting cilia development.

They tested this hypothesis using microarray analysis to determine what genes FoxJ1 activated. Indeed, FoxJ1 increased the levels of a host of genes involved in motile cilia development rather than just a small set relating to the basal body.

"This really suggests that at least in Xenopus, FoxJ1 is a master-regulator of ciliogenesis and doesn't just play a role in basal body docking," says Stubbs.

Kintner and colleagues are currently investigating in closer detail the suite of genes activated by FoxJ1 to further understand its mode of action.

“Doing so might help develop novel therapies to treat ciliopathies, whose symptoms range from respiratory defects to infertility. In many diseases such as chronic asthmas and cystic fibrosis, trouble clearing mucus causes defects where the ciliated cells begin to die," says Kintner, "and knowing about the dominant pathways that drive differentiation of ciliated cells types might allow us to do something prevent that situation."

"It may provide a way of repairing ciliated cells that are already there, enabling them to regrow their cilia," says Jennifer Stubbs.
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Tiny Sacs from Cells Carry Information about Tumours

Exosomes deliver factors that promote tumour growth and may serve as blood biomarkers
Monday, 17 November 2008

Microvesicles – tiny membrane-covered sacs – released from glioblastoma cells contain molecules that may provide data that can guide treatment of the deadly brain tumour. In their report in the December 2008 Nature Cell Biology, which is receiving early online release, Massachusetts General Hospital (MGH) researchers describe finding tumour-associated RNA and proteins in membrane microvesicles called exosomes in blood samples from glioblastoma patients. Detailed analysis of exosome contents identified factors that could facilitate a tumour's growth through delivery of genetic information or proteins, or signify its vulnerability to particular medications.

"Glioblastomas release exosomes in sufficient quantities to pass the blood-brain barrier. We were able to isolate them, analyze the RNA transcripts and show how they might be used as biomarkers to guide targeted therapy and monitor treatment response," says Johan Skog, PhD, the study's lead author, who works in the laboratory of Xandra Breakefield, PhD, at the MGH Neuroscience Center.

"Exosomes also may someday be used to deliver therapeutic molecules to the site of a tumour," he added.

Many types of cells release exosomes as part of normal cell-to-cell communication, and several types of tumours are known to shed exosomes containing proteins that can alter the cellular environment to favour tumour growth. The current investigation is believed to be the first to carefully analyze the contents of exosomes shed from glioblastoma cells and characterize their contents.

The investigators first analyzed tumour cells from three glioblastomas and verified that the cells released exosomes containing RNA and protein molecules. Some messenger RNAs related to activities such as cell proliferation and migration, angiogenesis, and immune response were highly abundant in the exosomes. When glioblastoma exosomes were cultured with normal cells, tumour RNA was delivered into the normal cells and generated its encoded protein, supporting the role of exosome-delivered RNA in manipulating the cellular environment.

To study the potential of glioblastoma exosomes as markers of a tumour's genetic makeup, the researchers analyzed tumour tissue and blood serum from 25 glioblastoma patients and were able both to find tumour exosomes and to identify, in some tissue samples, a mutation in the epidermal growth factor receptor (EGFR) gene that characterizes a tumour subtype. In two patients, an EGFR mutation that did not appear in the tumour tissue sample was identified by exosome analysis, reflecting how a surgical biopsy can miss tissue conveying critical information because of the often-chaotic diversity of cells within a tumour.

"It is known that the effects of some anticancer drugs depend on a tumour's genetic mutational profile, so our results have broad implications for personalized medicine," explains Skog, who is an instructor in Neurology at Harvard Medical School.

"Detecting mutational profiles through a non-invasive blood test could allow us to monitor how a tumour's genetic makeup changes in response to therapy, which may necessitate changes in treatment strategy."

Skog, Breakefield and their colleagues are also investigating the role of exosomes in other solid tumours and how they may help monitor additional tumour-associated mutations.

The current study was supported by grants from the Wenner-Gren Foundation, Stiftelsen Olle Engkvist Byggmästare, the National Cancer Institute, the Brain Tumour Society and the American Brain Tumour Association. The MGH's provisional patent on the work described in this study has been exclusively licensed to Exosome Diagnostics, Inc.. Subsequent to the completion of this work, Skog was appointed the company's director of Research, while maintaining his position at MGH.
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Newborn Neurons in the Adult Brain Can Settle in the Wrong Neighbourhood

Newborn Neurons in the Adult Brain Can Settle in the Wrong Neighbourhood
Wednesday, 12 November 2008

A new study published in this week's PLoS Biology could have significant consequences for neural tissue transplantation to treat brain injuries or neural degeneration. Researchers at the Salk Institute for Biological Studies report that inactivating a specific gene in adult neural stem cells makes nerve cells emerging from those precursors form connections in the wrong part of the adult brain.

The research team, led by Professor Fred H. Gage, Ph.D., discovered that a protein called cdk5 is necessary for both correct elaboration of highly branched and complex dendrites, a kind of antennae, which are extended by neurons. Cdk5 is also involved in the proper migration of cells bearing those antennae.

Previously described functions of cdk5 are manifold, among them neuronal migration and dendritic path-finding of neurons born during embryonic development.

"The surprising element was that the dendrites of newborn granule cells in the adult hippocampus lacking cdk5 stretched in the wrong direction and actually formed synapses with the wrong cells," explains Gage. Synapses are the specialized contact points where dendrites receive input from the long processes, or axons, of neighbouring neurons. The investigators injected retroviruses into the hippocampus of adult mice to tag and knock out cdk5 activity in newborn granule cell neurons.

These findings offer extremely valuable, unanticipated insight.

"Our data shows that cells that fail to find their 'right spot' might actually become integrated into the brain and possibly interfere with normal information processing," says the study's lead author Sebastian Jessberger, M.D., a former postdoc in the Gage lab and now an assistant professor at the Swiss Federal Institute of Technology in Zurich, Switzerland.

"We found that dendrites of cells lacking cdk5 seemed to integrate into the brain no matter what direction they grew in," he says.

Gage notes that the findings "reflect the need for therapeutic approaches that will assure that cells used in regenerative medicine are strategically placed so that they will make appropriate rather than promiscuous connections."

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Newborn neurons deficient in cdk5 (green) extend aberrant dendrites that nonetheless synaptically integrate into the pre-existing dentate circuitry containing neurons (red) and glial cells (blue). From: For New Neurons in an Old Brain, cdk5 Shows the Way Robinson R PLoS Biology Vol. 6, No. 11, e291 doi:10.1371/journal.pbio.0060291.

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In fact, the inappropriate synaptic connections made by cdk5-deficient cells persisted for months after the treatment with cdk5-antagonizing retroviruses.

"One might have predicted that aberrant maturing nerve cells would get kicked out of the circuitry later on," reports Jessberger, who followed the behaviour of newborn granule cells in treated mice long after cdk5 activity was eliminated.

"Even after one year, some of those cells remained in the wrong part of the hippocampus."

"The nice part of this story is that it emerged from a systems genetics approach we used in a previous study," says Gage.

"It continues our effort to apply genetic analysis to find chromosomal regions harbouring genes that may play a critical role in neurogenesis."

Reference:
Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus
Sebastian Jessberger, Stefan Aigner, Gregory D. Clemenson Jr., Nicolas Toni, D. Chichung Lie, Özlem Karalay, Rupert Overall, Gerd Kempermann, Fred H. Gage
PLoS Biol (2008) 6(11): e272. doi:10.1371/journal.pbio.0060272

See also:
For New Neurons in an Old Brain, cdk5 Shows the Way
Richard Robinson
PLoS Biol (2008), 6(11): e291 doi:10.1371/journal.pbio.0060291
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Teeth Stem Cells Can Stimulate Growth of Brain Cells in Monkeys

Teeth Stem Cells Can Stimulate Growth of Brain Cells in Monkeys
Wednesday, 12 November 2008

Researchers at the Yerkes National Primate Research Center, Emory University, have discovered dental pulp stem cells can stimulate growth and generation of several types of neural cells. Findings from this study, available in the October issue of the journal Stem Cells, suggest dental pulp stem cells show promise for use in cell therapy and regenerative medicine, particularly therapies associated with the central nervous system.

Dental stem cells are adult stem cells, one of the two major divisions of stem cell research. Adult stem cells have the ability to regenerate many different types of cells, promising great therapeutic potential, especially for diseases such as Huntington's and Parkinson's. Already, dental pulp stem cells have been used for regeneration of dental and craniofacial cells.

Yerkes researcher Anthony Chan, DVM, PhD, and his team of researchers placed dental pulp stem cells from the tooth of a rhesus macaque into the hippocampal areas of mice. The dental pulp stem cells stimulated growth of new neural cells, and many of these formed neurons.

"By showing dental pulp stem cells are capable of stimulating growth of neurons, our study demonstrates the specific therapeutic potential of dental pulp stem cells and the broader potential for adult stem cells," says Chan, who also is assistant professor of human genetics in Emory School of Medicine.

Because dental pulp stem cells can be isolated from anyone at any age during a visit to the dentist, Chan is interested in the possibility of dental pulp stem cell banking.

"Being able to use your own stem cells for therapy would greatly decrease the risk of cell rejection that we now experience in transplant medicine," says Chan.

Chan and his research team next plan to determine if dental pulp stem cells from monkeys with Huntington's disease can enhance brain cell development in the same way dental pulp stem cells from healthy monkeys do.

Reference:
Putative Dental Pulp-Derived Stem/Stromal Cells Promote Proliferation and Differentiation of Endogenous Neural Cells in the Hippocampus of Mice
Anderson Hsien-Cheng Huang, Brooke R. Snyder, Pei-Hsun Cheng, Anthony W.S. Chan
Stem Cells Vol. 26 No. 10 October 2008, pp. 2654 -2663, doi:10.1634/stemcells.2008-0285
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Biological Reaction Essential to Life Takes 2.3B Years Without Enzyme

Biological Reaction Essential to Life Takes 2.3B Years Without Enzyme
Tuesday, 11 November 2008

All biological reactions within human cells depend on enzymes. Their power as catalysts enables biological reactions to occur usually in milliseconds. But how slowly would these reactions proceed spontaneously, in the absence of enzymes – minutes, hours, days? And why even pose the question?

One scientist who studies these issues is Richard Wolfenden, Ph.D., Alumni Distinguished Professor Biochemistry and Biophysics and Chemistry at the University of North Carolina at Chapel Hill. Wolfenden holds posts in both the School of Medicine and in the College of Arts and Sciences and is a member of the National Academy of Sciences.

In 1995, Wolfenden reported that without a particular enzyme, a biological transformation he deemed "absolutely essential" in creating the building blocks of DNA and RNA would take 78 million years.

"Now we've found a reaction that – again, in the absence of an enzyme – is almost 30 times slower than that," Wolfenden said.

"Its half-life – the time it takes for half the substance to be consumed – is 2.3 billion years, about half the age of the Earth. Enzymes can make that reaction happen in milliseconds."

With co-author Charles A. Lewis, Ph.D., a postdoctoral scientist in his lab, Wolfenden published a report of their new findings recently in the online early edition of the Proceedings of the National Academy of Science. The study is also due to appear in the Nov. 11 print edition.

The reaction in question is essential for the biosynthesis of haemoglobin and chlorophyll, Wolfenden noted. But when catalyzed by the enzyme uroporphyrinogen decarboxylase, the rate of chlorophyll and haemoglobin production in cells "is increased by a staggering factor, one that's equivalent to the difference between the diameter of a bacterial cell and the distance from the Earth to the sun."

"This enzyme is essential for both plant and animal life on the planet," Wolfenden said.

"What we're defining here is what evolution had to overcome, that the enzyme is surmounting a tremendous obstacle, a reaction half-life of 2.3 billion years."

Knowing how long reactions would take without enzymes allows biologists to appreciate their evolution as prolific catalysts, Wolfenden said. It also enables scientists to compare enzymes with artificial catalysts produced in the laboratory.

"Without catalysts, there would be no life at all, from microbes to humans," he said.

"It makes you wonder how natural selection operated in such a way as to produce a protein that got off the ground as a primitive catalyst for such an extraordinarily slow reaction."

Experimental methods for observing very slow reactions can also generate important information for rational drug design based on cellular molecular studies.

"Enzymes that do a prodigious job of catalysis are, hands-down, the most sensitive targets for drug development," Wolfenden said.

"The enzymes we study are fascinating because they exceed all other known enzymes in their power as catalysts."

Wolfenden has carried out extensive research on enzyme mechanisms and water affinities of biological compound. His work has also influenced rational drug design, and findings from his laboratory helped spur development of ACE inhibitor drugs, now widely used to treat hypertension and stroke. Research on enzymes as proficient catalysts also led to the design of protease inhibitors that are used to treat HIV infection.

"We've only begun to understand how to speed up reactions with chemical catalysts, and no one has even come within shouting distance of producing, or predicting the magnitude of, their catalytic power," Wolfenden said.

Reference:
Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes
Charles A. Lewis, Jr. and Richard Wolfenden
PNAS, November 6, 2008, doi: 10.1073/pnas.0809838105
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Umbilical Cord Blood May Help Build New Heart Valves

Umbilical Cord Blood May Help Build New Heart Valves
Tuesday, 11 November 2008

Children with heart defects may someday receive perfectly-matched new heart valves built using stem cells from their umbilical cord blood, according to research presented at the American Heart Association's Scientific Sessions 2008.

When infants are born with malfunctioning heart valves that cannot be surgically repaired, they rely on replacements from animal tissue, compatible human organ donations or artificial materials. These replacements are lifesaving, but do not grow and change shape as a child develops; so two or more surgeries may be needed to replace outgrown valves. The animal tissue may also stiffen over time as well and be less durable than a normal human valve. With artificial valves, children also must be treated with blood thinners.

"In our concept, if prenatal testing shows a heart defect, you could collect blood from the umbilical cord at birth, harvest the stem cells, and fabricate a heart valve that is ready when the baby needs it," said Ralf Sodian, M.D., lead author of the study and a cardiac surgeon at the University Hospital of Munich.

The tissue engineering of heart valves is still in its infancy, with various researchers investigating the possibility of using cells from blood, bone marrow or amniotic fluid.

In the study, the research team used stem cells (CD133+ cells) derived from umbilical cord blood. The cord blood was frozen to preserve it. After 12 weeks, the cells were seeded onto eight heart valve scaffolds constructed of a biodegradable material and then grown in a laboratory.

Afterwards, examination using electron microscopes revealed that the cells had grown into pores of the scaffolding and formed a tissue layer. Biochemical examination indicated that the cells had not only survived and grown, but had produced important elements of the "extracellular matrix," the portion of body tissue that functions outside of cells and is essential to tissue function and structure. Compared with human tissue from pulmonary heart valves, the tissue-engineered valves formed:

• 77.9 percent as much collagen (the main protein in connective tissue);
• 85 percent as much glycosaminoglycan, a carbohydrate important in connective tissue); and
• 67 percent as much elastin (a protein in connective tissue)
  

Furthermore, using antibodies to detect various proteins, the researchers found the valves contained desmin (a protein in muscle cells), laminin (a protein in all internal organs), alpha-actin (a protein that helps muscle cells contract) and CD31, VWF and VE-cadherin (components of blood vessel linings).

"These markers all indicate that human cardiovascular tissue was grown in the lab," Sodian said.

Several important questions remain to be solved regarding tissue-engineered functional heart valves, including identifying the optimal scaffold material and learning how to condition the valves in the laboratory so they work properly after being implanted, Sodian said.

"Tissue engineering provides the prospect of an ideal heart valve substitute that lasts throughout the patient's lifetime and has the potential to grow with the recipient and to change shape as needed," he said. .........


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Mechanism that Regulates the Development of Stem Cells into Neurons

Findings provide insight into potential therapies for neurodegenerative disorders and cancers
Tuesday, 11 November 2008

Researchers at the University of Southern California (USC) have identified a novel mechanism in the regulation and differentiation of neural stem cells.

Researchers found that the protein receptor Ryk has a key role in the differentiation of neural stem cells, and demonstrated a signalling mechanism that regulates neuronal differentiation as stem cells begin to grow into neurons. The study will be published in the Nov. 11 issue of the journal Developmental Cell.

The findings could have important implications for regenerative medicine and cancer therapies, says Wange Lu, Ph.D., assistant professor of biochemistry and molecular biology at the Keck School of Medicine of USC, and the principal investigator on the study.

"Neural stem cells can potentially be used for cell-replacement therapy for neurodegenerative diseases such as Alzheimer's and Parkinson's Disease, as well as spinal cord injury," Lu says.

"Knowledge gained from this study will potentially help to generate neurons for such therapy. This knowledge can also be used to inhibit the growth of brain cancer stem cells."

During brain development, neural stem cells respond to the surrounding environment by either proliferation or differentiation, but the molecular mechanisms underlying the development of neural stem cells and neurons are unclear, Lu notes.

Ryk functions as a receptor of Wnt proteins required for cell-fate determination, axon guidance and neurite outgrowth in organisms. Researchers at the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC analyzed sections of the forebrain in animal model embryos to investigate Ryk's function in vivo.

They found that during neurogenesis, when neural stem cells start to grow into neurons, Ryk protein is cleaved and translocates to the cell nucleus to regulate neuronal differentiation.

This finding is extremely important for understanding the regulation of self-renewal and differentiation of neural stem cells, Lu says. Previous research has shown that Ryk functions as a receptor of Wnt proteins. However, the role of Ryk in neural stem cells and the molecular mechanism of Ryk signalling have not previously been known.

"This study will help in our efforts to produce nerve cells from embryonic stem cells, and may lead to the development of new strategies for the repair of the nervous system, using protein or small molecule therapeutic agents," says Martin Pera, Ph.D., director of the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC.

Further research is needed to explore how Ryk regulates neuronal gene expression, Lu says. Researchers are now expanding their research to studies of differentiation of human embryonic stem cells into neural stem cells and neurons. These studies are very important for regenerative medicine and drug discovery for therapy of neurodegenerative diseases.

Reference:
Cleavage of Wnt Receptor Ryk Regulates Neuronal Differentiation during Cortical Neurogenesis
Jungmook Lyu, Vicky Yamamoto and Wange Lu.
Developmental Cell, Volume 15, Issue 5, 773-780, 11 Nov. 2008, doi:10.1016/j.devcel.2008.10.004
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Genetic Blueprint for Kidney Design and Formation

Cincinnati Children's-led study opens way for new research of development, disease
Tuesday, 11 November 2008

Researchers have generated the first comprehensive genetic blueprint of a forming mammalian organ, shedding light on the genetic and molecular dynamics of kidney development.

Part of an international consortium sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), a research team led by Cincinnati Children's Hospital Medical Center reports the creation of a detailed genome-based atlas for understanding healthy and abnormal kidney development and disease. Published in the Nov. 11 Developmental Cell and featured on the journal's cover, the research provides a molecular genetic map detailing "gene expression analysis of all the major elements of kidney formation," according to the investigators.

The study, involving embryonic mice, shows how the entire genome is regulated to produce thousands of specific genes that are mixed and re-mixed to form genetic teams. The teams work together to direct formation of 15 embryonic compartments in the developing kidney – from the earliest phases when stem cells are told how to differentiate into specific kidney cells to the development of nephrons, the kidney's primary functioning unit.

"This study establishes a baseline for what changing gene expression looks like in a normal developing kidney in a very global way," said Steven Potter, Ph.D., a researcher in the division of Developmental Biology at Cincinnati Children's and the study's senior author.

"Now we have molecular insights that will allow us to understand specific interactions throughout all stages of kidney development."

Dr. Potter explained this will let researchers analyze kidney abnormalities in mutant mice "in a much more complete and profound way than ever before. Given the mouse's genetic similarities with people, this should help us understand the underpinnings of human disease," he said.

The researchers conducted their multi-step analysis of mouse embryonic kidneys that were aged 15.5 days. This developmental time point in a mouse's normal 19- to 21-day gestation allows multiple stages of kidney formation to be studied at once because of how the organ develops. The organ's outer layers contain early stem cells that are still differentiating to become specific cell types, while inside the organ structures are forming at intermediate and more mature stages. This enabled measurement of varied gene expression stage-by-stage, compartment by compartment, the researchers said.

One of the study's more unexpected discoveries is overlapping gene expression between the kidney's different structures, according to Eric Brunskill, Ph.D., the study's lead author. Most of the thousands of genes involved in making a mammalian kidney are expressed at some level in every compartment. Previously it had been thought each kidney compartment would have unique genes driving its development, and those genes would not be expressed in the cells of other structures. This is not the case, as the research team found only a small number of genes expressed exclusively in specific kidney structures.

"Instead of it being a digital on-off pattern, where you might have many unique genes expressed in one part of the kidney but not in the other structures, we instead see a more analogue picture, where almost all of the genes are expressed in the different parts but at varied levels," Dr. Brunskill said.

Helping make this discovery possible is the study's use of microarray technology to measure relative expression levels of every gene in each unique structure and developmental stage. Combined with two other technologies to precisely isolate specific types of cell populations (laser capture micro-dissection and florescent activated cell sorting), microarray analysis allowed a more quantitative and sensitive measure of varied gene expression than ever observed in a developing organ system.

Computational biology analysis (bioinformatics) then let the researchers see how different sets of genes cooperate through circuits or pathways, some already defined and others defined in this study for the first time. The genes cooperate by signalling each other, telling cells when to grow, when to make tubes, when to turn on pumps or perform other critical functions, said Bruce Aronow, Ph.D., study co-author and scientific director of the Center for Computational Medicine at Cincinnati Children's. The study also makes new headway into identifying different transcription factors – "boss" genes that regulate the activity of other genes – and the target genes they may activate or repress.

Given that about one in every 500 births results in a kidney development abnormality, this provides insight into genetic programs that are critical to deciding how kidney stem cells form structures in the adult kidney. The researchers identified genes that regulate DNA transcription, establish functioning developmental processes, and are involved in pattern specification, cell differentiation and organ compartment shaping.

About Cincinnati Children's Hospital Medical Center:
Cincinnati Children's Hospital Medical Center is one of America's top three children's hospitals for general paediatrics and is highly ranked for its expertise in digestive diseases, respiratory diseases, cancer, neonatal care, heart care and neurosurgery, according to the annual ranking of best children's hospitals by U.S. News & World Report. One of the three largest children's hospitals in the U.S., Cincinnati Children's is affiliated with the University of Cincinnati College of Medicine and is one of the top two recipients of paediatric research grants from the National Institutes of Health. For its achievements in transforming healthcare, Cincinnati Children's is one of six U.S. hospitals since 2002 to be awarded the American Hospital Association-McKesson Quest for Quality Prize ® for leadership and innovation in quality, safety and commitment to patient care. The hospital is a national and international referral centre for complex cases, so that children with the most difficult-to-treat diseases and conditions receive the most advanced care leading to better outcomes.

Reference:
Atlas of Gene Expression in the Developing Kidney at Microanatomic Resolution
Eric W. Brunskill, Bruce J. Aronow, Kylie Georgas, Bree Rumballe, M. Todd Valerius, Jeremy Aronow, Vivek Kaimal, Anil G. Jegga, Sean Grimmond, Andrew P. McMahon, Larry T. Patterson, Melissa H. Little, S. Steven Potter
Developmental Cell, Volume 15, Issue 5, 781-791, 11 Nov. 2008, doi:10.1016/j.devcel.2008.09.007
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Iran to Invest Heavily in Stem Cell Research

Iran to Invest $2.5B in Stem Cell Research
Saturday, 08 November 2008

Iranian news organisations report that Iran's Cord Blood Bank is to invest $2.5B in the country's stem cell research over the next five years.

Iranian scientists developed human embryonic stem cell lines already in 2003. This was done with the highest Islamist approval, from the Ayatollah Seyed Ali Khamenei himself. Some Muslim clerics acknowledge that human life begins only three months after conception, thus granting scientists access to blastocysts, early embryos and human embryonic stem cells left over from fertilization trials. Iran has therefore some of the most liberal laws providing grounds for such studies.

According to Mohammad Reza Mohammad Hassani, the general secretary of the 10th National Congress on Cardiovascular Updates, Iran's achievements in this field have led to a successful stem cell heart transplant of an 11-year-old boy in 2003.

The Royan Institute is the main place behind Iran's fast progress in human stem cell technology. Initially being an infertility clinic in Tehran established in 1991, they produced Iran’s first human embryonic stem cell line in 2003. So far, they have established six different human embryonic stem cell lines and numerous mouse ESC lines as well. They have also used adult stem cells to treat corneal injuries and heart muscles after myocardial infarct in humans, and a diverse set of animal models of diseases. In 2006 they succeeded to clone a sheep; and in 2004 induce hESCs to produce insulin.

Iran’s Health ministry have also established a stem cell network in 2005, the Iranian Stem Cell Network, in an ambitious attempt to promote stem cell research between scientists, clinicians and business people from the 17 participating institutes and research centres.

See also:
Iranian Scientists Produce A Human ESC Line
CellNEWS - Tuesday, 09 September 2003
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Key Trigger of Embryonic Stem Cell Differentiation

Key Trigger of Embryonic Stem Cell Differentiation
Thursday, 06 November 2008

Clusters of mouse embryonic stem cells called embryoid bodies more closely approximate true embryos in organization and structure than previously thought, according to researchers at the Stanford University School of Medicine. Harnessing the signals that influence the cells' fate may help researchers more accurately direct the differentiation of embryonic stem cells for use in therapy.

The researchers found that embryoid bodies have hallmarks of gastrulation - a remarkable developmental step that launches a hollow ball of cells toward becoming an organism with three distinct types of precursor cells. The scientists showed that this process is initiated by a single signalling pathway in embryoid bodies and in real embryos. Enhancing or blocking this signal affects what the cells become, the scientists found.

"A lot of embryonic stem cell research is aimed at devising ways to help the cells differentiate along a particular path," said Roeland Nusse, PhD, professor of developmental biology.

"But it's very difficult to know how to do this. We're learning that they do more things in culture than we previously thought; at the same time, we're developing more tools to control what they become."

Nusse is the senior author of the research, which will be published in the Nov. 6 issue of the journal Cell Stem Cell. He is also a Howard Hughes Medical Institute investigator and a member of Stanford's Cancer Center. The study was funded in part by a grant from the California Institute of Regenerative Medicine intended to clarify the role of a common group of cell signalling molecules called the Wnt family in the differentiation of embryonic stem cells.

Nusse and the first authors of the paper, postdoctoral scholar Derk ten Berge, PhD, and undergraduate student Wouter Koole, used easily tracked reporter genes that are expressed only when cells are responding to Wnt signals to figure out when and where Wnt is active in mouse embryos and embryoid bodies. Embryoid bodies are clumps of embryonic stem cells that can to begin to differentiate into different tissues but they are not true embryos.

Using this system, the researchers learned that Wnt-responsive cells first appear in 6.5-day-old embryos in an area called the primitive streak that forms on what will become the posterior side of the embryo. It is the first step toward gastrulation, in which an outer layer of cells dimples inward at what will be either the mouth or anus to form the three distinct precursor cell types shared by most animals: the ectoderm, or outer layer, which forms neurons, skin cells and pigment; the endoderm, or inner layer, which forms many of the organs; and mesoderm, or middle layer, which forms muscle and red blood cells.

More importantly, Nusse and his colleagues determined that Wnt-responsive cells in the embryoid bodies also spontaneously form a primitive streak, though they never truly gastrulate. Supplementing the naturally occurring Wnt signal with "extra" Wnt protein accelerated the formation of the primitive streak, and adding proteins that blocked Wnt activity inhibited it.

"We knew that embryoid bodies did exhibit some self-organization," said Nusse.

"They form a hollow cavity with inner and outer cell layers. But the primitive streak is the first indication we have that they can develop the kind of asymmetry that is seen in embryos."

Furthermore, the extra Wnt caused the Wnt-responsive cells to differentiate primarily into mesendodermal precursors (which can become either mesoderm or endoderm and is associated with the posterior of the embryo) and inhibited the formation of neurectoderm (ectoderm destined to become cells of the nervous system that are mostly associated with the embryo's anterior). Blocking Wnt activity tipped the balance in the other direction, causing the cells to shun mesendoderm and become mainly neurectoderm.

The first step to controlling cell fate is to understand which protein in the normal cocktail of growth factors used to maintain the embryoid bodies is responsible for triggering the cells' Wnt-responsive pathways. The researchers identified one specific factor, called BMP, that gets the ball rolling. Inhibiting this factor stops the spontaneous formation of the primitive streak in the embryoid bodies and gives the researcher more precise control over the cells' differentiation.

"Differentiation is a step-wise process," said Nusse.

"To get to a particular endpoint, you need to know all the steps along the way. Our research indicates that embryoid bodies are a better-than-expected model of what happens in the embryo, and suggests how we may be able to manipulate those steps to our advantage to get pure populations of certain types of cells for research or therapy."

Reference:
Wnt Signaling Mediates Self-Organization and Axis Formation in Embryoid Bodies
Derk ten Berge, Wouter Koole, Christophe Fuerer, Matt Fish, Elif Eroglu and Roel Nusse
Cell Stem Cell, Volume 3, Issue 5, 508-518, 6 November 2008, doi:10.1016/j.stem.2008.09.013
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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DNA Chunks in Chimps and Humans

Marks of differences between human and chimp genomes
Thursday, 06 November 2008

Researchers have carried out the largest study of differences between human and chimpanzee genomes, identifying regions that have been duplicated or lost during evolution of the two lineages. The study, published in Genome Research, is the first to compare many human and chimpanzee genomes in the same fashion.

The team show that particular types of genes - such as those involved in the inflammatory response and in control of cell proliferation - are more commonly involved in gain or loss. They also provide new evidence for a gene that has been associated with susceptibility to infection by HIV.

"This is the first study of this scale, comparing directly the genomes of many humans and chimpanzees," says Dr Richard Redon, from the Wellcome Trust Sanger Institute, a leading author of the study.

"By looking at only one 'reference' sequence for human or chimpanzee, as has been done previously, it is not possible to tell which differences occur only among individual chimpanzees or humans and which are differences between the two species.”

"This is our first view of those two important legacies of evolution."

Rather than examining single-letter differences in the genomes (so-called SNPs), the researchers looked at copy number variation (CNV) - the gain or loss of regions of DNA. CNVs can affect many genes at once and their significance has only been fully appreciated within the last two years. The team looked at genomes of 30 chimpanzees and 30 humans: a direct comparison of this scale or type has not been carried out before.

The comparison uncovered CNVs that are present in both species as well as copy number differences (CNDs) between the two species. CNDs are likely to include genes that have influenced evolution of each species since humans and chimpanzees diverged some six million years ago.

"Broadly, the two genomes have similar patterns and levels of CNVs - around 70-80 in each individual - of which nearly half occur in the same regions of the two species' genomes," continues Dr Redon.

"But beyond that similarity we were able to find intriguing evidence for key sets of genes that differ between us and our nearest relative."

One of the genes affected by CNVs is CCL3L1, for which lower copy numbers in humans have been associated with increased susceptibility to HIV infection. Remarkably, the study of 60 human and chimpanzee genomes found no evidence for fixed CNDs between human and chimp and no within-chimp CNV. Rather, they found that a nearby gene called TBC1D3 was reduced in number in chimpanzee compared to human: typically, there were eight copies in human, but apparently only one in all chimpanzees.

The authors suggest that it might be evolutionary selection of CNDs in TBC1D3 that have driven the population differences. Consistent with this novel observation, TBC1D3 is involved in cell proliferation (favoured category) and is on a core region for duplication - a focal point for large regions of duplication in human genome.

"It is evident that there has been striking turnover in gene content between humans and chimpanzees, and some of these changes may have resulted from exceptional selection pressures," explains Dr George Perry from Arizona State University and Brigham and Women's Hospital, another leading author of the study.

"For example, a surprisingly high number of genes involved in the inflammatory response - APOL1, APOL4, CARD18, IL1F7, IL1F8 - are completely deleted from chimp genome. In humans, APOL1 is involved in resistance to the parasite that causes sleeping sickness, while IL1F7 and CARD18 play a role in regulating inflammation: therefore, there must be different regulations of these processes in chimpanzees.”

"We already know that inactivation of an immune system gene from the human genome is being positively selected: now we have an example of similar consequences in the chimpanzee."

CNVs in humans and chimpanzees often occur in equivalent genomic locations: most lie in regions of the genomes, called segmental duplications, which are particularly 'fragile'. However, one in four of the 355 CNDs that the team found do not overlap with CNVs within either species - suggesting that they are variants that are 'fixed' in each species and might mark significant differences between human and chimpanzee genomes.

DNA Samples and analysis
The project used DNA samples from 30 chimpanzees (29 from W Africa, one from E Africa): the chimpanzee reference was produced using DNA from Clint, the chimpanzee whose DNA was used for the genome sequence.

Human DNA samples were obtained from following participants: ten Yoruba (Ibadan, Nigeria), ten Biaka rainforest hunter-gatherers (Central African Republic) and ten Mbuti rainforest hunter-gatherers (Democratic Republic of Congo). The human reference is a European-American male from the HapMap Project (NA10852).

Participating Centres
School of Human Evolution & Social Change, Arizona State University, Tempe, AZ, USA
Department of Pathology, Brigham & Women's Hospital, Boston, MA, USA
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
Department of Genome Sciences, University of Washington School of Medicine and the Howard Hughes Medical Institute, Seattle, WA, USA
Harvard Medical School, Boston, MA, USA

Websites
http://www.sanger.ac.uk/Teams/Team29/
http://www.sanger.ac.uk/Teams/Team70/
http://www.sanger.ac.uk/humgen/cnv/


Reference:
Copy number variation and evolution in humans and chimpanzees
George H. Perry, Fengtang Yang, Tomas Marques-Bonet, Carly Murphy, Tomas Fitzgerald, Arthur S. Lee, Courtney Hyland, Anne C. Stone, Matthew E. Hurles, Chris Tyler-Smith, Evan E. Eichler, Nigel P. Carter, Charles Lee, and Richard Redon
Genome Research 18: 1698-1710 (2008)
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Scientists Improve iPS Cell Method

Scientists Improve iPS Cell Method
Thursday, 06 November 2008

Scientists from the Scripps Research Institute have identified a combination of drugs that can be used to reprogram cells from human tissue to become similar to embryonic stem cells so that they may develop into all the cell types in the body.

The Scripps method, documented in a story published Thursday in the journal Cell Stem Cell, improves upon a process that until now involved using genes and viruses to coax human cells backward down the development pathway until they are pluripotent, meaning they can become many different cell types. One of the genes used, known as Sox2, had previously been regarded as essential for the reprogramming process.

In June, the Scripps team, led by Sheng Ding, associate professor in the Department of Chemistry at The Scripps Research Institute, showed that it could use drugs to create pluripotent cells from the brain cells of mice. This recent Scripps study appears to be the first published showing success with an alternative method for creating human pluripotent cells.

Ding said the work of his team, which included scientists from the Max Planck Institute for Molecular Biomedicine in Germany, could be used to identify other drugs and small molecules that might also be used in the process for different results.

Reference:
Induction of Pluripotent Stem Cells from Mouse Embryonic Fibroblasts by Oct4 and Klf4 with Small-Molecule Compounds
Yan Shi, Caroline Desponts, Jeong Tae Do, Heung Sik Hahm, Hans R. Schöler and Sheng Ding
Cell Stem Cell, Volume 3, Issue 5, 568-574, 6 November 2008, doi:10.1016/j.stem.2008.10.004

See also:
Embryo-free Stem Cell Research Gets an Advance
CellNEWS - Thursday, 05 June 2008

Scientists Identify Synthetic Compound that Keeps Stem Cells Young
CellNEWS - Wednesday, 08 November 2006
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ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Three More Complete Human Genomes Published

Number of sequenced human genomes doubles
Thursday, 06 November 2008

This week, three reports describe the first African, the first Asian, and the first cancer patient to have their entire DNA deciphered. The sequences provide clues about genome variation and disease; they also demonstrate the potential of a relatively new sequencing technique to mass-produce human genomes.

Scientists have sequenced the entire gene maps of two men, one Chinese and the other African, for a fraction of the price that such exercises used to cost. The Chinese project cost US$500,000, down from what had cost hundreds of millions of dollars when the sequencing of the first human genome was completed in 2004. Another team of scientists, led by David Bentley of Illumina Cambridge Ltd, sequenced the gene map of the African man and described the process as "low cost."

The third group sequenced the complete DNA of a female suffering from acute myeloid leukaemia (AML) and identified a limited, but new set of genes responsible for this severe type of cancer. This is the second comlete female genome to be reported.

The teams published their findings in separate articles in the science journal Nature.

See more:
Scientists Decode Cancer Patient's Genome
CellNEWS -Wednesday, 05 November 2008

First Human Female DNA Sequenced
CellNEWS - Monday, 26 May 2008

Chinese Scientists Map Out First Asian Genome
CellNEWS - Friday, 12 October 2007

References:
Accurate whole human genome sequencing using reversible terminator chemistry
David R. Bentley, Shankar Balasubramanian, Harold P. Swerdlow, Geoffrey P. Smith, John Milton, Clive G. Brown, Kevin P. Hall, Dirk J. Evers, Colin L. Barnes, Helen R. Bignell, Jonathan M. Boutell, Jason Bryant, Richard J. Carter, R. Keira Cheetham, Anthony J. Cox, Darren J. Ellis, Michael R. Flatbush, Niall A. Gormley, Sean J. Humphray, Leslie J. Irving, Mirian S. Karbelashvili, Scott M. Kirk, Heng Li, Xiaohai Liu, Klaus S. Maisinger, Lisa J. Murray, Bojan Obradovic, Tobias Ost, Michael L. Parkinson, Mark R. Pratt, Isabelle M. J. Rasolonjatovo, Mark T. Reed, Roberto Rigatti, Chiara Rodighiero, Mark T. Ross, Andrea Sabot, Subramanian V. Sankar, Aylwyn Scally, Gary P. Schroth, Mark E. Smith, Vincent P. Smith, Anastassia Spiridou, Peta E. Torrance, Svilen S. Tzonev, Eric H. Vermaas, Klaudia Walter, Xiaolin Wu, Lu Zhang, Mohammed D. Alam, Carole Anastasi, Ify C. Aniebo, David M. D. Bailey, Iain R. Bancarz, Saibal Banerjee, Selena G. Barbour, Primo A. Baybayan, Vincent A. Benoit, Kevin F. Benson, Claire Bevis, Phillip J. Black, Asha Boodhun, Joe S. Brennan, John A. Bridgham, Rob C. Brown, Andrew A. Brown, Dale H. Buermann, Abass A. Bundu, James C. Burrows, Nigel P. Carter, Nestor Castillo, Maria Chiara E. Catenazzi, Simon Chang, R. Neil Cooley, Natasha R. Crake, Olubunmi O. Dada, Konstantinos D. Diakoumakos, Belen Dominguez-Fernandez, David J. Earnshaw, Ugonna C. Egbujor, David W. Elmore, Sergey S. Etchin, Mark R. Ewan, Milan Fedurco, Louise J. Fraser, Karin V. Fuentes Fajardo, W. Scott Furey, David George, Kimberley J. Gietzen, Colin P. Goddard, George S. Golda, Philip A. Granieri, David E. Green, David L. Gustafson, Nancy F. Hansen, Kevin Harnish, Christian D. Haudenschild, Narinder I. Heyer, Matthew M. Hims, Johnny T. Ho, Adrian M. Horgan, Katya Hoschler, Steve Hurwitz, Denis V. Ivanov, Maria Q. Johnson, Terena James, T. A. Huw Jones, Gyoung-Dong Kang, Tzvetana H. Kerelska, Alan D. Kersey, Irina Khrebtukova, Alex P. Kindwall, Zoya Kingsbury, Paula I. Kokko-Gonzales, Anil Kumar, Marc A. Laurent, Cynthia T. Lawley, Sarah E. Lee, Xavier Lee, Arnold K. Liao, Jennifer A. Loch, Mitch Lok, Shujun Luo, Radhika M. Mammen, John W. Martin, Patrick G. McCauley, Paul McNitt, Parul Mehta, Keith W. Moon, Joe W. Mullens, Taksina Newington, Zemin Ning, Bee Ling Ng, Sonia M. Novo, Michael J. O'Neill, Mark A. Osborne, Andrew Osnowski, Omead Ostadan, Lambros L. Paraschos, Lea Pickering, Andrew C. Pike, Alger C. Pike, D. Chris Pinkard, Daniel P. Pliskin, Joe Podhasky, Victor J. Quijano, Come Raczy, Vicki H. Rae, Stephen R. Rawlings, Ana Chiva Rodriguez, Phyllida M. Roe, John Rogers, Maria C. Rogert Bacigalupo, Nikolai Romanov, Anthony Romieu, Rithy K. Roth, Natalie J. Rourke, Silke T. Ruediger, Eli Rusman, Raquel M. Sanches-Kuiper, Martin R. Schenker, Josefina M. Seoane, Richard J. Shaw, Mitch K. Shiver, Steven W. Short, Ning L. Sizto, Johannes P. Sluis, Melanie A. Smith, Jean Ernest Sohna Sohna, Eric J. Spence, Kim Stevens, Neil Sutton, Lukasz Szajkowski, Carolyn L. Tregidgo, Gerardo Turcatti, Stephanie vandeVondele, Yuli Verhovsky, Selene M. Virk, Suzanne Wakelin, Gregory C. Walcott, Jingwen Wang, Graham J. Worsley, Juying Yan, Ling Yau, Mike Zuerlein, Jane Rogers, James C. Mullikin, Matthew E. Hurles, Nick J. McCooke, John S. West, Frank L. Oaks, Peter L. Lundberg, David Klenerman, Richard Durbin & Anthony J. Smith
Nature 456, 53-59, 6 November 2008, doi:10.1038/nature07517

The diploid genome sequence of an Asian individual
Jun Wang, Wei Wang, Ruiqiang Li, Yingrui Li, Geng Tian, Laurie Goodman, Wei Fan, Junqing Zhang, Jun Li, Juanbin Zhang, Yiran Guo, Binxiao Feng, Heng Li, Yao Lu, Xiaodong Fang, Huiqing Liang, Zhenglin Du, Dong Li, Yiqing Zhao, Yujie Hu, Zhenzhen Yang, Hancheng Zheng, Ines Hellmann, Michael Inouye, John Pool, Xin Yi, Jing Zhao, Jinjie Duan, Yan Zhou, Junjie Qin, Lijia Ma, Guoqing Li, Zhentao Yang, Guojie Zhang, Bin Yang, Chang Yu, Fang Liang, Wenjie Li, Shaochuan Li, Dawei Li, Peixiang Ni, Jue Ruan, Qibin Li, Hongmei Zhu, Dongyuan Liu, Zhike Lu, Ning Li, Guangwu Guo, Jianguo Zhang, Jia Ye, Lin Fang, Qin Hao, Quan Chen, Yu Liang, Yeyang Su, A. san, Cuo Ping, Shuang Yang, Fang Chen, Li Li, Ke Zhou, Hongkun Zheng, Yuanyuan Ren, Ling Yang, Yang Gao, Guohua Yang, Zhuo Li, Xiaoli Feng, Karsten Kristiansen, Gane Ka-Shu Wong, Rasmus Nielsen, Richard Durbin, Lars Bolund, Xiuqing Zhang, Songgang Li, Huanming Yang & Jian Wang
Nature 456, 60-65, 6 November 2008, doi:10.1038/nature07484

DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome
Timothy J. Ley, Elaine R. Mardis, Li Ding, Bob Fulton, Michael D. McLellan, Ken Chen, David Dooling, Brian H. Dunford-Shore, Sean McGrath, Matthew Hickenbotham, Lisa Cook, Rachel Abbott, David E. Larson, Dan C. Koboldt, Craig Pohl, Scott Smith, Amy Hawkins, Scott Abbott, Devin Locke, LaDeana W. Hillier, Tracie Miner, Lucinda Fulton, Vincent Magrini, Todd Wylie, Jarret Glasscock, Joshua Conyers, Nathan Sander, Xiaoqi Shi, John R. Osborne, Patrick Minx, David Gordon, Asif Chinwalla, Yu Zhao, Rhonda E. Ries, Jacqueline E. Payton, Peter Westervelt, Michael H. Tomasson, Mark Watson, Jack Baty, Jennifer Ivanovich, Sharon Heath, William D. Shannon, Rakesh Nagarajan, Matthew J. Walter, Daniel C. Link, Timothy A. Graubert, John F. DiPersio & Richard K. Wilson
Nature 456, 66-72, 6 November 2008, doi:10.1038/nature07485
.........


ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Scientists Decode Cancer Patient's Complete Genome

Scientists Decode Cancer Patient's Complete Genome
Wednesday, 05 November 2008

For the first time, scientists have decoded the complete DNA of a cancer patient and traced her disease - acute myelogenous leukaemia - to its genetic roots. A large research team at the Genome Sequencing Center and the Siteman Cancer Center at Washington University School of Medicine in St. Louis sequenced the genome of the patient - a woman in her 50s who ultimately died of her disease - and the genome of her leukaemia cells, to identify genetic changes unique to her cancer.

The study is reported in the Nov. 6 issue of the journal Nature.

The pioneering work sets the stage for using a more comprehensive, genome-wide approach to unravel the genetic basis of cancer.

"Our work demonstrates the power of sequencing entire genomes to discover novel cancer-related mutations," says senior author Richard K. Wilson, Ph.D., director of Washington University's Genome Sequencing Center.

"A genome-wide understanding of cancer, which is now possible with faster, less expensive DNA sequencing technology, is the foundation for developing more effective ways to diagnose and treat cancer."

The researchers discovered just 10 genetic mutations in the patient's tumour DNA that appeared to be relevant to her disease; eight of the mutations were rare and occurred in genes that had never been linked to AML. They also showed that virtually every cell in the tumour sample had nine of the mutations, and that the single genetic alteration that occurred less frequently was likely the last to be acquired. The scientists suspect that all the mutations were important to the patient's cancer.

Like most cancers, AML – a cancer of blood-forming cells in the bone marrow – arises from mutations that accumulate in people's DNA over the course of their lives. However, little is known about the precise nature of those changes and how they disrupt biological pathways to cause the uncontrolled cell growth that is the hallmark of cancer.

Previous efforts to decode individual human genomes have looked at common points of DNA variation that may be relevant for disease risk. What is striking about the new research is that the scientists were able to sift through the 3 billion pairs of chemical bases that make up the human genome to pull out the mutations that contributed to the patient's cancer.

"Until now, no one has sequenced a patient's genome to find all the mutations that are unique to that person's disease," says lead author Timothy Ley, M.D., a haematologist and the Alan A. and Edith L. Wolff Professor of Medicine.

"We didn't know what we would find, but we felt that the answers to why this patient had AML had to be embedded in her DNA."

To date, scientists involved in large-scale genetic studies of cancer have not gone so far as to do a full side-by-side comparison of the genomes of normal cells and tumour cells from the same patient. Rather, most earlier studies have involved the sequencing of genes with known or suspected relationships to cancer, a method that likely misses key mutations.

"The determination of the first complete DNA sequence of a human cancer genome, and its comparison to normal tissues of the same individual, is a true landmark in cancer research," says geneticist Francis Collins, M.D., Ph.D., former director of the National Human Genome Research Institute.

"In the past, cancer researchers have been 'looking under the lamppost' to find the causes of malignancy – but now the team from Washington University has lit up the whole street. This achievement ushers in a new era of comprehensive understanding of the fundamental nature of cancer, and offers great promise for the development of powerful new approaches to diagnosis, prevention and treatment."

An estimated 13,000 cases of AML will be diagnosed in the United States this year, and some 8,800 will die of the disease. It occurs most often among those age 60 or older and becomes more difficult to treat as patients age. According to the American Cancer Society, the five-year survival rate for AML is 21 percent.

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Acute myelogenous leukaemia cells. Credit: Washington University.

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Despite advances in the genetic understanding of many cancers, scientists have learned very little about the genetic basis of AML.

"After years of genetic studies of AML looking at genes of interest, we were getting no closer to uncovering the molecular underpinnings of the disease," Ley says.

"We felt that with new genome sequencing technology, now was the time to take a whole-genome approach."

Based on genetic testing with traditional methods at the study's outset, the patient was known to have two mutations that are common among AML patients, an indicator she had a typical subtype of the disease, and one of the many reasons why her genome was selected for sequencing.

The researchers sequenced the patient's full genome, meaning DNA from both sets of chromosomes, using genetic material obtained from a skin sample. This gave the scientists a reference DNA sequence to which they could compare genetic alterations in the patient's tumour cells, taken from a bone marrow sample that was comprised only of tumour cells. Both samples were obtained before the patient received cancer treatment, which can further damage DNA.

The scientists then looked for genetic differences – points of single base changes in the DNA – in the patient's tumour genome compared with her normal genome. Of the nearly 2.7 million single nucleotide variants in the patient's tumour genome, almost 98 percent also were detected in DNA from the patient's skin sample, thus narrowing the number of variants that required further study to about 60,000.

Using sophisticated software and analytical tools, some of which the researchers developed specifically for this project, they identified the 10 mutations (including the two previously known genetic mutations that are common to her leukaemia subtype but do not directly cause the disease) by looking for single base DNA changes that altered the instructions for making proteins.

Of the eight novel mutations discovered, three were found in genes that normally act to suppress tumour growth. One of these mutations is in the PTPRT tyrosine phosphatase gene, which is frequently altered in colon cancer.

Four other mutated genes appear to be involved in molecular pathways that promote cancer growth. In particular, one mutation was found in a gene family that also is expressed in embryonic stem cells and may be involved with cell self-renewal. Interestingly, the researchers note, self-renewal is thought to be an essential feature of leukaemia cells.

Another gene alteration appears to affect the transport of drugs into the cell, and may have contributed to the patient's chemotherapy resistance.

"We're still analyzing the patient's non-coding DNA and expect to find a number of additional relevant mutations in this portion of the genome," says Elaine Mardis, Ph.D., co-lead author of the study and co-director of the Genome Sequencing Center.

"But the role of these non-coding mutations will be more of a challenge to elucidate because we do not yet fully understand the function of this part of the genome."

The team also looked to see if the eight novel mutations in the patient's tumour genome also occurred in the DNA of tumour samples from 187 additional AML patients. None of those tumours had any of the eight mutations.

"This suggests that there is a tremendous amount of genetic diversity in cancer, even in this one disease," Wilson says.

"There are probably many, many ways to mutate a small number of genes to get the same result, and we're only looking at the tip of the iceberg in terms of identifying the combinations of genetic mutations that can lead to AML."

Based on their current understanding of cancer, the researchers suspect that the mutations occurred sequentially. The first mutation gave the cell a slight tendency toward cancer, and then one by one, the other genetic alterations were acquired, with each contributing something to the cancer. One mutation, in the FLT3 gene, was not present in all of the tumour cells, and they suspect that it was the last one to occur.

"The final mutation may represent a tipping point that causes the cancer cells to become more dangerous," Ley says.

The team is now sequencing the genomes of additional patients with AML, and they are also planning to expand the whole-genome approach to breast and lung cancers.

This type of approach is exactly what is needed to understand the genetic basis of cancer, an essential first step to developing targeted therapies, says Brian Druker, M.D., whose research helped identify the targeted drug Gleevec as a promising therapy for chronic myelogenous leukaemia. Druker, the director of the Oregon Health & Science University Cancer Institute and a Howard Hughes Medical Institute investigator, was not involved in the current study.

"This tour-de-force effort identified a small number of mutations in genes that no one predicted, and their uniqueness for this patient begins to give us a glimmer of the genetic complexity and diversity of this disease," he says.

"Although this information doesn't yet tell us how to treat patients, it is a critical first step along that path. It sets the stage for large scale sequencing of cancer genomes and unravelling the mystery of cancer."

Reference:
DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome
Timothy J. Ley, Elaine R. Mardis, Li Ding, Bob Fulton, Michael D. McLellan, Ken Chen, David Dooling, Brian H. Dunford-Shore, Sean McGrath, Matthew Hickenbotham, Lisa Cook, Rachel Abbott, David E. Larson, Dan C. Koboldt, Craig Pohl, Scott Smith, Amy Hawkins, Scott Abbott, Devin Locke, LaDeana W. Hillier, Tracie Miner, Lucinda Fulton, Vincent Magrini, Todd Wylie, Jarret Glasscock, Joshua Conyers, Nathan Sander, Xiaoqi Shi, John R. Osborne, Patrick Minx, David Gordon, Asif Chinwalla, Yu Zhao, Rhonda E. Ries, Jacqueline E. Payton, Peter Westervelt, Michael H. Tomasson, Mark Watson, Jack Baty, Jennifer Ivanovich, Sharon Heath, William D. Shannon, Rakesh Nagarajan, Matthew J. Walter, Daniel C. Link, Timothy A. Graubert, John F. DiPersio & Richard K. Wilson
Nature 456, 66-72, 6 November 2008, doi:10.1038/nature07485
.........


ZenMaster

---

For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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More 'Junk' DNA Proves Functional

Helps explain human differences from other species
Tuesday, 04 November 2008

In a paper published in Genome Research on Nov. 4, scientists at the Genome Institute of Singapore (GIS) report that what was previously believed to be "junk" DNA is one of the important ingredients distinguishing humans from other species.

More than 50 percent of human DNA has been referred to as "junk" because it consists of copies of nearly identical sequences. A major source of these repeats is internal viruses that have inserted themselves throughout the genome at various times during mammalian evolution.

Using the latest sequencing technologies, GIS researchers showed that many transcription factors, the master proteins that control the expression of other genes, bind specific repeat elements. The researchers showed that from 18 to 33% of the binding sites of five key transcription factors with important roles in cancer and stem cell biology are embedded in distinctive repeat families.

Over evolutionary time, these repeats were dispersed within different species, creating new regulatory sites throughout these genomes. Thus, the set of genes controlled by these transcription factors is likely to significantly differ from species to species and may be a major driver for evolution.

This research also shows that these repeats are anything but "junk DNA," since they provide a great source of evolutionary variability and might hold the key to some of the important physical differences that distinguish humans from all other species.

The GIS study also highlighted the functional importance of portions of the genome that are rich in repetitive sequences.

"Because a lot of the biomedical research use model organisms such as mice and primates, it is important to have a detailed understanding of the differences between these model organisms and humans in order to explain our findings," said Guillaume Bourque, Ph.D., GIS Senior Group Leader and lead author of the Genome Research paper.

"Our research findings imply that these surveys must also include repeats, as they are likely to be the source of important differences between model organisms and humans," added Dr. Bourque.

"The better our understanding of the particularities of the human genome, the better our understanding will be of diseases and their treatments."

"The findings by Dr. Bourque and his colleagues at the GIS are very exciting and represent what may be one of the major discoveries in the biology of evolution and gene regulation of the decade," said Raymond White, Ph.D., Rudi Schmid Distinguished Professor at the Department of Neurology at the University of California, San Francisco, and chair of the GIS Scientific Advisory Board.

"We have suspected for some time that one of the major ways species differ from one another – for instance, why rats differ from monkeys – is in the regulation of the expression of their genes: where are the genes expressed in the body, when during development, and how much do they respond to environmental stimuli," he added.

"What the researchers have demonstrated is that DNA segments carrying binding sites for regulatory proteins can, at times, be explosively distributed to new sites around the genome, possibly altering the activities of genes near where they locate. The means of distribution seem to be a class of genetic components called 'transposable elements' that are able to jump from one site to another at certain times in the history of the organism. The families of these transposable elements vary from species to species, as do the distributed DNA segments which bind the regulatory proteins."

Dr. White also added:

"This hypothesis for formation of new species through episodic distributions of families of gene regulatory DNA sequences is a powerful one that will now guide a wealth of experiments to determine the functional relationships of these regulatory DNA sequences to the genes that are near their landing sites. I anticipate that as our knowledge of these events grows, we will begin to understand much more how and why the rat differs so dramatically from the monkey, even though they share essentially the same complement of genes and proteins."

Genome Institute of Singapore:
The Genome Institute of Singapore (GIS) is a member of the Agency for Science, Technology and Research (A*STAR). It is a national initiative with a global vision that seeks to use genomic sciences to improve public health and public prosperity. Established in 2001 as a centre for genomic discovery, the GIS will pursue the integration of technology, genetics and biology towards the goal of individualized medicine. The key research areas at the GIS include Systems Biology, Stem Cell & Developmental Biology, Cancer Biology & Pharmacology, Human Genetics, Infectious Diseases, Genomic Technologies, and Computational & Mathematical Biology. The genomics infrastructure at the GIS is utilized to train new scientific talent, to function as a bridge for academic and industrial research, and to explore scientific questions of high impact.

Agency for Science, Technology and Research (A*STAR):
A*STAR is Singapore's lead agency for fostering world-class scientific research and talent for a vibrant knowledge-based Singapore. A*STAR actively nurtures public sector research and development in Biomedical Sciences, Physical Sciences and Engineering, with a particular focus on fields essential to Singapore's manufacturing industry and new growth industries. It oversees 22 research institutes, consortia and centres, and supports extramural research with the universities, hospital research centres and other local and international partners. At the heart of this knowledge intensive work is human capital. Top local and international scientific talent drive knowledge creation at A*STAR research institutes. The agency also sends scholars for undergraduate, graduate and post-doctoral training in the best universities, a reflection of the high priority A*STAR places on nurturing the next generation of scientific talent.

Reference:
Evolution of the mammalian transcription factor binding repertoire via transposable elements
Guillaume Bourque, Bernard Leong, Vinsensius B. Vega, Xi Chen, Yen Ling Lee, Kandhadayar G. Srinivasan, Joon-Lin Chew, Yijun Ruan, Chia-Lin Wei, Huck Hui Ng, and Edison T. Liu
Genome Research, Nov. 4, 2008

See also:
Study Finds Value in 'Junk' DNA
CellNEWS - Friday, 17 October 2008
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ZenMaster

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Tuesday 4 November, 2008

Effective HPV Vaccine Needed for Both Men and Women

Effective HPV Vaccine Needed for Both Men and Women
Monday, 03 November 2008

A call to explore a broader use of HPV (human papillomavirus) vaccines and the validation of a simple oral screening test for HPV-caused oral cancers are reported in two studies by a Johns Hopkins Kimmel Cancer Center investigator.

Leading HPV expert Maura Gillison, M.D., Ph.D., reports her latest work in the November 3, 2008, journal Clinical Cancer Research and in a Centers for Disease Control and Prevention (CDC) monograph. The CDC report on HPV-associated cancers appears on line November 3 and in the November 15, 2008, supplement edition of Cancer. She was the first to identify HPV infection as the cause of certain oral cancers and who identified multiple sex partners as the most important risk factor for these cancers.

In the CDC report, believed to be the first and most comprehensive assessment of HPV-associated cancer data in the United States, investigators analyzed cancer registry data from 1998-2003 and found 25,000 cancer cases each year occurred at cancer sites associated with HPV infection. In additional analysis, Gillison and colleagues at the National Cancer Institute identified HPV infection as the underlying cause of approximately 20,000 of these cancers.

Gillison and team found approximately 20,000 cases of cancer in the United States each year are caused by HPV infection. Oral cancers are the second most common type of HPV-associated cancers and are increasing in incidence in the U.S., particularly among men. Add to that anal, penile, vaginal, and vulvar cancers that are also linked to HPV infection, and Gillison says these cancers, when combined, equal the number of cervical cancers, the most common and well known of the cancers caused by HPV.

While about one-quarter of HPV-linked cancers occur in men, vaccines are currently approved only for use in girls and young women for cervical cancer prevention.

"We need to have a more comprehensive discussion of the potential impact the HPV vaccine could have on cancer rates among men and women in this country," says Gillison, associate professor of oncology.

"Currently available HPV vaccines have the potential to reduce the rates of HPV-associated cancers, like oral and anal cancers, that are currently on the rise and for which there no effective or widely-applied screening programs." Gillison notes, however, that studies are needed to confirm that the vaccine effectively prevents HPV infections that lead to oral and anal cancers.

Gillison's findings were part of a project known as ABHACUS (Assessing the Burden of Human Papillomavirus-Associated Cancers). The data studied came from the CDC's National Program of Cancer Registries and the National Cancer Institute's Surveillance, Epidemiology, and End Results program. More than 80 investigators from across the country participated in the project, which addressed a variety of HPV-cancer associated issues, including racial disparity, economic impact, behavioural risk factors, and cancer mortality.

Other than prevention, early detection is held by cancer experts as the best way to control cancer. In the Clinical Cancer Research study, the first to track the disease and related oral infections over an extended period, Gillison found that simple "swish and spit" oral rinses can successfully track oral HPV infection over time. These findings open the door to a potential, non-invasive screening test to detect the disease and monitor for tumour recurrence. Head and neck cancer is the broad term for a variety of cancers of the oral cavity, including the tonsils, base of the tongue, and the side and back wall of the throat.

The study found that oral rinses successfully detected high-risk HPV infections in patients with HPV 16-positive head and neck cancers for up to five years after treatment for their cancer. Gillison says the findings indicate a high rate of persistent infection and reaffirms the connection between high-risk types of HPV and HPV-positive head and neck cancers.

In the study, the researchers used oral rinses to collect cells shed from inside the mouths of 135 head and neck cancer patients. The researchers genetically sequenced the DNA obtained from the rinses and tumour samples to identify those with HPV-positive cancers and determine the HPV type. There are approximately 120 types of HPV, but HPV 16 is one of the two most common associated with cancer.

The analysis revealed 44 patients with HPV 16-positive tumours and found that these patients were more likely to have continuing oral HPV 16 infections both before and after cancer treatment. While this study did not link the continued post-treatment infections to tumour recurrence, it was noted that patients with high-risk oral HPV infections prior to therapy, maintained high rates of infection after completing therapy. The team plans further, long-term research to determine if this continued infection leads to cancer recurrence.

In 2000, Gillison identified HPV-positive head and neck cancer as a distinct subtype of the disease and linked it to improved survival.

"There is no question of cause," says Gillison.

"It has now become a question of tracking the infection over time to identify those at risk of developing cancer or cancer recurrence."

See also:
CDC Releases First Estimate of Human Papillomavirus-Associated Cancer Data
CDC News - November 3, 2008
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
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Human Genes Sing Different Tunes in Different Tissues

Biologists find almost all genes express multiple messenger RNAs
Sunday, 02 November 2008

Scientists have long known that it's possible for one gene to produce slightly different forms of the same protein by skipping or including certain sequences from the messenger RNA. Now, an MIT team has shown that this phenomenon, known as alternative splicing, is both far more prevalent and varies more between tissues than was previously believed.

Nearly all human genes, about 94 percent, generate more than one form of their protein products, the team reports in the Nov. 2 online edition of Nature. Scientists' previous estimates ranged from a few percent 10 years ago to 50-plus percent more recently.

"A decade ago, alternative splicing of a gene was considered unusual, exotic … but it turns out that's not true at all — it's a nearly universal feature of human genes," said Christopher Burge, senior author of the paper and the Whitehead Career Development Associate Professor of Biology and Biological Engineering at MIT.

Burge and his colleagues also found that in most cases the mRNA produced depends on the tissue where the gene is expressed. The work paves the way for future studies into the role of alternative proteins in specific tissues, including cancer cells.

They also found that different people's brains often differ in their expression of alternative spliced mRNA isoforms.

Human genes typically contain several "exons," or DNA sequences that code for amino acids, the building blocks of proteins. A single gene can produce multiple protein sequences, depending on which exons are included in the mRNA transcript, which carries instructions to the cell's protein-building machinery.

Two different forms of the same protein, known as isoforms, can have different, even completely opposite functions. For example, one protein may activate cell death pathways while its close relative promotes cell survival.

The researchers found that the type of isoform produced is often highly tissue-dependent. Certain protein isoforms that are common in heart tissue, for example, might be very rare in brain tissue, so that the alternative exon functions like a molecular switch. Scientists who study splicing have a general idea of how tissue-specificity may be achieved, but they have much less understanding of why isoforms display such tissue specificity, Burge said.

Scientists have also observed that cells express different isoforms during embryonic development and at different stages of cellular differentiation. Burge's team is now studying cells at various stages of differentiation to see when different isoforms are expressed.

Isoform switching also occurs in cancer cells. One such switch involves a metabolic enzyme and contributes to cancer cells burning large amounts of glucose and growing more rapidly. Learning more about such switches could lead to potential cancer therapies, Burge said.

Until now, it has been difficult to study isoforms on a genome-wide scale because of the high cost of sequencing and technical issues in discriminating similar mRNA isoforms using microarrays. The team took mRNA samples from 10 types of tissue and five cell lines from a total of 20 individuals, and generated more than 13 billion base pairs of sequence, the equivalent of more than four entire human genomes.

The sequencing was done by researchers at biotech firm Illumina, using a new high-throughput sequencing machine.

Reference:
Alternative isoform regulation in human tissue transcriptomes
Eric T. Wang, Rickard Sandberg, Shujun Luo, Irina Khrebtukova, Lu Zhang, Christine Mayr, Stephen F. Kingsmore, Gary P. Schroth & Christopher B. Burge
Nature advance online publication 2 November 2008, doi:10.1038/nature07509

See also:
Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing
Qun Pan, Ofer Shai, Leo J Lee, Brendan J Frey & Benjamin J Blencowe
Nature Genetics, Published online: 2 November 2008, doi:10.1038/ng.259
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Panoramic View of Protein-RNA Interactions in Living Cells

Panoramic View of Protein-RNA Interactions in Living Cells
Sunday, 02 November 2008

DNA, it has turned out, is not all it was cracked up to be. In recent years we learned that the molecule of life, the discovery of the 20th century, did not – could not – by itself explain the huge differences in complexity between a human and a worm. Forced to look elsewhere, scientists turned to RNA, a direct yet more complex transcript of DNA. But methodological problems have historically plagued the study of RNA regulation in living cells, limiting not only the accuracy of results but also our understanding of RNA's role in human disease.

But now, in research to appear in the November 2 advance online issue of Nature, Robert B. Darnell, head of the Laboratory of Molecular Neuro-oncology at Rockefeller University and a Howard Hughes Medical Institute investigator, and his team have changed all that.

By adapting techniques mastered in the test tube and combining them with high throughput technology, the team has developed a genome-wide platform to study how specialized proteins regulate RNA in living, intact cells. The platform allows researchers to identify, in a single experiment, every sequence within every strand of RNA to which proteins bind. The result is an unbiased and unprecedented look at how differences in RNA can explain how a worm and a human can each have 25,000 genes yet be so different.

"RNA offers a way to make the cell much more complex than what this limited set of genes can offer," says Darnell, who is Robert and Harriet Heilbrunn Professor at Rockefeller.

"But how is RNA being regulated in different conditions and diseases, and in different cell types? With this platform, we now have a way to address all these questions."

Traditional methods used molecules to extract protein-RNA complexes from living tissue. But often the molecule only extracted the RNA. Other times, the protein bound too weakly to survive the purification process, which involved stripping the complex of unwanted debris. To address the issue, Darnell and his team used a trick from test-tube biochemistry that molecularly cements these regulatory proteins to RNA at the moment they touch. The technique, when applied to high throughput sequencing, is called high throughput sequencing-cross linking immunoprecipitation, or HITS-CLIP for short.

Since the RNA and RNA-binding protein are fused together, the researchers can really beat up the extract and rigorously purify the protein without fear of losing the RNA. At the end of the day, they are left with the RNA sequence to which the protein was bound. They can then take these sequences to Rockefeller's high throughput sequence facility, and with the help of Research Support Specialist Scott Dewell, overlay them onto the genome and see where they match. What they get is a map of every position on every transcribed RNA where the RNA binding protein is binding.

When DNA is transcribed into RNA, the primary transcript is divided into many blocks called exons, which are separated by empty spaces. In order to convert the transcript into some sort of message, all the spaces need to be removed; but if an exon is dropped, a different version of that protein, which could carry a very different message, is created.

"That's RNA splicing," says first author Donny Licatalosi, a postdoctoral associate in the lab.

"It is what gives rise to this massive pool of diverse and complex tissues with a relatively small number of genes."

In the past, the group used a sophisticated process of evidence and inference to make predictions of the points of regulation along the transcript.

"Now, we have direct biochemical validation that these interactions occur in the brain to regulate splicing," says Licatalosi.

"The observed map – and this was amazing – looked just like our predicted map," says Darnell.

Darnell, Licatalosi and their colleagues Aldo Mele, a research assistant, John Fak, a research assistant, Sung-Wook Chi, a graduate fellow in computational biology and medicine, Xuning Wang, assistant director of biocomputing and Jennifer Darnell, a research associate professor, looked at an RNA-binding protein called Nova2 that is found exclusively in neurons. They found that depending on where Nova2 binds to RNA, they could predict and directly observe whether an exon would be included or excluded in the final transcript, and which protein version it created.

"The cell seems to be going through great trouble to regulate these RNAs in different conditions and different cell types," says Darnell.

"When RNA developed the ability to make a more stable copy of itself – DNA – it didn't write itself off as a relic for the textbooks. It stayed at the core of complex processes in the cell."

Reference:
HITS-CLIP yields genome-wide insights into brain alternative RNA processing
Donny D. Licatalosi, Aldo Mele, John J. Fak, Jernej Ule, Melis Kayikci, Sung Wook Chi, Tyson A. Clark, Anthony C. Schweitzer, John E. Blume, Xuning Wang, Jennifer C. Darnell & Robert B. Darnell
Nature advance online publication 2 November 2008, doi:10.1038/nature07488
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Mending Broken Hearts with Tissue Engineering

New scaffold approach could also aid engineering of other tissues
Sunday, 02 November 2008

Broken hearts could one day be mended using a novel scaffold developed by MIT researchers and colleagues.

The idea is that living heart cells or stem cells seeded onto such a scaffold would develop into a patch of cardiac tissue that could be used to treat congenital heart defects, or aid the recovery of tissue damaged by a heart attack. The biodegradable scaffold would be gradually absorbed into the body, leaving behind new tissue.

The accordion-like honeycomb scaffold, to be reported in the Nov. 2 online edition of Nature Materials, is the first to be explicitly designed to match the structural and mechanical properties of native heart tissue. As a result, it has several advantages over previous cardiac tissue engineering scaffolds.

Further, the MIT team's general approach has applications to other types of engineered tissues.

"In the long term we'd like to have a whole library of scaffolds for different tissues in need of repair," said Lisa E. Freed, corresponding author of the paper and a principal research scientist in the Harvard-MIT Division of Health Sciences and Technology (HST). Each scaffold could be tailor-made with specific structural and mechanical properties.

"We're already on the way to a few other examples," Freed said.

With respect to the current work, "previous scaffolds did not necessarily possess structural or mechanical properties consistent with the native myocardial [heart muscle] structure," said George C. Engelmayr Jr., lead author of the paper and an HST postdoctoral fellow. Heart muscle, he explained, is "directionally dependent" — meaning its cells are aligned in specific directions.

The researchers reasoned that "borrowing more closely from nature's lessons," as they write in Nature Materials, might lead to a tissue with properties closer to the real thing. So, using a laser similar to that used for eye surgery, they created a scaffold with directionally dependent structural and mechanical properties.

The scaffold has three principal advantages over its predecessors. First, its mechanical properties closely match those of native heart tissue. For example, it is stiffer when stretched circumferentially as compared to longitudinally.

Engelmayr found that he could essentially "dial in" specific mechanical properties for the polymer scaffold by varying the time it is allowed to set, or cure. He noted that with this ability, coupled with the flexibility of the laser technique, "we might be able to come up with even better pore shapes with better mechanical properties."

In a second advantage, the team found that a patch of tissue created from neonatal rat heart cells cultured on the scaffold showed directionally dependent electrophysiological properties similar to native tissue. In other words, when an electrical field was applied the engineered patch contracted more readily in one direction than in another.

In a third advantage, "the scaffold itself has an intrinsic ability to guide the orientation of cultured heart cells," Freed said. Freed was part of another MIT team that, in 2004, showed that heart cells cultured on a traditional scaffold could also be coaxed into alignment, but only with electrical stimulation.

The researchers note that the scaffold used in the experiments described above has some limitations. For example, they write, it is "too thin to address reconstruction of full-thickness myocardium." However, as they report in Nature Materials, they have already begun addressing those problems by creating new honeycomb scaffolds that, among other things, allow much thicker, multi-layered tissue structures.

Reference:
Accordion-like honeycombs for tissue engineering of cardiac anisotropy
George C. Engelmayr, Jr, Mingyu Cheng, Christopher J. Bettinger, Jeffrey T. Borenstein, Robert Langer & Lisa E. Freed
Nature Materials, Published online: 2 November 2008, doi:10.1038/nmat2316
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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