Promoting Healing by Keeping Skeletal Stem Cells 'Young'

Kicking Notch up a notch could aid treatment of osteoporosis, fractures, arthritis
Wednesday, 31 March 2010

Scientists seeking new ways to fight maladies ranging from arthritis and osteoporosis to broken bones that will not heal have cleared a formidable hurdle, pinpointing and controlling a key molecular player to keep stem cells in a sort of extended infancy. It's a step that makes treatment with the cells in the future more likely for patients.

Controlling and delaying development of the cells, known as mesenchymal stem cells, is a long-sought goal for researchers. It's a necessary step for doctors who would like to expand the number of true skeletal stem cells available for a procedure before the cells start becoming specific types of cells that may – or may not – be needed in a patient with, say, weak bones from osteoporosis, or an old knee injury.

"A big problem has been that these stem cells like to differentiate rapidly – oftentimes too rapidly to make them very useful," said Matthew J. Hilton, Ph.D., the leader of the team at the University of Rochester Medical Center.

"It's been very hard to get a useful number of stem cells that can still become any one of several types of tissue a patient might need. Having a large population of true skeletal stem cells available is a key consideration for new therapies, and that's been a real roadblock thus far."

In a study published online in the journal Development, Hilton's team discussed how it was able to increase the number and delay the development of stem cells that create bones, cartilage, muscle and fat. The first authors of the paper are Yufeng Dong, Ph.D., senior instructor, and technician Alana Jesse, who worked in Hilton's laboratory at the Center for Musculoskeletal Research.

Hilton's team showed in mice that a molecule called Notch, which is well known for the influence it wields on stem cells that form the blood and the nervous system, is a key factor in the development of mesenchymal stem cells, which make up a tiny fraction of the cells in the bone marrow and other tissues.

The team showed that Notch prevents stem cells from maturing. When the scientists activated the Notch pathway, the stem cells did not progress as usual. Instead, they remained indefinitely in an immature state and did not go on to become bone cells, cartilage cells, or cells for connective tissue.

The team also settled a long-standing question, fingering the molecule RBPJ-kappa as the molecule through which Notch works in mesenchymal stem cells. That knowledge is crucial for scientists trying to understand precisely how Notch works in bone and cartilage development. A few years ago, Hilton was part of a team that showed that Notch is a critical regulator of the development of bone and cartilage. The latest study extends those observations, providing important details that suggest appropriate activation and manipulation of the Notch pathway may provide doctors with a tool to maintain and expand mesenchymal stem cells for use in treating disease.

The work is part of ongoing research around the world aimed at harnessing the promise of stem cells for human health. Unfortunately, stem cell therapy has been slow to actually make a difference in the lives of patients with problems of the bones and cartilage, Hilton notes, largely because so many questions are currently unanswered.

"To really make stem-cell medicine work, we need to understand where the stem cells have come from and how to get them to become the cell you want, when and where you want it. We are definitely in the infancy of learning how to manipulate stem cells and use them in treatment," said Hilton, assistant professor of Orthopaedics and Rehabilitation.

"This research helps set the foundation for ultimately trying new therapies in patients," he added.

"For instance, let's say a patient has a fracture that simply won't heal. The patient comes in and has a sample of bone marrow drawn. Their skeletal stem cells are isolated and expanded in the laboratory via controlled Notch activation, then put back into the patient to create new bone in numbers great enough to heal the fracture. That's the hope."
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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/

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Human Gene Patent Invalidated by New York Judge

Patents On Breast Cancer Genes Ruled Invalid In ACLU/PubPat Case
Tuesday, 30 March 2010

Patents on genes associated with hereditary breast and ovarian cancer are invalid, ruled a New York federal court today. The precedent-setting ruling marks the first time a court has found patents on genes unlawful and calls into question the validity of patents now held on approximately 2,000 human genes. The ruling follows a lawsuit brought by a group of patients and scientists represented by the American Civil Liberties Union and the Public Patent Foundation (PUBPAT), a not-for-profit organization affiliated with Benjamin N. Cardozo School of Law.

"Today's ruling is a victory for the free flow of ideas in scientific research," said Chris Hansen, a staff attorney with the ACLU First Amendment Working Group.

"The human genome, like the structure of blood, air or water, was discovered, not created. There is an endless amount of information on genes that begs for further discovery, and gene patents put up unacceptable barriers to the free exchange of ideas."

The ACLU's and PUBPAT's lawsuit against Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the BRCA genes, as well the U.S. Patent and Trademark Office (USPTO), charged that the challenged patents are illegal and restrict both scientific research and patients' access to medical care, and that patents on human genes violate the First Amendment and patent law because genes are "products of nature."

The court today granted the U.S. Patent and Trademark Office's (USPTO) request that it be released as a defendant in the lawsuit. The court found that it was unnecessary to reach the First Amendment claims against the USPTO because it had already ruled in favour of the plaintiffs.

The lawsuit, Association for Molecular Pathology, et al. v. U.S. Patent and Trademark Office, et al., was filed on May 12 in the U.S. District Court for the Southern District of New York on behalf of breast cancer and women's health groups, individual women, geneticists and scientific associations representing approximately 150,000 researchers, pathologists and laboratory professionals.

Because the ACLU's lawsuit challenges the whole notion of gene patenting, its outcome could have far-reaching effects beyond the patents on the BRCA genes. Approximately 20 percent of all human genes are patented, including genes associated with Alzheimer's disease, muscular dystrophy, colon cancer, asthma and many other illnesses.

The court recognized the far-reaching impact of the case on medical research and public health. The opinion stated, "…the resolution of the issues presented to this Court deeply concerns breast cancer patients, medical professionals, researchers, caregivers, advocacy groups, existing gene patent holders and their investors, and those seeking to advance public health."

"The court correctly saw that companies should not be able to own the rights to a piece of the human genome," said Daniel B. Ravicher, Executive Director of PUBPAT and co-counsel in the lawsuit.

"No one invented genes. Inventions are specific tests or drugs, which can be patented, but genes are not inventions."

The specific patents the ACLU had challenged are on the BRCA1 and BRCA2 genes. Mutations along the BRCA1 and 2 genes are responsible for most cases of hereditary breast and ovarian cancers. Many women with a history of breast and ovarian cancer in their families opt to undergo genetic testing to determine if they have the mutations on their BRCA genes that put them at increased risk for these diseases. This information is critical in helping these women decide on a plan of treatment or prevention, including increased surveillance or preventive mastectomies or ovary removal.

"We are extremely gratified by this groundbreaking decision," said Sandra Park, staff attorney with the ACLU Women's Rights Project.

"This is the beginning of the end to patents that restrict women's access to their own genetic information and interfere with their medical care."

The patents granted to Myriad give the company the exclusive right to perform diagnostic tests on the BRCA1 and BRCA2 genes and to prevent any researcher from even looking at the genes without first getting permission from Myriad. Myriad's monopoly on the BRCA genes makes it impossible for women to access alternate tests or gets a comprehensive second opinion about their results and allows Myriad to charge a high rate for their tests.

Several major organizations, including the American Medical Association, the March of Dimes and the American Society for Human Genetics, filed friend-of-the-court briefs in support of the challenge to the patents on the BRCA genes.

Attorneys on the case include Hansen and Aden Fine of the ACLU First Amendment Working Group; Park and Lenora Lapidus of the ACLU Women's Rights Project; and Ravicher and Sabrina Hassan of PUBPAT.

Today's decision can be found online at:
www.aclu.org/free-speech-technology-and-liberty-womens-rights/association-molecular-pathology-et-al-v-uspto-et-al.

More information about the case, including an ACLU video featuring breast cancer patients, plaintiff and supporter statements and declarations and the legal complaint, can be found online at: www.aclu.org/brca.

Published on American Civil Liberties Union (http://www.aclu.org/)

Source URL: http://www.aclu.org/free-speech-womens-rights/patents-breast-cancer-genes-ruled-invalid-aclupubpat-case.
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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/

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How much is a human egg worth?

How much is a human egg worth?

It is still illegal in the UK, as in most other European countries, to pay a human egg donor. However, in sunny California, and the rest of the US, it is OK to put a price on an egg. Now, the UK government regulatory agency HFEA is expected to approve payment for donating human eggs for scientific research. Should this kind of merchandise take place, or should it be done on a ‘non-profit’ basis? What do you all think? I think it’s the same question as with transplants – so feel free to include that topic too in the discussion!


In the US ‘big bucks’:
Increase in egg donors raises concerns
AP - Sun Feb 18, 1:47 PM ET
Human egg donation was a rarity not so long ago. But heightened demands for eggs — and rising compensation for donors — are prompting more young women to consider it. Jennifer Dziura, a 28-year-old New Yorker, is one of them.She received $8,000 to donate her eggs in the fall of 2005 and hopes she'll be chosen again before the private egg broker she's registered with considers her too old. She realizes prospective parents who view her profile might think it a minus that her father is adopted, allowing for little medical history from his side. She also figures some are looking for a blonde, instead of a brunette.


… and in the UK:
Women will be paid to donate eggs for science
£250 payment to aid disease research. Fears over landmark medical ruling
The Observer - Sunday February 18, 2007

Women will be paid to donate their eggs for scientific research in a landmark decision that will prompt a fierce backlash from leading figures in the medical world. The Human Fertility and Embryology Authority (HFEA), the government regulator of this highly sensitive area, is expected to approve the policy when it meets on Wednesday. At present, clinics are not allowed to accept eggs donated for scientific research unless they are a byproduct of either IVF treatment or sterilisation. Campaigners for change say that this has led to a chronic shortage of eggs for scientific use.



See also:
Asian women command premium prices for egg donation in U.S. 
LA Times - May 5, 2012 
Egg donors offered up to $50,000
Fees far exceed ethics guidelines, study finds
MSNBC - March 26, 2010
Human Ovulation Caught on Camera
CellNEWS - Wednesday, 18 June 2008
Most egg cells in a female body die naturally by programmed cell death
CellNEWS - Tuesday, 24 July 2007
Back to the question: How much is a human egg worth?
CellNEWS - Tuesday, 09 October 2007
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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/

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Novel Parkinson's Treatment Strategy Involves Cell Transplantation

Novel Parkinson's Treatment Strategy Involves Cell Transplantation

Thursday, 25 March 2010

UCSF scientists have used a novel cell-based strategy to treat motor symptoms in rats with a disease designed to mimic Parkinson's disease.

The strategy suggests a promising approach, the scientists say, for treating symptoms of Parkinson's disease and other neurodegenerative diseases and disorders, including epilepsy.

The scientists transplanted embryonic neurons from foetal rats into an area of the adult rat brain known as the striatum, which integrates excitatory and inhibitory neurotransmitter signals to control movement. In Parkinson's disease, cells that produce the neurotransmitter dopamine are damaged, and thus unable to project their communication wires, or axons, to the region. As a result, the balance of excitation and inhibition in the striatum is lost, causing the motor deficits that are a primary symptom of the disease.

In the study, the transplanted embryonic neurons migrated and integrated into the correct neural circuitry of the striatum, matured into so-called GABA-ergic inhibitory interneuron’s, and dampened the over-excitation in the region. The rats had improved motor function, as seen in their balance, speed, and length of stride during walking. Moreover, the healthy "control" rats in which the cells had been transplanted took longer strides and ran faster on a runway test.

The results, the scientists say, demonstrate that the transplanted cells, known as embryonic medial ganglionic eminence (MGE) cells, can very precisely modify the balance of excitation and inhibition in neural circuits to influence behaviour. As overactive neural circuits are associated with other neurodegenerative diseases – a result of non-functioning or missing cells or abnormal synaptic transmission -- the finding may have broad implications.

"This strategy represents a whole new approach to treating nervous system disorders," says neurologist Arnold Kriegstein, MD, PhD, the senior author of the study and director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

The study, featured on the cover of the journal Cell Stem Cell (vol. 6, issue 3, 2010), was led by Verónica Martínez-Cerdeño, PhD, at the time a postdoctoral fellow in the Kriegstein lab, and was a collaboration involving Arturo Alvarez-Buylla, PhD, UCSF Heather and Melanie Muss Professor of Neurological Surgery and Krys Bankiewicz, MD, PhD, UCSF professor of neurological surgery.

The approach used by the team differs from another cell-based strategy for Parkinson's disease currently being explored by other research teams. This traditional transplantation strategy involves attempting to replace the dopamine-producing cells that are lost in the disease, by grafting precursors for these cells directly in the striatum. The loss of these cells is thought to account for most of the disease's symptoms – motor deficits, cognitive and autonomic dysfunction and disturbances in mood.

This traditional strategy has shown severe drawbacks, including that the grafted dopaminergic cells show little, if any, dispersion when grafted into the striatum, and that patients have developed disabling spontaneous movements in preliminary trials, prompting early suspension of the trials.

The ability to modify the neural circuitry of the striatum, part of a larger region known as the basal ganglia, is a function only cells can perform, says Kriegstein. The nervous system is a complex system of neural networks composed of highly individualized cells that relay electrochemical signals between regions of the brain and spinal cord at millisecond speeds, accounting for every behaviour, emotion, and thought.

"Each cell has its own role to play based on the circuits in which it is embedded," he says.

"It has to carry out its role at exactly the right time, with exactly the right partners, and the activity pattern changes moment by moment.”

"Once MGE cells were integrated into striatal neural circuitry, they would be able to modify circuit activity, in a way no other therapies can."

Current treatment approaches – drugs, surgery and electrical stimulation – are relatively blunt instruments, he says. Drugs, for instance, generally act indiscriminately, affecting whole areas of the nervous system, so there often are multiple side effects.

The new study findings complement two other recent UCSF studies using MGE cells to modify neural circuits. In a collaborative study among the laboratories of Scott Baraban, PhD, professor of neurological surgery; John Rubenstein, MD, PhD, professor of psychiatry, and Alvarez-Buylla, the cells were grafted into the neocortex of juvenile rodents, where they reduced the intensity and frequency of epileptic seizures. (Proceedings of the National Academy of Sciences, vol. 106, no. 36, 2009). Other teams are exploring this tactic, as well.

In the other study (Science, Vol. 327. no. 5969, 2010), UCSF scientists reported the first use of MGEs to broaden the period of plasticity, or capacity to change, in the mouse visual cortex. The finding, reported by the labs of Alvarez-Buylla and Michael Stryker, PhD, professor of physiology, might some day be used, they say, to create a new period of plasticity of limited duration for repairing damaged brains.

Looking ahead, the team studying MGE cells in the rat model of Parkinson's disease plans to target a more specific sub region of the striatum, with the goal of getting a more precise effect. They also want to see if the cells could be genetically modified to produce dopamine, thus more directly addressing the biochemical changes of Parkinson's disease, and they plan to attempt to prompt human embryonic stem cells to differentiate, or specialize, into MGE cells in the lab, with the goal of establishing a mechanism for creating a sufficient supply of the cells for clinical use.
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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/

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New Period of Brain 'Plasticity' Created with Transplanted Embryonic Cells

New Period of Brain 'Plasticity' Created with Transplanted Embryonic Cells

Thursday, 25 March 2010

UCSF scientists report that they were able to prompt a new period of "plasticity," or capacity for change, in the neural circuitry of the visual cortex of juvenile mice. The approach, they say, might some day be used to create new periods of plasticity in the human brain that would allow for the repair of neural circuits following injury or disease.

The strategy – which involved transplanting a specific type of immature neuron from embryonic mice into the visual cortex of young mice – could be used to treat neural circuits disrupted in abnormal foetal or postnatal development, stroke, traumatic brain injury, psychiatric illness and aging.

Like all regions of the brain, the visual cortex undergoes a highly plastic period during early life. Cells respond strongly to visual signals, which they relay in a rapid, directed way from one appropriate cell to the next in a process known as synaptic transmission. The chemical connections created in this process produce neural circuitry that is crucial for the function of the visual system. In mice, this critical period of plasticity occurs around the end of the fourth week of life.

The catalyst for the so-called critical period plasticity in the visual cortex is the development of synaptic signalling by neurons that release the inhibitory neurotransmitter GABA. These neurons receive excitatory signals from other neurons, thus helping to maintain the balance of excitation and inhibition in the visual system.

In their study, published in the journal Science, (Vol. 327. no. 5969, 2010), the scientists wanted to see if the embryonic neurons, once they had matured into GABA-producing inhibitory neurons, could induce plasticity in mice after the normal critical period had closed.

The team first dissected the immature neurons from their origin in the embryonic medial ganglionic eminence (MGE) of the embryonic mice. Then they transplanted the MGE cells into the animals' visual cortex at two different juvenile stages. The cells, targeted to the visual cortex, dispersed through the region, matured into GABA-ergic inhibitory neurons, and made widespread synaptic connections with excitatory neurons.

The scientists then carried out a process known as monocular visual deprivation, in which they blocked the visual signals to one eye in each of the animals for four days. When this process is carried out during the critical period, cells in the visual cortex quickly become less responsive to the eye deprived of sensory input, and become more responsive to the non-deprived eye, creating alterations in the neural circuitry. This phenomenon, known as ocular dominance plasticity, greatly diminishes as the brain matures past this critical postnatal developmental period.

The team wanted to see if the transplanted cells would affect the visual system's response to the visual deprivation after the critical period. They studied the cells' effects after allowing them to mature for varying lengths of time. When the cells were as young as 17 days old or as old as 43 days old, they had little impact on the neural circuitry of the region. However, when they were 33-39 days old, their impact was significant. During that time, monocular visual deprivation shifted the neural responses away from the deprived eye and toward the non-deprived eye, revealing the state of ocular dominance plasticity.

Naturally occurring, or endogenous, inhibitory neurons are also around 33-39 days old when the normal critical period for plasticity occurs. Thus, the transplanted cells' impact occurred once they had reached the cellular age of inhibitory neurons during the normal critical period.

The finding, the team says, suggests that the normal critical period of plasticity in the visual cortex is regulated by a developmental program intrinsic to inhibitory neurons, and that embryonic inhibitory neuron precursors can retain and execute this program when transplanted into the postnatal cortex, thereby creating a new period of plasticity.

"The findings suggest it ultimately might be possible to use inhibitory neuron transplantation, or some factor that is produced by inhibitory neurons, to create a new period of plasticity of limited duration for repairing damaged brains," says author Sunil P. Gandhi, PhD.. He is a postdoctoral fellow in the lab of Michael Stryker, PhD, professor of physiology and a member of the Keck Center for Integrative Neurosciences at UCSF.

"It will be important to determine whether transplantation is equally effective in older animals."

Likewise, "the results raise a fundamental question: how do these cells, as they pass through a specific stage in their development, create these windows of plasticity?" says author Derek G. Southwell, PhD, a student in the lab of Arturo Alvarez-Buylla, PhD, Heather and Melanie Muss Professor of Neurological Surgery and a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF.

The findings could be relevant to understanding why learning certain behaviors, such as language, occurs with ease in young children but not in adults, says Alvarez-Buylla.

"Grafted MGE cells may some day provide a way to induce cortical plasticity and learning later in life."

The findings also complement two other recent UCSF studies using MGE cells to modify neural circuits. In a collaborative study among the laboratories of Scott Baraban, PhD, professor of neurological surgery; John Rubenstein, MD, PhD, professor of psychiatry, and Alvarez-Buylla, the cells were grafted into the neocortex of juvenile rodents, where they reduced the intensity and frequency of epileptic seizures. (Proceedings of the National Academy of Science, vol. 106, no. 36, 2009). Other teams are exploring this tactic, as well.

In the other study (Cell Stem Cell, vol. 6, issue 3, 2010), UCSF scientists reported the first use of MGEs to treat motor symptoms in mice with a condition designed to mimick Parkinson's disease. The finding was reported by the lab of Arnold Kriegstein, MD, PhD, UCSF professor of neurology and director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF, in collaboration with Alvarez-Buylla and Krys Bankiewicz, MD, PhD, UCSF professor of neurological surgery.

Reference:
Cortical Plasticity Induced by Inhibitory Neuron Transplantation
Derek G. Southwell, Robert C. Froemke, Arturo Alvarez-Buylla, Michael P. Stryker, Sunil P. Gandhi
Science 26 February 2010, Vol. 327. no. 5969, pp. 1145 – 1148, DOI: 10.1126/science.1183962
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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/

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Your Fat May Help You Heal

Rice researcher extracts natural scaffold for tissue growth
Thursday, 25 March 2010

It frequently happens in science that what you throw away turns out to be most valuable. It happened to Deepak Nagrath, but not for long.

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Adipogel forms a viscous droplet when isolated on a Petri dish. After further processing, it can be used as a natural extracellular matrix to support new tissue growth. Credit: N. Sharma.

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The Rice assistant professor in chemical and biomolecular engineering was looking for ways to grow cells in a scaffold, and he discarded the sticky substance secreted by the cells.

"I thought it was contamination, so I threw the plates away," said Nagrath, then a research associate at Harvard Medical School.

That substance, derived from adipose cells – aka body fat – turned out to be a natural extracellular matrix, the very thing he was looking for.

Nagrath, who joined Rice in 2009, and his co-authors have since built a biological scaffold that allows cells to grow and mature. He hopes the new material, when suffused with stem cells, will someday be injected into the human body, where it can repair tissues of many types without fear of rejection.

The research by Nagrath and his co-authors appeared last week in the Federation of American Societies for Experimental Biology (FASEB) Journal.

The basic idea is simple: Prompt fat cells to secrete what bioengineers call "basement membrane." This membrane mimics the architecture tissues naturally use in cell growth, literally a framework to which cells attach while they form a network. When the cells have matured into the desired tissue, they secrete another substance that breaks down and destroys the scaffold.

Structures that support the growth of living cells into tissues are highly valuable to pharmaceutical companies for testing drugs in vitro. Companies commonly use Matrigel, a protein mixture secreted by mouse cancer cells, but for that reason it can't be injected into patients.

"Fat is one thing that is in excess in the body. We can always lose it," Nagrath said. The substance derived from the secretions, called Adipogel, has proven effective for growing hepatocytes, the primary liver cells often used for pharmaceutical testing.

"My approach is to force the cells to secrete a natural matrix," he said. That matrix is a honey-like gel that retains the natural growth factors, cytokines (substances that carry signals between cells) and hormones in the original tissue.

Nagrath's strategy for growing cells is not the only approach being pursued, even at Rice: Another method reported last week in Nature Nanotechnology uses magnetic levitation to grow three-dimensional cell cultures.

However, Nagrath is convinced his strategy is ultimately the most practical for rebuilding tissue in vivo, and not only because it may cost significantly less than Matrigel.

"The short-term goal is to use this as a feeder layer for human embryonic stem cells. It's very hard to maintain them in the pluripotent state, where they keep dividing and are self-renewing," he said.

Once that goal is achieved, Adipogel may be just the ticket for transplanting cells to repair organs.

"You can use this matrix as an adipogenic scaffold for stem cells and transplant it into the body where an organ is damaged. Then, we hope, these cells and the Adipogel can take over and improve their functionality."

Reference:

Adipocyte-derived basement membrane extract with biological activity: applications in hepatocyte functional augmentation in vitro

Nripen S. Sharma, Deepak Nagrath, and Martin L. Yarmush

The FASEB Journal, March 16, 2010, doi: 10.1096/fj.09-135095

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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/

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miRNA and Down Syndrome

New theory of Down Syndrome cause may lead to new therapies
Tuesday, 23 March 2010

Conventional wisdom among scientists for years has suggested that because individuals with Down syndrome have an extra chromosome, the disorder most likely results from the presence of too many genes or proteins contained in that additional structure.

But a recent study reveals that just the opposite could be true – that a deficiency of a protein in the brain of Down Syndrome patients could contribute to the cognitive impairment and congenital heart defects that characterize the syndrome.

Scientists have shown in a series of experiments that there are lower levels of this protein in the brains of humans and mice with Down syndrome than are present in humans and mice without the disorder.

The researchers also showed that manually manipulating pieces of RNA that regulate the protein could increase protein levels in both human cell lines and mouse brains. In fact, an experimental drug that acts on those RNA segments returned this protein to normal levels in mice that model the syndrome.

When this RNA segment is over-expressed – meaning that more of it is present than needed in a cell – the protein level goes down, or is under-expressed. A total of at least five of these RNA segments are naturally over-expressed in persons with Down syndrome because the segments are housed on chromosome 21 – the chromosome that causes the disorder.

Terry Elton.

“We’re talking about a paradigm-shifting idea that maybe we should look for under-expressed proteins and not over-expressed proteins in Down syndrome,” said Terry Elton, senior author of the study and a professor of pharmacology at Ohio State University.

“What this offers to the Down syndrome community is the potential for at least five new therapeutic targets to pursue.”

The Centers for Disease Control and Prevention estimates that about 13 of every 10,000 babies born in the United States each year have Down syndrome, characterized primarily by a mild-to-moderate range of intellectual disabilities, possible delayed language development and difficulties with physical coordination.

Elton, also interim director of Ohio State’s Davis Heart and Lung Research Institute, stumbled upon this theory about Down syndrome while working on a different protein associated with cardiovascular disease. It turns out the protein he has studied for 25 years was regulated by one of these microRNAs that is known to be housed on chromosome 21.

A key role of RNA in a cell is to make protein, and proteins are the building blocks of all life. But the process has many steps. MicroRNAs are small pieces of RNA that bind to messenger RNA, which contains the actual set of instructions for building proteins. When that connection is made, however, the microRNA inhibits the building of the protein. Why that occurs is not completely understood, but increasingly microRNAs are considered tiny molecules that have a big impact in a number of physiological processes.

For his cardiovascular disease research, Elton found that a genetic trait in some people caused one specific microRNA to be bad at its job, leading to high protein levels that contribute to cardiovascular disease. This malfunctioning molecule is called microRNA-155, or miR-155.

“So we became interested in miR-155, and it is on chromosome 21. That’s how we jumped to Down syndrome,” Elton said.

There is also a strong link between the heart and Down syndrome. About half of those with the syndrome are born with congenital heart defects – problems with the heart’s anatomy, not coronary arteries. But they do not experience cardiovascular disease or high blood pressure.

The advent of biomedical informatics has allowed scientists to use supercomputers to explore the human genome in a search for genes and their various relationships in the context of human disease. Elton consulted a bioinformatics database and found that five microRNAs sit on chromosome 21, and he and colleagues demonstrated in previous research that all five of them are over-expressed in the tissues, brains and hearts of Down syndrome patients.

“That means that whatever proteins these microRNAs work with are under-expressed,” Elton said.

Further database exploration suggested that these five microRNAs target 1,695 proteins, all of which could cause problems in Down syndrome because they are under-expressed. To narrow that to a more manageable number, Elton’s group had to make an educated guess based on a variety of data, including which proteins that are connected to these microRNAs are made by cells in the brain and heart – two areas most commonly affected by Down syndrome.

A protein surfaced as an attractive target to study: methyl-CpG-binding protein 2, known as MeCP2. Among the reasons it seemed important: A mutation in this protein is already known to lead to Rett syndrome, a cognitive disorder.

“So we thought that it was more than a coincidence that this protein plays a role in normal brain development, and if the protein doesn’t function right, you’re going to have cognitive impairment. Maybe this is the connection,” Elton said.

“We still don’t know if this is the most important protein related to Down syndrome. But we were able to go on and prove scientifically that MeCP2 is a target of these microRNAs on chromosome 21.”

The researchers used just two of the five microRNAs on chromosome 21 for the experiments in this study, miR-155 and miR-802, to match the only microRNAs available in the genetically engineered mouse model of Down syndrome.

First, the researchers made copies of the relevant microRNAs. In human brain cell lines, they manipulated levels of those two molecules to show the inverse relationship with the protein. If the microRNAs were more active, the level of the MeCP2 protein went down. When the microRNAs were under-expressed, the protein levels went up.

Next, the researchers examined adult and foetal human brain tissue from healthy and Down syndrome samples obtained from a national tissue bank.

“In both adult and foetal Down syndrome brain samples, it didn’t matter which area of the brain we were looking at, the MeCP2 proteins were down. These are just observations with no manipulation on our part, and the MeCP2 is almost non-existent in the Down syndrome brain,” Elton said.

“We marked the protein with a fluorescent molecule, and by comparison, we could visualize and appreciate how much MeCP2 was being made by neurons in the control samples.”

MeCP2 is a transcription factor, meaning it turns genes on and off. If its levels are too low in the brain, this suggests that genes influenced by its presence should be malfunctioning, too. Based on previous research by another group, Elton and colleagues focused on two genes affected by the MeCP2 protein for their next set of experiments.

Looking again at the human brain tissue samples, they found that the genes were indeed affected by the lowered protein level in Down syndrome brains – one gene that MeCP2 normally silences was in abundance, and the gene that should have been activated was under-expressed. Because the two genes examined have known roles in neural development, Elton said the results suggested even more strongly that the lowered protein’s effects on the genes likely contribute to cognitive problems associated with Down syndrome.

Finally, the researchers tested an experimental drug called an antagomir on mice that serve as models for Down syndrome research. Antagomirs are relatively new agents that render microRNAs inactive. The scientists injected an antagomir into the brains of these mice to silence the miR-155 with the intent to increase levels of the MeCP2 protein. Seven days after the injection, the level of the protein in the treated mouse brains resembled levels in normal mouse brains.

“We showed that we can fix the protein abnormality in mice that model Down syndrome. But we can’t undo the pathology that has already occurred,” Elton said.

“It’s a starting point, but it appears that we have new therapeutic targets to consider.”

The study is published in a recent issue of the Journal of Biological Chemistry.
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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/

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Newly Identified Growth Factor Promotes Stem Cell Growth, Regeneration

Newly Identified Growth Factor Promotes Stem Cell Growth, Regeneration
Sunday, 21 March 2010

Scientists at Duke University Medical Center have identified a new growth factor that stimulates the expansion and regeneration of hematopoietic (blood-forming) stem cells in culture and in laboratory animals. The discovery, appearing in the journal Nature Medicine, may help researchers overcome one of the most frustrating barriers to cellular therapy: the fact that stem cells are so few in number and so stubbornly resistant to expansion.

Researchers believe that umbilical cord blood could serve as a universal source of stem cells for all patients who need a stem cell transplant, but the numbers of stem cells in cord blood units are limited, so there is a clinical need to develop a method to expand cord blood stem cells for transplantation purposes.

"Unfortunately, there are no soluble growth factors identified to date that have been proven to expand human stem cells for therapeutic purposes," said John Chute, M.D., a stem cell transplant physician and cell biologist at Duke and senior author of the paper.

Chute, working with Heather Himburg, a post-doctoral fellow in his laboratory, discovered that adding pleiotrophin, a naturally-occurring growth factor, stimulated a ten-fold expansion of stem cells taken from the bone marrow of a mouse.

They also found that pleiotrophin increased the numbers of human cord blood stem cells in culture that were capable of engraftment in immune-deficient mice. When they injected pleiotrophin into mice that had received bone marrow-suppressive radiation, they observed a 10-fold increase in bone marrow stem cells compared to untreated mice.

"These results confirmed that pleiotrophin induces stem cell regeneration following injury," said Chute.

Chute says the finding could lead to broader application of cord blood transplants for the large numbers of patients who do not have an immune-matched donor.

"Perhaps more importantly, systemic treatment with pleiotrophin may have the potential to accelerate recovery of the blood and immune system in patients undergoing chemotherapy or radiotherapy," he said.

Given the potency of the effect of pleiotrophin on stem cell expansion, the authors examined whether pleiotrophin provoked blood-forming cells to become malignant. So far, Chute says they have not seen any evidence of cancer in mice up to six months after treatment with pleiotrophin.

The Duke team is already conducting further experiments to determine if pleiotrophin is necessary for normal stem cell growth and development, and Chute says it will be important to conduct additional animal studies before moving into human clinical trials.

"At this point, any progress we can make that helps us better understand which biological pathways are activated in stem cells in response to pleiotrophin will help move the discovery forward."

Reference:

Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells
Heather A Himburg, Garrett G Muramoto, Pamela Daher, Sarah K Meadows, J Lauren Russell, Phuong Doan, Jen-Tsan Chi, Alice B Salter, William E Lento, Tannishtha Reya, Nelson J Chao & John P Chute
Nature Medicine, Published online: 21 March 2010, doi:10.1038/nm.2119
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ZenMaster

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siRNA Nanoparticles Can Silence Cancer Genes in Humans

Caltech-led researchers unveil scientific results from siRNA Phase I clinical trial in cancer patients
Sunday, 21 March 2010

A California Institute of Technology-led team of researchers and clinicians has published the first proof that a targeted nanoparticles — used as an experimental therapeutic and injected directly into a patient's bloodstream—can traffic into tumours, deliver double-stranded small interfering RNAs (siRNAs), and turn off an important cancer gene using a mechanism known as RNA interference (RNAi). Moreover, the team provided the first demonstration that this new type of therapy, infused into the bloodstream, can make its way to human tumours in a dose-dependent fashion — i.e., a higher number of nanoparticles sent into the body leads to a higher number of nanoparticles in the tumour cells.

These results, published in the March 21 advance online edition of the journal Nature, demonstrate the feasibility of using both nanoparticles and RNAi-based therapeutics in patients, and open the door for future "game-changing" therapeutics that attack cancer and other diseases at the genetic level, says Mark Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering at Caltech, and the research team's leader.

The discovery of RNA interference, the mechanism by which double strands of RNA silence genes, won researchers Andrew Fire and Craig Mello the 2006 Nobel Prize in Physiology or Medicine. The scientists first reported finding this novel mechanism in worms in a 1998 Nature paper. Since then, the potential for this type of gene inhibition to lead to new therapies for diseases like cancer has been highly touted.

"RNAi is a new way to stop the production of proteins," says Davis. What makes it such a potentially powerful tool, he adds, is the fact that its target is not a protein. The vulnerable areas of a protein may be hidden within its three-dimensional folds, making it difficult for many therapeutics to reach them. In contrast, RNA interference targets the messenger RNA (mRNA) that encodes the information needed to make a protein in the first place.

"In principle, that means every protein now is druggable because its inhibition is accomplished by destroying the mRNA. And we can go after mRNAs in a very designed way given all the genomic data that are and will become available," says Davis.

Still, there have been numerous potential roadblocks to the application of RNAi technology as therapy in humans. One of the most problematic has been finding a way to ferry the therapeutics, which are made up of fragile siRNAs, into tumour cells after direct injection into the bloodstream. Davis, however, had a solution. Even before the discovery of RNAi, he and his team had begun working on ways to deliver nucleic acids into cells via systemic administration. They eventually created a four-component system — featuring a unique polymer — that can self-assemble into a targeted, siRNA-containing nanoparticle. The siRNA delivery system is under clinical development by Calando Pharmaceuticals, Inc., a Pasadena-based nanobiotech company.

"These nanoparticles are able to take the siRNAs to the targeted site within the body," says Davis. Once they reach their target — in this case, the cancer cells within tumours — the nanoparticles enter the cells and release the siRNAs.

The scientific results described in the Nature paper are from a Phase I clinical trial of these nanoparticles that began treating patients in May 2008. Phase I trials are, by definition, safety trials; the idea is to see if and at what level the drug or other therapy turns harmful or toxic. These trials can also provide an in-human scientific proof of concept — which is exactly what is being reported in the Nature paper.

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This electron micrograph shows the presence of numerous siRNA-containing targeted nanoparticles both entering and within a tumour cell. Credit: Caltech/Swaroop Mishra.

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Using a new technique developed at Caltech, the team was able to detect and image nanoparticles inside cells biopsied from the tumours of several of the trial's participants. In addition, Davis and his colleagues were able to show that the higher the nanoparticle dose administered to the patient, the higher the number of particles found inside the tumour cells — the first example of this kind of dose-dependent response using targeted nanoparticles.

Even better, Davis says, the evidence showed the siRNAs had done their job. In the tumour cells analyzed by the researchers, the mRNA encoding the cell-growth protein ribonucleotide reductase had been degraded. This degradation, in turn, led to a loss of the protein.

More to the point, the mRNA fragments found were exactly the length and sequence they should be if they had been cleaved in the spot targeted by the siRNA, notes Davis.

"It's the first time anyone has found an RNA fragment from a patient's cells showing the mRNA was cut at exactly the right base via the RNAi mechanism," he says.

"It proves that the RNAi mechanism can happen using siRNA in a human."

"There are many cancer targets that can be efficiently blocked in the laboratory using siRNA, but blocking them in the clinic have been elusive," says Antoni Ribas, associate professor of medicine and surgery at UCLA's Jonsson Comprehensive Cancer Center.

"This is because many of these targets are not amenable to be blocked by traditionally designed anti-cancer drugs. This research provides the first evidence that what works in the lab could help patients in the future by the specific delivery of siRNA using targeted nanoparticles. We can start thinking about targeting the un-targetable."

"Although these data are very early and more research is needed, this is a promising study of a novel cancer agent, and we are proud of our contribution to the initial clinical development of siRNA for the treatment of cancer," says Anthony Tolcher, director of clinical research at South Texas Accelerated Research Therapeutics (START).

"Promising data from the clinical trials validates our years of research at City of Hope into ribonucleotide reductase as a target for novel gene-based therapies for cancer," adds co-author Yun Yen, associate director for translational research at City of Hope.

"We are seeing for the first time the utility of siRNA as a cancer therapy and how nanotechnology can target cancer cells specifically."

The Phase I trial — sponsored by Calando Pharmaceuticals — is proceeding at START and UCLA's Jonsson Comprehensive Cancer Center, and the clinical results of the trial will be presented at a later time.

"At the very least, we've proven that the RNAi mechanism can be used in humans for therapy and that the targeted delivery of siRNA allows for systemic administration," Davis says.

"It is a very exciting time."

Reference:
Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticlesMark E. Davis, Jonathan E. Zuckerman, Chung Hang J. Choi, David Seligson, Anthony Tolcher, Christopher A. Alabi, Yun Yen, Jeremy D. Heidel & Antoni Ribas
Nature advance online publication 21 March 2010, doi:10.1038/nature08956
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ZenMaster

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What Makes You Unique?

Not genes so much as surrounding sequences, says Stanford study
Thursday, 18 March 2010

The key to human individuality may lie not in our genes, but in the sequences that surround and control them, according to new research by scientists at the Stanford University Medical Center and Yale University. The interaction of those sequences with a class of key proteins, called transcription factors, can vary significantly between two people and are likely to affect our appearance, our development and even our predisposition to certain diseases, the study found.

The discovery suggests that researchers focusing exclusively on genes to learn what makes people different from one another have been looking in the wrong place.

"We are rapidly entering a time when nearly anyone can have his or her genome sequenced," said Michael Snyder, PhD, professor and chair of genetics at Stanford.

"However, the bulk of the differences among individuals are not found in the genes themselves, but in regions we know relatively little about. Now we see that these differences profoundly impact protein binding and gene expression."

Snyder is the senior author of two papers — one in Science Express and one in Nature — exploring these protein-binding differences in humans, chimpanzees and yeast. Snyder, the Stanford W. Ascherman, MD, FACS, Professor in Genetics, came to Stanford in July 2009 from Yale, where much of the work was conducted.

Genes, which carry the specific instructions necessary to make proteins do the work of the cell, vary by only about 0.025 percent across all humans. Scientists have spent decades trying to understand how these tiny differences affect who we are and what we become. In contrast, non-coding regions of the genome, which account for approximately 98 percent of our DNA, vary in their sequence by about 1 to 4 percent. But until recently, scientists had little, if any, idea what these regions do and how they contribute to the "special sauce" that makes me, me, and you, you.

Now Snyder and his colleagues have found that the unique, specific changes among individuals in the sequence of DNA affect the ability of "control proteins" called transcription factors to bind to the regions that control gene expression. As a result, the subsequent expression of nearby genes can vary significantly.

"People have done a lot of work over the years to characterize differences in gene expression among individuals," said Snyder.

"We're the first to look at differences in transcription-factor binding from person to person."

What's more, by selectively breeding, or crossing, yeast strains, Snyder and his colleagues found that many, but not all, of these differences in binding and expression levels are heritable.

In the Science Express paper, which will be published online March 18, Snyder and his colleagues compared the binding patterns of two transcription factors in 10 people and one chimpanzee. They identified more than 15,000 binding sites across the genome for the transcription factor called NF-kB and more than 19,000 sites for another factor called RNA PolII. They then looked to see if every site was bound equally strongly by the proteins, or if there were variations among individuals.

They found that about 25 percent of the PolII sites and 7.5 percent of the NF-kB sites exhibited significant binding differences among individuals — in some cases greater than two orders of magnitude from one person to another. (For comparison, the binding differences between the humans and the chimpanzee were about 32 percent.) Many of these binding differences could be traced to differences in sequences or structure in the protein binding sites, and several were directly correlated to changes in gene expression levels.

"These binding regions, or chunks, vary among individuals, and they have a profound impact on gene expression," Snyder said.

In particular, the researchers found that several of the variable binding regions were near genes involved in such diseases as type-1 diabetes, lupus, leukaemia and schizophrenia.

The researchers confirmed and extended their findings in the Nature paper, which will be published online March 17. In this study, they used yeast to determine that many of the binding differences and variations in gene expression levels in individuals are passed from parent to progeny, and they identify several control proteins that vary — a study that would have been impossible to perform in humans.

"We conducted the two studies in parallel and found the same thing. Many of the binding sites differed. When we mapped the areas of difference, we found that they were associated with key regulators of variation in the population. Together these two studies tell us a lot about the so-called regulatory code that controls variation among individuals," Snyder said.

References:
Variation in Transcription Factor Binding Among Humans
Maya Kasowski, Fabian Grubert, Christopher Heffelfinger, Manoj Hariharan, Akwasi Asabere, Sebastian M. Waszak, Lukas Habegger, Joel Rozowsky, Minyi Shi, Alexander E. Urban, Mi-Young Hong, Konrad J. Karczewski, Wolfgang Huber, Sherman M. Weissman, Mark B. Gerstein, Jan O. Korbel, and Michael Snyder
Science Published online March 18 2010; 10.1126/science.1183621

Genetic analysis of variation in transcription factor binding in yeast
Wei Zheng, Hongyu Zhao, Eugenio Mancera, Lars M. Steinmetz & Michael Snyder
Nature advance online publication 17 March 2010, doi:10.1038/nature08934
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ZenMaster

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Dogs Likely Originated in the Middle East

Findings based on analysis of largest set of genetic markers ever studied
Thursday, 18 March 2010

Dogs likely originated in the Middle East, not Asia or Europe, according to a new genetic analysis by an international team of scientists led by UCLA biologists. The research, funded by the National Science Foundation and the Searle Scholars Program, appears March 17 in the advance online edition of the journal Nature.

"Dogs seem to share more genetic similarity with Middle Eastern gray wolves than with any other wolf population worldwide," said Robert Wayne, UCLA professor of ecology and evolutionary biology and senior author of the Nature paper.

"Genome-wide analysis now directly suggests a Middle East origin for modern dogs. We have found that a dominant proportion of modern dogs' ancestry derives from Middle Eastern wolves, and this finding is consistent with the hypothesis that dogs originated in the Middle East.”

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This evolutionary tree shows dog breeds and gray wolves. Credit: UCLA.

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"This is the same area where domestic cats and many of our livestock originated and where agriculture first developed," Wayne noted.

Previous genetic research suggested an East Asian origin for dogs, "which was unexpected," Wayne said, "because there was never a hint in the archaeological record that dogs evolved there."

"We were able to study a broader sampling of wolves globally than has ever been done before, including Middle Eastern wolves," said the paper's lead author, Bridgett vonHoldt, a UCLA graduate student of ecology and evolutionary biology in Wayne's laboratory who studies the genetics of dog domestication.

"In our analysis of the entire genome, we found that dogs share more unique markers with Middle Eastern wolves than with East Asian wolves. We used a genome-wide approach, which avoids the bias of single genome region."

The biologists report genetic data from more than 900 dogs from 85 breeds (including all the major ones) and more than 200 wild gray wolves (the ancestor of domestic dogs) worldwide, including populations from North America, Europe, the Middle East and East Asia. They used molecular genetic techniques to analyze more than 48,000 genetic markers. No previous study has ever analyzed anywhere near that many markers.

The biologists have samples from Israel, Saudi Arabia and Iran — but they have not pinpointed a specific location in the Middle East where dogs originated.

"This study is unique in using a particular technology called a single nucleotide polymorphism, or SNP, genotyping chip; these chips interrogate the nucleotides at 48,000 locations in the genome," said John Novembre, UCLA assistant professor of ecology and evolutionary biology and a member of UCLA's Interdepartmental Program in Bioinformatics.

"We are able to compare dogs looking at not just one small part of the genome, but at 48,000 different locations. That gives us the fine-scale resolution to analyze how these breeds are related to one another and how they are related to wolves."

Previous genetic research had suggested an East Asian origin based on the higher diversity of mitochondrial sequences in East Asia and China than anywhere else in the world. (Mitochondria are tiny cellular structures outside the nucleus that produce energy and have their own small genome.) However, that research was based on only one sequence, a small part of the mitochondrial genome, Wayne noted.

"That research made extrapolations about how the domestic dog has evolved from examination of one region in the mitochondrial genome," Wayne said.

"This new Nature paper is a much more comprehensive analysis because we have analyzed 48,000 markers distributed throughout the nuclear genome to try to conclude where the most likely ancestral population is.”

"What we found is much more consistent with the archaeological record," he said.

"We found strong kinship to Middle Eastern gray wolves and, to some extent, European gray wolves — but much less so to any wolves from East Asia. Our findings strongly contradict the conclusions based on earlier mitochondrial DNA sequence data."

Eighty percent of dog breeds are modern breeds that evolved in the last few hundred years, Wayne said. But some dog breeds have ancient histories that go back thousands of years.

"We sampled both groups, the modern explosion of dog breeds and some of the ancient lineages," he said.

"Our data were aimed at resolving questions about the origin of domestic dogs, the evolution of dog breeds, and the history of dog breeds and relationships to their closest wild progenitor, the gray wolf."

The first dogs that appeared in the Middle Eastern archaeological record date back some 12,000 to 13,000 years, Wayne said. Wolves have been in the Old World for hundreds of thousands of years. The oldest dogs from the archaeological record come from Europe and Western Russia. A dog from Belgium dates back 31,000 years, and a group of dogs from Western Russia is approximately 15,000 years old, Wayne said.

"We know that dogs from the Middle East were closely associated with humans because they were found in ancient human burial sites," Wayne said.

"In one case, a puppy is curled up in the arms of a buried human."

Some very old strains of dogs, with a history dating back more than several thousand years, may be mixed with modern breeds, enhancing their diversity in certain areas such as East Asia, Wayne said, interpreting the higher mitochondrial DNA diversity in that area of the globe.

There is one small set of East Asian breeds that does not indicate a strong Middle East origin, showing instead a high level of genetic sharing with Chinese wolves. This finding suggests there was some intermixing between East Asian dog breeds and East Asian wolves; the data do not make clear how long ago this occurred.

"However, the vast majority of dogs that we studied show significant levels of sharing with Middle Eastern wolves," said Novembre, a population geneticist who studies genetic diversity and the lessons that can be learned from it.

Co-authors on the Nature paper include a group of researchers from the National Institutes of Health/National Human Genome Research Institute led by Elaine Ostrander; a group led by Carlos Bustamante, formerly of the Cornell University Department of Biological Statistics and Computational Biology and now a professor of genetics at the Stanford University School of Medicine; and scientists from China, Israel, Australia, Europe and Canada.

UCLA co-authors include Eunjung Han, a UCLA graduate student of Novembre's in biostatistics; John Pollinger, director of UCLA's Conservation Genetics Resource Center and associate director of the Center for Tropical Research at UCLA's Institute of the Environment; and James Knowles, a graduate student from Canada's University of Alberta working in Wayne's laboratory. Wayne and Novembre's research is federally funded by the National Science Foundation. Novembre's research is also funded by the Searle Scholars Program.

"By analyzing a sea of scientific data, Bob Wayne and John Novembre are at the forefront of the 'new life sciences' — which represents new ways to make discovery," said Victoria Sork, dean of the UCLA Division of Life Sciences.

"Their integration of genomic data with bioinformatics approaches illustrates how integration has enhanced our ability to analyze biological systems. Integration of knowledge is changing how we think about how life works. We are no longer limited to studying just one piece of a puzzle."

Toy dogs and an evolutionary framework for dog domestication
The biologists have also found that when one looks at a relationship tree of modern and ancient dog breeds, there is surprising structure to it, and the structure mimics the classifications of dogs by breeders into herding dogs, retrievers, sight hounds, small terriers and others.

"We found there is a surprising genetic structure that accords with functional classifications — suggesting that new breeds are developed from crosses within specific breed groups that share particular traits," Wayne said.

"If they want a new sight hound, they tend to cross sight hounds with each other, and the same with herding dogs and retrieving dogs. That may not seem so surprising, but we had no reason to think beforehand that these groups would be strongly genealogical.”

"There are some notable exceptions, such as 'toy dogs.' In this grouping, there are many different kinds of lineages represented, including traces of herding dogs and retrievers. When it comes to miniaturizing a dog, breeders start with a larger breed and cross that with a miniature dog to make a dwarfed breed on a new genetic background, causing the mixing of various lineages. It is a mix-and-match approach for some of these breed groupings. But in other cases, new breeds have been based on combinations of breeds that have specific traits."

New insights into the evolution of dogs have emerged from this Nature paper and several other recent studies by biologists, including Wayne and his colleagues.

"A framework about dog evolution is emerging," Wayne said.

"Even though dogs have an almost infinite variety of forms, geneticists have been discovering that much of this diversity has a simple genetic basis. Short-legged dogs — there are at least 19 such breeds, including dachshunds, corgis and basset hounds — have short legs due to the appearance of just one unique gene, a mutant growth-factor gene."

Recent research by Wayne and his colleagues has identified genes responsible for short legs, small size, different fur types and different coat patterns and colours.

"It seems that in dogs, unlike other domesticated species, many of these different phenotypes distil to just a handful of genes," Wayne said.

"These genes have been mixed with retrievers, herding dogs and sight hounds to create new breeds."

In humans, by contrast, most differences in height and weight involve many genes, each of which has only a small effect; most of the genes account for only about 1 or 2 percent of variability. Even in agricultural plants, most genes have only a small influence on a single trait.

In dogs, however, one gene that is responsible for differences in size accounts for more than 50 percent of the variation in body size, Wayne said. A small number of genes, he said, have been moved around in dogs to create the appearance of amazing diversity.

"Because we analyzed 48,000 locations in the genome, we can ask which regions are the most different between dogs and wolves," said Novembre, whose research group investigated whether specific regions of the genome have changed under domestication.

"We identified a few regions that are exceptionally different between dogs and wolves; these might be places in the genome where some of the changes occurred that make dogs and wolves different from each other today. These are good candidate regions for follow-up research."

In a separate paper, Melissa Gray, who earned her Ph.D. from UCLA in Wayne's laboratory, reported in February, along with Wayne and colleagues, on an important gene known as IGF1, which is responsible for small size in dogs, and analyzed which wolf populations are closest evolutionarily to this gene. The findings, published in the journal BMC Biology show that the gene appears to have arisen in Middle Eastern wolves, giving further support to the major claim in the new Nature paper.

Reference:
Genome-wide SNP and haplotype analyses reveal a rich history underlying dog domestication
Bridgett M. vonHoldt, John P. Pollinger, Kirk E. Lohmueller, Eunjung Han, Heidi G. Parker, Pascale Quignon, Jeremiah D. Degenhardt, Adam R. Boyko, Dent A. Earl, Adam Auton, Andy Reynolds, Kasia Bryc, Abra Brisbin, James C. Knowles, Dana S. Mosher, Tyrone C. Spady, Abdel Elkahloun, Eli Geffen, Malgorzata Pilot, Wlodzimierz Jedrzejewski, Claudia Greco, Ettore Randi, Danika Bannasch, Alan Wilton, Jeremy Shearman, Marco Musiani, Michelle Cargill, Paul G. Jones, Zuwei Qian, Wei Huang, Zhao-Li Ding, Ya-ping Zhang, Carlos D. Bustamante, Elaine A. Ostrander, John Novembre & Robert K. Wayne
Nature advance online publication 17 March 2010, doi:10.1038/nature08837
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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/

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Regenerative Medicine: One Gene Lost Equals One Limb Regained

Wistar scientists demonstrate mammalian regeneration through a single gene deletion Tuesday, 16 March 2010 A quest that began over a decade ago with a chance observation has reached a milestone: the identification of a gene that may regulate regeneration in mammals. The absence of this single gene, called p21, confers a healing potential in mice long thought to have been lost through evolution and reserved for creatures like flatworms, sponges, and some species of salamander. In a report published today in the Proceedings of the National Academy of Sciences, researchers from The Wistar Institute demonstrate that mice that lack the p21 gene gain the ability to regenerate lost or damaged tissue. Unlike typical mammals, which heal wounds by forming a scar, these mice begin by forming a blastema, a structure associated with rapid cell growth and de-differentiation as seen in amphibians. According to the Wistar researchers, the loss of p21 causes the cells of these mice to behave more like embryonic stem cells than adult mammalian cells, and their findings provide solid evidence to link tissue regeneration to the control of cell division. "Much like a newt that has lost a limb, these mice will replace missing or damaged tissue with healthy tissue that lacks any sign of scarring," said the project's lead scientist Ellen Heber-Katz, Ph.D., a professor in Wistar's Molecular and Cellular Oncogenesis program. "While we are just beginning to understand the repercussions of these findings, perhaps, one day we'll be able to accelerate healing in humans by temporarily inactivating the p21 gene." Heber-Katz and her colleagues used a p21 knockout mouse to help solve a mystery first encountered in 1996 regarding another mouse strain in her laboratory. MRL mice, which were being tested in an autoimmunity experiment, had holes pierced in their ears to create a commonly used life-long identification marker. A few weeks later, investigators discovered that the ear-holes had closed without a trace. While the experiment was ruined, it left the researchers with a new question: Was the MRL mouse a window into mammalian regeneration? The discovery set the Heber-Katz laboratory off on two parallel paths. Working with geneticists Elizabeth Blankenhorn, Ph.D., at Drexel University, and James Cheverud, Ph.D., at Washington University, the laboratory focused on mapping the critical genes that turn MRL mice into healers. Meanwhile, cellular studies ongoing at Wistar revealed that MRL cells behaved very differently than cells from "non-healer" mouse strains in culture. Khamilia Bedebaeva, M.D., Ph.D., having studied genetic effects following the Chernobyl reactor radiation accident, noticed immediately that these cells were atypical, showing profound differences in cell cycle characteristics and DNA damage. This led Andrew Snyder, Ph.D., to explore the DNA damage pathway and its effects on cell cycle control. Snyder found that p21, a cell cycle regulator, was consistently inactive in cells from the MRL mouse ear. The tumour suppressor p53, another regulator of cell division and a known factor in many forms of cancer tightly control P21 expression. The ultimate experiment was to show that a mouse lacking p21 would demonstrate a regenerative response similar to that seen in the MRL mouse. And this indeed was the case. As it turned out, p21 knockout mice had already been created, were readily available, and widely used in many studies. What had not been noted was that these mice could heal their ears. "In normal cells, p21 acts like a brake to block cell cycle progression in the event of DNA damage, preventing the cells from dividing and potentially becoming cancerous," Heber-Katz said. "In these mice without p21, we do see the expected increase in DNA damage, but surprisingly no increase in cancer has been reported." In fact, the researchers saw an increase in apoptosis in MRL mice – also known as programmed cell death – the cell's self-destruct mechanism that is often switched on when DNA has been damaged. According to Heber-Katz, this is exactly the sort of behaviour seen in naturally regenerative creatures. "The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous," Heber-Katz said. "In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle." ......... ZenMaster

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