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Week Ending FRIDAY April 3, 2009---------------------------News Archive

Gene Discovery Could Lead to Male Contraceptive
A newly discovered genetic abnormality that appears to prevent some men from conceiving children could be the key for developing a male contraceptive, according to University of Iowa researchers reporting their findings in the April 2 online edition of the American Journal of Human Genetics.

Although female oral contraceptives were developed over 40 years ago and have proven very effective for family planning, no similar pharmacological contraceptive has been developed for males. Surveys conducted by the Medical Research Council Reproductive Biology Unit in the United Kingdom, suggest that men would be willing to use a pharmacological contraceptive if one was available. Presently the only contraceptives available for men are condoms or a vasectomy.

"We have identified CATSPER1 as a gene that is involved in non-syndromic male infertility in humans, a finding which could lead to future infertility therapies that replace the gene or the protein. But, perhaps even more importantly, this finding could have implications for male contraception," said Michael Hildebrand, Ph.D., co-lead author of the study and a UI postdoctoral researcher in otolaryngology at the UI Roy J. and Lucille A. Carver College of Medicine.

The research team, which included scientists from the University of Social Welfare and Rehabilitation Sciences in Tehran, Iran, discovered the male infertility gene while studying the genetics of families from Iran - a population that has relatively high rates of disease-causing gene mutations.

Although the team's research with these Iranian families focuses on identifying genetic causes of deafness, collecting genetic information from this population allowed the researchers to identify two families where male infertility that was not part of a syndrome appeared to be inherited. The affected men's infertility was diagnosed with a routine semen analysis.

Focusing on a group of genes that have been implicated in male infertility in mice, the researchers found that mutations in both Iranian families occurred in a single gene called CATSPER1. DNA analysis revealed two different mutations - one in each family - but both mutations would likely lead to either a very truncated, non-functional version of the protein, or no protein at all. Neither mutation was found in the DNA of 576 Iranian individuals who were screened as controls.

Harvard University studies on mouse models that lack the CATSPER1 gene reveal how sperm is affected when the protein is missing or abnormal. These studies show that CATSPER1 mutations affect sperm motility, specifically the very vigorous hyperactive motion the sperm uses when it is entering the egg during fertilization.

"Our research suggests that the defect in sperm hyperactivity that is seen in mice without CATSPER1 will also occur in humans with the genetic mutation," Hildebrand said. "Identification of targets such as the CATSPER1 gene that are involved in the fertility process and are specific for sperm -- potentially minimizing side effects of a drug targeting the protein's function -- provide new targets for a pharmacological male contraceptive."

Several approaches to male contraception are currently under investigation at other institutions. One approach that could potentially target CATSPER1 is immunocontraception where antibodies are developed that bind to a targeted protein and block its function. Immunocontraception is still in early stages of development and in order to be useful it will need to be proven effective, safe and reversible.

The study was funded by grants from the National Institutes of Health.

Gene Protects Against Neurotoxins that Spur Inflammation and Parkinson's disease
A new study in the April 3rd issue of the journal Cell, a Cell Press publication, helps to explain why people who carry mutations in a gene known as Nurr1 develop a rare, inherited form of Parkinson's disease, the most prevalent movement disorder in people over the age of 65.

They have found evidence that the gene normally acts to suppress an inflammatory response and, in turn, the production of neurotoxins in the brain. Those neurotoxins can otherwise spawn the damage to dopaminergic neurons that is characteristic of Parkinson's disease. The findings not only offer new insight into the causes of the disease, but also may point to new avenues for therapy, according to the researchers.

In its normal form, "the gene protects against Parkinson's," said Christopher Glass of the University of California, San Diego. "This system functions in the brain, and probably in other parts of the body, to protect from the deleterious effects of excessive inflammation." When the Nurr1 gene is disabled, as it is in those with the rare familial form of Parkinson's disease, it leads to a pattern of inflammation that is exaggerated in both magnitude and duration, he added.

The causes of most common forms of Parkinson's remain poorly understood, but the disease is generally associated with an inflammatory component involving cells known as microglia, the researchers explained. Those microglia act as sentinel cells, keeping a lookout for potential infection or tissue injury in the central nervous system.

As for Nurr1, studies had found it plays an important role in dopaminergic neurons and that people with a rare mutant form of the gene produce too little of the protein it encodes, leading them to develop Parkinson's disease late in life. Earlier reports also showed that Nurr1 operates in cells other than neurons, where its activity is increased by inflammatory factors.

Glass and his colleague Kaoru Saijo, also of UCSD, suspected that Nurr1's roles outside of neurons might also be involved in Parkinson's disease. Indeed, they've now shown that Nurr1 limits the activity of pro-inflammatory neurotoxic mediators in microglia and in cells known as astrocytes, which serve as support cells to neurons. When Nurr1's activity is reduced, microglia launch an exaggerated inflammatory response that is amplified further by astrocytes. It is this overreaction that leads to the production of factors that ultimately kill dopaminergic neurons.

The findings suggest that inflammation may be an important general contributor to Parkinson's disease, which in the vast majority of cases has not been traced to any genetic cause, Saijo said. The researchers noted that while experts have grown to appreciate that Parkinson's disease has an inflammatory component, questions still remain about its role as a cause or consequence of the disease.

"We think if inflammation is not an initiating event, it is definitely a part of the process that could amplify the disease," Glass said. That's a key point moving forward, he said, because it suggests there should be further efforts to evaluate and test anti-inflammatory therapies in the treatment of Parkinson's. Treatments designed to interrupt the signals between microglia and astrocytes might hold additional promise for fighting the disease.

The new results may also have implications for the ultimate success or failure of stem cell therapies, Glass said. If the progression of Parkinson's disease is significantly influenced by inflammation as the researchers suggest, then any cell-based therapies designed to replace the dopaminergic neurons that are lost with new ones will also "have to deal with this process."

Protein is Key to Embryonic Stem Cell Differentiation
Investigators at Burnham Institute for Medical Research (Burnham) have learned that a protein called Shp2 plays a critical role in the pathways that control decisions for differentiation or self-renewal in both human embryonic stem cells (hESCs) and mouse embryonic stem cells (mESCs).

The research, led by Gen-Sheng Feng, Ph.D., differs with some earlier findings that suggested hESCs and mESCs differentiate as a result of different signaling mechanisms. The discovery that Shp2 has a conserved role between mice and humans suggests an interesting common signaling mechanism between mESCs and hESCs, despite the known distinct signaling paths and biological properties between the two types of pluripotent stem cells. The study was published online in the journal PLoS ONE on March 17, 2009.

Embryonic stem cells (ESCs) are pluripotent cells that can differentiate to become more than 200 different cell types. Because of their plasticity, ESCs have been suggested as potential therapies for numerous diseases and conditions, including neurodegenerative diseases, spinal cord injury and tissue damage. Development of such therapies is largely dependent on fully understanding and controlling the processes that lead to differentiation of hESCs into specialized cell types.

“There are many signaling pathways that help embryonic stem cells decide their fate,” said Dr. Feng. “We found that the Shp2 protein acts as a coordinator that fine-tunes the signal strength of multiple pathways and gives us a better understanding of the fundamental signaling methods that determine whether a stem cell’s fate will be self-renewal or differentiation.”

In the study, the Feng lab created mutant Shp2 mESCs and showed that differentiation was dramatically impaired as the cells self-renewed as stem cells. The researchers also demonstrated small interfering RNAs in hESCs reduce Shp2 expression and subsequent cell differentiation. Feng and colleagues screened chemical libraries and identified a small-molecule inhibitor of Shp2 that, in small doses, partially inhibits differentiation in both mESCs and hESCs. Taken together, these results suggest a conserved role for Shp2 in ESC differentiation and self-renewal in both mice and humans.

“This opens the door for new experimental reagents that will amplify the self-renewal process to create more stem cells for research and potential clinical use in the future,” Dr. Feng added. “This research also suggests that comparative analysis of mouse and human embryonic stem cells will provide fundamental insight into the cellular processes that determine ‘stemness,’ a critical question that remains to be answered in the stem cell biology field.”


Nuclear Hormone Receptors, MicroRNAs Form Cellular Switch
A particular nuclear hormone receptor called DAF-12 and molecules called microRNAs in the let-7 family form a molecular switch that encourages cells in the larvae of a model worm to shift to a more developed state, said a consortium led by researchers from Baylor College of Medicine (www.bcm.edu) in a report that appears online today in the journal Science (www.sciencemag.org).

As organisms go through the stages of life, hormones coordinate the changes. Nuclear receptors respond to hormones to coordinate stage transitions, but how they do so is not well understood.

GOING FROM STAGE 2 TO STAGE 3
"We knew that nuclear hormone receptors were involved in stage 2 to stage 3 transitions in Caenorhabditis elegans," said Dr. Adam Antebi (http://www.bcm.edu/mcb/?PMID=8411), associate professor in the Huffington Center on Aging at BCM and the report's senior author. "Another class of molecules called microRNAs is also involved in that transition. We hypothesized that maybe if they are involved in the same process, one turns on the other."

That turns out to be the case in C. elegans and may be true in more advanced organisms as well, he said.

A MODEL WORM ENABLES STUDIES
Scientists use the tiny worm called Caenorhabditis elegans to study such processes because it has a simple anatomy and life cycle. C. elegans develops from embryo through four larval stages into adulthood.

Each "stage" has specific programs of cell division, migration, differentiation and death that are crucial to the organism's final development. Particular master regulators in the worm determine each stage transition and are responsible for organizing developmental time.

"Expression of the let-7 family of microRNAs is dependent on the nuclear receptor and its hormone," Antebi said. "We can show in the worm and in cell culture that DAF-12 and its steroid hormone are directly activating these microRNAs."

HOW TRANSITIONS OCCUR
But how does this cause stage transitions? The tiny molecules called microRNAs work as switches to turn off other genes. In this case, the nuclear hormone receptor DAF-12 and its ligand turn on the microRNAs, which then turn off the earlier developmental "programming" of the cell (stage 2), allowing the later programming (stage 3) to take over.

Specifically, the microRNAs dial-back the activity of a protein called "hunchback," which specifies that the cells are in stage 2. That enables stage 3 to start and development to continue.

PROVIDES CANCER INSIGHTS
"We think this could also give insight into cancers," Antebi said, "particularly those that are hormone-dependent, such as breast or prostate cancer. When worm skin cells go from stage 2 to stage 3 they reduce their cell proliferation. When they fail this transition, skin cells overproliferate (grow uncontrollably)."

It is known that both nuclear receptors and microRNAs play a role in human cancers. These studies could help bridge understanding of the effects of the two.

LINKING DEVELOPMENT AND ENVIRONMENT
Antebi also thinks that this system links development to the environment. DAF-12 plays a role in a long-lived quiescent stage called the dauer diapause, which the worms enter in times of starvation and overcrowding.

"In good times, the DAF-12 steroid ligand is made, the microRNAs are turned on, and the worm goes through all stages of development to adult," said Antebi. (A ligand is a molecule that binds to the receptor to form a biologically active complex.)

"In bad times, the ligand is not made and the nuclear receptor (DAF-12) causes the animals to go into the long lived dauer stage, shutting down the microRNAs and the developmental clock," he said.

In this way, environmental signals actually affect the worm's rate of development, and perhaps even its aging, said Antebi.

Funding for this work came from the National Institutes of Health, the Ellison Medical Foundation, the Howard Hughes Medical Institute and the Robert Welch Foundation.

Alzheimer’s Disease Linked to Mitochondrial Damage
Investigators at the Burnham Institute for Medical Research (Burnham) have demonstrated that attacks on the mitochondrial protein Drp1 by the free radical nitric oxide—which causes a chemical reaction called S-nitrosylation—mediates neurodegeneration associated with Alzheimer’s disease.

Prior to this study, the mechanism by which beta-amyloid protein caused synaptic damage to neurons in Alzheimer’s disease was unknown. These findings suggest that preventing S-nitrosylation of Drp1 may reduce or even prevent neurodegeneration in Alzheimer’s patients. The paper was published in the April 3 issue of the journal Science.

The team of scientists, led by neuroscientist and clinical neurologist Stuart A. Lipton, M.D., Ph.D., director of the Del E. Webb Center for Neuroscience, Aging and Stem Cell Research, showed that S-nitrosylated Drp1 (SNO-Drp1) facilitates mitochondrial fragmentation, damaging regions of nerve cell communication called synapses.

Mitochondria are the energy storehouses of the cell, and their compromise by excessive fragmentation causes synaptic injury and eventual nerve cell death. Synapses are critical for learning and memory and their impairment leads to the dementia seen in Alzheimer’s patients.

“We now have a better understanding of the mechanism by which beta-amyloid protein causes neurodegeneration in Alzheimer’s disease,” said Dr. Lipton. “We found that beta-amyloid can generate nitric oxide that reacts with Drp1. By identifying Drp1 as the protein responsible for synaptic injury, we now have a new target for developing drugs that may slow or stop the progression of Alzheimer’s.”

Drp1 is an enzyme that mediates fission or fragmentation of mitochondria. The Burnham researchers showed that excessive production of nitric oxide caused S-nitrosylation of Drp1 and induced excessive fragmentation of mitochondria in cultured nerve cells or neurons.

The scientists also showed that beta-amyloid protein multimers, which had been previously implicated in Alzheimer’s disease, induced formation of SNO-Drp1. Importantly, elevated SNO-Drp1 levels were also found in human brains of Alzheimer’s patients, but not in those with Parkinson’s disease or controls who didn’t have neurodegenerative diseases.

Molecular modeling performed by the team suggested that S-nitrosylation of Drp1 causes dimerization of the protein and activation of enzymatic activity that induces mitochondrial fragmentation. To confirm this hypothesis, the scientists showed that RNA interference to knock down Drp1 or a mutation that prevented Drp1 activity inhibited excess mitochondrial fragmentation and protected the neurons.

Finally, the researchers showed that a mutated Drp1, lacking the nitrosylation site, did not induce mitochondrial fragmentation and also prevented neuronal damage. Taken together, these findings suggest that multimers of beta-amyloid protein induce generation of nitric oxide, which reacts with Drp1 to cause excessive mitochondrial fragmentation and in turn neuronal damage.

Cancer Stem Cells Generated by Cancer Outgrowth
Scientists have discovered that growing mouse skin cells in spheres can lead to generation of cells with properties of cancer stem cells, even without genetic manipulation of stem cell genes

This unexpected finding, published by Cell Press in the April 3rd issue of the journal Cell Stem Cell, provides a potential pathway for generation of cancer stem cells from differentiated cells and may even eventually lead to safer strategies for creation of induced pluripotent stem cells for use in regenerative therapies.

"A hallmark of all solid tumors is the outgrowth of cancer cells into three-dimensional structures," explains senior study author, Dr. Douglas C. Dean, from the University of Louisville Health Sciences Center in Louisville, Kentucky. Dr. Dean and colleagues examined whether abnormal cell configurations might trigger reprogramming of differentiated cells into cells that resembled cancer stem cells.

The researchers observed that mutation of all of the retinoblastoma tumor suppressor gene (RB1) family members, known to be critical for regulating cell-contact inhibition and restricting growth of normal cells into three-dimensional tumor-like structures, led to an outgrowth of cells into spheres that triggered generation of cells similar to cancer stem cells.

Surprisingly, the cancer stem cell-like cells expressed key genes expressed in embryonic stem cells and gave rise to a variety of differentiated cells.

Interestingly, cells with only one RB1 mutation remained contact inhibited, but when mechanically scraped off the dish and forced to form spheres, they also exhibited cancer stem-like characteristics. Even cells with intact RB1 genes could be forced to form spheres, suggesting that the reprogramming did not require the loss of RB1.

The researchers went on to show that the cancer stem-like cells isolated from the spheres with disrupted RB1 genes formed tumors when injected into mice and differentiated into mature cells in advancing cancers.

These results using cultured cells lead the authors to hypothesize that cancer stem cells may be generated as a direct function of the outgrowth of cells in the animal. "To our knowledge, this is the first example that silenced endogenous embryonic stem cell genes can be spontaneously reactivated in differentiated cells," says Dr. Dean. "We propose that the loss of cell contact inhibition when the RB1 pathway is inhibited leads to outgrowth into sphere-like structures, and these conditions in the advancing cancer trigger reprogramming of differentiated cells to cells with properties of cancer stem cells."



THURSDAY April 2, 2009---------------------------News Archive

Evidence Explains Poor Infant Vaccine Immune Reactions
For years, researchers and physicians have known that infants' immune systems do not respond well to certain vaccines, thus the need for additional boosters as children develop

Now, in a new study from the University of Missouri, one researcher has found an explanation for that poor response. In the study, the MU scientist found evidence that the immune systems of newborns might require some time after birth to mature to a point where the benefits of vaccines can be fully realized.

Habib Zaghouani, a professor of molecular microbiology and immunology and child health at the MU School of Medicine, recently found that a slowly maturing component of the immune system might explain why newborns contract infections easily. In his work, Zaghouani studied newborn mice and how their immune systems reacted when they were repeatedly exposed to an antigen that simulates a virus.

Zaghouani found that while the antigen would prompt a response of the immune system, it was not the expected response. In the adult immune system, two major types of cells, known as T-helper 1 (Th-1) and T-helper 2 (Th-2) cells, are instrumental in the development of an effective immune response. Typically, Th-1 cells respond when dangerous microbes enter the body. The Th-1 cells then work to help destroy the foreign microbes. When an antigen from a vaccine enters a body with a mature immune system, Th-1 cells respond and, after destroying the invader, the Th-1 cells "remember" how to fight the antigen for future battles. Th-2 cells typically develop when the body is exposed to allergens. The responses of Th-2 cells are usually strong and manifest in the form of allergic reactions.

When Zaghouani gave the newborn mice an antigen shortly after birth, he noticed the presence of both Th-1 and Th-2 cells. However, when he gave the antigen a second time, he noticed an abundance of Th-2 cells that responded to the antigen instead of Th-1 cells. Zaghouani was surprised to notice that the Th-2 cells worked to destroy the small contingent of Th-1 cells that had responded to the antigen given at birth.

"Perhaps we should test vaccines at a very early age in animals to establish a regimen with the most effectiveness," Zaghouani said.

When a baby first gets an infection, the immature immune system responds with both types of T cells. Unfortunately, Th-1 cells have an unusual receptor that binds to a specific hormone, which is deadly to the Th-1 cells. Ironically, this particular hormone is produced by the Th-2 cells. This results in an overabundance of Th-2 cells during the first few days of life.

We found that after six days, the immune systems in the mice matured enough to stop the death of the Th-1 cells," Zaghouani said. "After those initial days, the immune system is producing Th-1 cells with diminished hormonal receptors, thus surviving the effect of the compound that the Th-2 cells make."

Zaghouani's publication, "Delayed maturation of an IL-12-producing dendritic cell subset explains the early Th2 bias in neonatal immunity," was published in The Journal of Experimental Medicine.

Disappearing Before Dawn - Why We Sleep
A type of brain cell that was long overlooked by researchers embodies one of very few ways in which the human brain differs fundamentally from that of a mouse or rat, according to researchers who published their findings as the cover story in the March 11 issue of the Journal of Neuroscience

At 10 a.m. on a frigid January, the lights automatically flicker on in a rat room at the University of Wisconsin–Madison's Research Park. Postdoc Erin Hanlon strolls in, still wearing her scarf from the trip to the lab, where she will spend the next hour or so with Telito, a rat.

Telito's cage is tucked away in a television cabinet–like enclosure. He's freely moving but connected to a nearby computer by a bundle of wires emanating from the four tiny electrodes implanted into his cortex, held in place with screws and dental cement. She'll teach him to extend one paw through a plastic slot to grab a food reward—a task that will exercise a specific region of his brain. After 92 trials, she'll close the door behind her, let him nod off, and wait as the computer records the electrical brain waves of his slumber.

Hanlon is trying to replicate a similar 2004 experiment in humans performed by the same group, led by Chiara Cirelli and Giulio Tononi, which produced data that researchers are interpreting in two very different ways.

In the experiment, the group asked human subjects to complete a motor task using a computer mouse while wearing a snug-fitting, high-density electroencephalogram (EEG) cap. After the participants performed the task, the researchers measured their sleep patterns. They noticed an interesting pattern in subjects' slow waves, electrical patterns of less than four waves per second that are thought to reflect the need for sleep.

In general, people who are sleep-deprived tend to have more slow waves, and those waves are larger in amplitude than the slow waves of people who aren't sleep-deprived. In this experiment, slow waves were larger and occurred more often in the specific brain region used in the task, compared to other areas even within the same immediate brain region. And those subjects with the most active slow waves in that region seemed to perform better on the task the next day.

It was one of many experiments designed to answer one of life's biggest unsolved puzzles: Why do we sleep? For some researchers who study memory, the findings support a popular theory that the purpose of sleep is to replay and consolidate memories from the previous day. To them, sleep is important for memory, and the deep, slow waves seen in the same part of the brain used in a task indicate that the brain circuits involved in the task are reactivating. Such reactivation, or "replay," could explain why participants perform the task with greater accuracy after a night of sleep.

But for Cirelli and Tononi, their findings suggested an entirely different—and controversial—theory was perhaps true.

Sleep's core function, Cirelli and Tononi say, is to prune the strength or number of synapses formed during waking hours, keeping just the strongest neuronal connections intact. Synapse strength increases throughout the day, with stronger synapses creating better contact between neurons. Stronger synapses also take up more space and consume more energy, and if left unchecked, this process—which Cirelli and Tononi believe occurs in many brain regions—would become unsustainable.

Downscaling at night would reduce the energy and space requirement of the brain, eliminate the weakest synapses, and help keep the strongest neuronal connections intact. This assumption is based on the principle in neuroscience that if one neuron doesn't fire to another very often, the connection between the two neurons weakens. By eliminating some of the unimportant connections, the body, in theory, eliminates background connections and effectively sharpens the important connections. continued ...

New Strategy Developed to Diagnose Melanoma
A UCSF research team has developed a technique to distinguish benign moles from malignant melanomas by measuring differences in levels of genetic markers

Standard microscopic examinations of biopsied tissue can be ambiguous and somewhat subjective, the researchers say, and supplementing standard practice with the new technique is expected to help clarify difficult-to-diagnose cases.

In a large study of previously diagnosed cases, the new technique distinguished between benign, mole-like skin lesions and melanomas with a success rate higher than 90 percent. It also succeeded with most of the previously misdiagnosed cases, which were among the most difficult to distinguish.


This is the first large-scale study to demonstrate both the high diagnostic accuracy and practicality of a multi-biomarker approach to melanoma diagnosis, said Mohammed Kashani-Sabet, MD, professor of dermatology at UCSF and director of the Melanoma Center at the UCSF Helen Diller Family Comprehensive Cancer Center.

Kashani-Sabet is lead author on a paper reporting the new finding in the “Proceedings of the National Academy of Sciences,” which is scheduled for online publication the week of March 30, 2009. The paper also will appear in a future print issue of PNAS.

Melanoma is the deadliest form of skin cancer. It can spread to almost any organ of the body and is difficult to treat in its advanced stages. Progress in survival rates has been made principally through earlier diagnosis. The genomics-based approach combined with current diagnostic practice can aid earlier detection and contribute to more accurate assessment, report the UCSF scientists who developed the diagnostic tool.

The molecular diagnosis strategy is now being developed for clinical use by a diagnostics company
.

To develop the diagnostic tool, the researchers first used a microarray – a “gene chip”—to identify about 1,000 human genes that were present at different levels in malignant melanomas compared to benign moles. They narrowed their study down to five genes that all showed higher levels of activity in melanomas than in moles and could be studied with standard antibody techniques.

Focusing on the proteins produced by the five genes, they stained the proteins with antibodies to assess the level of gene expression in mole and melanoma tissues. The new diagnostic technique distinguished moles from melanomas by differences in both the level and the pattern of activity of the five proteins.

To develop and test the diagnostic technique, the researchers examined levels of the five biomarkers in 693 previously diagnosed tissue samples. To ensure that the diagnosis based on tissue examination had been correct, all samples were reviewed by the study’s pathologist. They analyzed the samples with the new procedure and found that the increased protein production by the melanomas compared with the moles was statistically significant, and thus a reliable diagnostic indicator. Unexpectedly, the proteins also showed different patterns of activity in the two types of tissue, yielding a second, even more discriminating diagnostic indicator.

“We hoped for clear diagnostic differences in the intensity of gene expression,” Kashani-Sabet said. “We found what we had hoped for, but then we got a bonus. The pattern of protein activity from the top to the bottom of the tissue was strikingly different between the benign and the malignant tissue, providing an additional trait valuable for diagnosis.”

Although some of the genes and their proteins were stronger indicators than others, the research team found that the combination of all five achieved the highest diagnostic accuracy. The multi-biomarker diagnostic correctly diagnosed 95 percent of the benign moles—a measure known as specificity. The accuracy rate was 91 percent for diagnosing malignant melanomas – the sensitivity rate. In addition, the strategy correctly diagnosed 75 percent of the most difficult cases, which had previously been misdiagnosed. The technique also accurately diagnosed other difficult-to-diagnose moles, known as dysplastic and Spitz nevi.

“We have a test that can help patients and help clinicians who treat melanoma,” said Kashani-Sabet. “With this added diagnostic tool we can shed light on lesions that are difficult to classify and diagnose.”

Co-authors on the paper and collaborators in the research, all at UCSF, are Javier Rangel, MD, resident in dermatology; Mehdi Nosrati, BS, staff research assistant; and Sima Torabian, MD, a former post-doctoral research fellow in the Kashani-Sabet lab.

Also, Jeff Simko, MD, associate professor of clinical pathology; Chris Haqq, MD, PhD, assistant adjunct professor of urology; James Miller, PhD, statistical consultant; Richard Sagebiel, MD, professor of dermatology and pathology; Dan Moore, PhD, statistical consultant; and David Jablons, MD, professor of surgery.

A patent has been filed by UCSF covering the use of these five genetic markers in melanoma diagnosis. The patent has been licensed to Melanoma Diagnostics, based in Fremont, Calif. Lead author Kashani-Sabet owns stock in this company. Co-author Miller has an ownership interest in MDMS, a software company in Arizona that provided the software to generate diagnostic algorithms.


The research was supported by the Auerback Melanoma Research Fund, the Herschel and Diana Zackheim Endowment Fund, the American Cancer Society and the National Institutes of Health.

UC Davis Researchers Identify Protein that May Help Spread Breast Cancer, Beat Cancer Drugs
New research from UC Davis Cancer Center shows that a protein called Muc4 may be the essential ingredient that allows breast cancer to spread to other organs and resist therapeutic treatment

The study, which appears in the April 1 issue of Cancer Research, is one of the first to characterize the role of Muc4 in the disease.

Kermit Carraway, senior author of the study, knew that Muc4 was not always expressed in primary breast cancer tumors, yet it could be present in lymph node metastases. He suspected that it may have a specialized function in the process of metastasis.

"Breast cancer deaths are caused by metastasis, not by the primary tumor," explained Carraway, an associate professor of biochemistry and molecular medicine. "It's at that point that the disease also becomes difficult to treat. We think that Muc4 may be packing a one-two punch by promoting the release of breast cancer cells from the primary tumor and then inhibiting their death."

Muc4 is member of a group of proteins called mucins, which are commonly found in fluids such as tears and mucus. They have a known role in protecting epithelial cells, from which breast cancer cells are derived. When separated from their surrounding cell matrix, epithelial cells tend to die. Metastasizing breast cancer cells, however, can survive this detachment.

"Because breast cancer cells can lose their adhesive properties and still thrive, we suspected that Muc4 may be somehow allowing them to leave their cellular framework, travel to secondary sites and withstand treatment," Carraway explained.

To test his suspicions, Carraway and his team conducted two experiments. They started by comparing breast cancer cells that express Muc4 with those for which Muc4 production is blocked. The researchers then exposed both types of cells to chemotherapy drugs. The Muc4-producing cells survived.

They repeated the experiment with breast cancer cells and epithelial cells that do not naturally express Muc4 but were engineered to do so. Both sets of cells avoided cell death and effectively resisted chemotherapy.

"Our results lead us to believe that Muc4 is somehow disrupting normal links between epithelial cells," said UC Davis graduate student Heather Workman, lead author of the study. "We now need to refine our understanding of this disruption process in order to find ways to interfere with it. There currently are no drugs that target Muc4, and this research will help change that."

Carraway is now preparing to test metastasizing breast cancer tumor cells for the overexpression of Muc4.

"If we find that Muc4 is all over metastasizing breast cancer cells, it will confirm that we are on the right track," he said.

While Carraway's current focus is on breast cancer, his findings could have relevance to other cancers that show aggressive properties. For example, Muc4 is also expressed in pancreatic, lung and ovarian tumor cells.

"Muc4 is likely a central cellular mechanism for metastasis of many cancers, and we will be continuing this important work to prove that," he said.

Health Benefits or Consequences of Folic Acid Depends on Circumstances
For the past several decades, evidence has shown that greater dietary intake of the B-vitamin, folate, offers protection against the development of certain common cancers and reduces neural tube defects in newborns, opening new avenues for public health interventions that have a great impact on health. However, folate’s central role as an essential factor in DNA synthesis also means that abundant availability of the vitamin can enhance the development of pre-cancerous and cancerous tumors

Further, the intake of folic acid that results from consuming foods that are voluntarily fortified (e.g.: ready-to-eat cereals) in combination with the additional intake received from mandatory fortification of flour means that supplementary intake of folic acid is unnecessary for many segments of the population, and may even present a risk.

Nevertheless, the issue is a complicated one since women of child-bearing age seem to benefit from supplemental folic acid in regard to its protection against birth defects. In the April issue of the journal Nutrition Reviews, two new articles by Omar Dary, Ph.D., and Joel B. Mason, M.D., assess the conditions under which folic acid can be beneficial and harmful and contribute to guidelines for the healthful intake of folic acid as a complement to dietary folate.

The consequences of inadequate folate intake remain prevalent in many countries, even in industrial countries where specific interventions of folic acid have not been implemented. Moreover, there continues to be some concern—which, to date, lacks compelling scientific evidence—that the synthetic form of the vitamin, folic acid, might have adverse effects that do not exist with natural sources of folate.

Under most circumstances, adequate intake of folate appears to assume the role of a protective agent against cancer, most notably colorectal cancer. However, in select circumstances in which an individual who harbors a pre-cancerous or cancerous tumor consumes too much folic acid, the additional amounts of folate may instead facilitate the promotion of cancer.

In countries in which the fortification of flour with folic acid is working well, additional supplementation in the form of vitamin pills can lead to excessive intakes of the vitamin, which can then have undesirable adverse effects. Thus, folate appears to assume different guises depending on the circumstances. The level of intake of this micronutrient that is safe for one person may be potentially harmful to another.

“These effects of folate on the risk of developing cancer have created a global dilemma in the efforts to institute nationwide folic acid fortification programs around the world,” Mason notes.

Most individuals in the U.S. population are now folate-replete, so one consideration would be to reduce the doses of the vitamin that are present in most over-the-counter supplements. Many people receive sufficient amounts of folate through their diet.

Now that the supply of folic acid in the diet is much larger than it was prior to mandatory fortification, food policies may need to be adjusted to the current knowledge and the new circumstances.

“The design of cogent public health policies that effectively optimize health for many while presenting no or minimal risk to others, must often occur in the absence of complete information,” Mason concludes. “However, we are nevertheless obliged to deliberate with as much of an in-depth understanding as the existing science allows.”

New Stem Cell Therapy May Lead To Treatment For Deafness
Deafness affects more than 250 million people worldwide. It typically involves the loss of sensory receptors, called hair cells, for their "tufts" of hair-like protrusions, and their associated neurons

The transplantation of stem cells that are capable of producing functional cell types might be a promising treatment for hearing impairment, but no human candidate cell type has been available to develop this technology.

A new study led by Dr. Marcelo N. Rivolta of the University of Sheffield has successfully isolated human auditory stem cells from fetal cochleae (the auditory portion of the inner ear) and found they had the capacity to differentiate into sensory hair cells and neurons.

The researchers painstakingly dissected and cultured cochlear cells from 9- to 11-week-old human fetuses. The cells were expanded and maintained in vitro for up to one year, with continued division for the first 7 to 8 months and up to 30 population doublings, which is similar to other non-embryonic stem cell populations, such as bone marrow. Gene expression analysis showed that all cell lines expressed otic markers that lead to the development of the inner ear as well as markers expressed by pluripotent embryonic stem cells, from which all tissues and organs develop.

They were able to formulate conditions that allowed for the progressive differentiation into neurons and hair cells with the same functional electrophysiological characteristics as cells seen in vivo.

"The results are the first in vitro renewable stem cell system derived from the human auditory organ and have the potential for a variety of applications, such as studying the development of human cochlear neurons and hair cells, as models for drug screening and helping to develop cell-based therapies for deafness," say the authors.

Although the hair cell-like cells did not show the typical formation of a hair bundle, the authors suggest that future studies will aim to improve the differentiation system. They are currently working on using the knowledge gleaned from this study to optimize the differentiation of human embryonic stem cells into ear cell types.

"Although considerable information has been obtained about the embryology of the ear using animal models, the lack of a human system has impaired the validation of such information," the authors note.
"Access to human cells that can differentiate should allow the exploration of features unique to humans that may not be applicable to animal models," says Donald G. Phinney, co-editor of the journal. The protocol they developed to expand and isolate human fetal auditory stem cells may be able to be adapted for deriving clinical-grade cells with potential therapeutic applications.

Dr Ralph Holme, director of biomedical research for Royal National Institute for Deaf and Hard of Hearing People, said: "There are currently no treatments to restore permanent hearing loss so this has the potential to make a difference to millions of deaf people."

The study is published in the April issue of Stem Cells.



WEDNESDAY April 1, 2009---------------------------News Archive

Lousy With Chromosomes
Scientists have found an unprecedented evolutionary modification deep within the cells of the lowly human body louse (Pediculus humanus): the tiny blood sucker contains not one but 18 separate mitochondrial chromosomes

"It's a big surprise to me and my colleagues," wrote Renfu Shao, lead author of the newly published Genome Research paper describing the discovery, in an email to The Scientist. "Since the human mitochondrial genome was sequenced in 1981, more than 1500 animals have been sequenced for complete mitochondrial genomes," he wrote. With the exception of some oddball ciliates, flagellates and cnidarians, virtually all animals have a single, circular mitochondrial chromosome that contains approximately 37 genes. "Thus, it has almost been taken for granted that any animals would have a single mitochondrial chromosome with all mitochondrial genes on it."

David Rand, a Brown University evolutionary geneticist who was not involved with the study, told The Scientist that multiple mitochondrial chromosomes in animals were exceedingly rare and that the presence of the 18 "mini chromosomes" in the mitochondria of the louse was "clearly a novel organization."

Shao, a postdoc in the lab of University of Queensland evolutionary geneticist Stephen Barker, and his collaborators used whole-genome shotgun sequencing to uncover the unique genetic structure of the body louse's mitochondrial genome after several years of failing to amplify the entire mitochondrial genome using traditional sequencing methods. Each of the 18 mini chromosomes found in the body louse's mitochondria were only 3-4 kb long and contained 1-3 of the total 37 mitochondrial genes.

Multiple mitochondrial chromosomes may allow the organism to streamline replication and transcription of individual genes, since generally, genes on a single strand of mitochondrial DNA are transcribed together, Shao explained. He added that if the DNA on multiple mitochondrial chromosomes is replicating in concert, replication of the entire mitochondrial genome would occur faster.

The evolution of this unique mitochondrial genome structure is not likely due to a single "big bang" event, the authors say. Where typical single mitochondrial chromosomes have only one copy of the sequences necessary to initiate replication and transcription, each of the louse's mini chromosomes has its own set of instructions. This, said Shao, points to a more complicated evolution towards multiple mitochondrial chromosomes.

"We think that the multiple minichromosomes were generated from a series of events that involved excision and rejoining of fragments of [mitochondrial] chromosome over a long period of time," he wrote. As non-coding, control regions from the single chromosome were copied and excised along with a few genes, the resulting mini chromosome may have functioned more efficiently than the region on the single chromosome; the redundant region on the larger chromosome would have then been deleted, explained Shao. It would take just a few more excision events to arrive at 18 mini chromosomes with all the functionality of their single-circle ancestor.

The findings generate many physiological questions about how the louse manages to keep track of separate mitochondrial chromosomes. During cell division, for example, all 18 mini chromosomes must be copied and transferred seamlessly to daughter mitochondria -- no easy task. If one is missing, said Shao, the new mitochondria would not function properly. "It is a mystery to us how these minimitochondrial chromosomes are 'herded' into daughter mitochondria and daughter cells at mitochondrial and cell division."

"Clearly the cell has solved this in some way," added Rand, "because [the lice] are extant."

Rand said that whole-genome sequencing may yet uncover more examples of such mitochondrial organization. "Certainly as we move to more whole-genome analyses of mitochondrial DNA, we may be more likely to pick this up," he said. Other parasites, he noted, might be a good place to look. Plasmodium falciparum, one of the protozoan parasites that causes malaria in humans, he said, has one of the smallest mitochondrial genomes known, though this reduction has left the single chromosome strategy intact.

Astrocytes Help Separate Man from Mouse
A type of brain cell that was long overlooked by researchers embodies one of very few ways in which the human brain differs fundamentally from that of a mouse or rat, according to researchers who published their findings as the cover story in the March 11 issue of the Journal of Neuroscience

Scientists at the University of Rochester Medical Center found that human astrocytes, cells that were long thought simply to support flashier brain cells known as neurons that send electrical signals, are bigger, faster, and much more complex than those in mice and rats.

“There aren’t many differences known between the rodent brain and the human brain, but we are finding striking differences in the astrocytes. Our astrocytes signal faster, and they’re bigger and more complex. This has big implications for how our brains process information,” said first author Nancy Ann Oberheim, Ph.D., a medical student who recently completed her doctoral thesis on astrocytes.

The study is one of the most extensive examinations yet of the astrocyte. Oberheim and co-authors discovered a previously unknown form of the cell, a varicose projection astrocyte, in the human brain but not in the rodent brain. The team also found that the most abundant type of astrocyte, protoplasmic astrocytes, are approximately 2.6 times larger than their rodent counterparts, and that the human cells have about 10 times as many “processes,” or structures designed to connect to other cells.

“We have not really been able to understand why the human brain is so much more capable than that of any other animal,” said neuroscientist Maiken Nedergaard, M.D., D.M.Sc., who led the study. “Some people have thought that it’s simply that a bigger brain is a better brain, but an elephant’s brain is bigger than a person’s, for example, but it’s not nearly as powerful. So that’s not the answer.

“It may be that humans have a much higher brain capacity in large part because our astrocytes are more sophisticated and have more complex processing power,” added Nedergaard, who spoke last week at a Gordon Research Conference on glial biology. “Studies in rodents show that non-neuronal cells are part of information processing, and our study suggests that astrocytes are part of the higher cognitive functioning that defines who we are as humans.”

Astrocytes had long been considered passive support cells, a means to hold the rest of the brain cells together, like glue. Medical students might spend a few minutes pondering the astrocyte before moving on to their flashy counterparts, the neurons that fire the electrical signals crucial to pretty much everything we do. It’s the electrical activity of neurons that constitutes what most scientists have considered to be brain activity, and it’s the neurons that are the target of every currently available drug aimed at brain cells. If astrocytes were important, scientists thought, it was most likely because they help create a healthy environment for the neurons.

I
t turns out that astrocytes, which are 10 times as plentiful as neurons, had been pushed to the boundaries of neuroscience because of a gap in the tools used to study the brain. Scientists measure signaling among brain cells mainly by looking at electrical activity. But astrocytes don’t fire in the same way as neurons, and so conventional techniques don’t record their activity. So when scientists “listened” with conventional techniques, they witnessed no activity.

Rather than realizing their tools were incomplete, scientists assumed that astrocytes were silent.

So Nedergaard devised a new way to “listen” for astrocyte activity, developing a sophisticated laser system to look at their activity by measuring the amount of calcium inside the cells. Her team has discovered what might be called the secret lives of astrocytes and has made a series of startling discoveries. Astrocytes use calcium to send signals to the neurons, and the neurons respond; neurons and astrocytes talk back and forth, indicating that astrocytes are full partners in the basic working of the brain; and astrocytes are central to conditions like stroke, Alzheimer’s, epilepsy, and spinal cord injury.

“Dogma is slow to change, and one of the dogmas of neuroscience is that astrocytes are support cells that don’t do much themselves,” said Oberheim. “The view is slow to change, but scientists are coming around. Astrocytes are now acknowledged as active participants in brain function and sensory processing.”

The brain’s two signaling systems – one composed of neurons, and one of astrocytes – complement each other, Nedergaard said. Neurons send signals extremely quickly over long distances – the hand touches a hot stove, for instance, and the brain detects the danger and moves the hand away, instantly. Astrocytes, in contrast, send slower signals whose function is still being worked out by scientists.

“The brain contains two communication networks using different languages,” said Nedergaard, director of the Division of Glial Disease and Therapeutics of the Center for Translational Neuromedicine. “You have a highly sophisticated electrical network embodied in the neurons, which send signals instantaneously. And then you have a much slower network composed of astrocytes whose signals are 10,000 times slower but which might be able to process the information in a more sophisticated manner and retrieve memories.

“There is no other tissue in the body that mixes up two different types of cells so completely as how astrocytes and neurons are interspersed throughout the brain,” Nedergaard added. “Both comprise extensive signaling networks. Where those networks interface and how they interact makes the brain so interesting.”

To do the study, the team studied human brain tissue taken from 30 people who had had surgery, mostly to treat epilepsy or brain tumors. They compared the astrocytes in human brains to those in mice and rats. In addition to the findings above, the team noted additional differences:

Astrocytes in people signal five times as fast as those in mice and rats.
Human astrocytes are organized into more complex units known as domains than are rodent astrocytes. A typical rodent domain includes tens of thousands of neuronal synapses, while the team found that a human domain might include up to 2 million synapses. These domains are highly organized groupings of cells that appear to be precisely situated, almost like atoms in a crystal. This organization is likely important for information processing, said Nedergaard, who notes that brain injury is associated with a loss of astrocytic domain organization and a decrease in cognitive function.

In people, cells known as fibrous astrocytes, which are mainly for structural support, are on average more than twice as large as their counterparts in mice and rats. Also, people have another type of cell known as interlaminar astrocytes, which are not present in rodents.

Another difference concerns the end feet of protoplasmic astrocytes, which wrap around blood vessels throughout the brain and are thought to play a role in the brain’s blood flow. In humans, these end feet cover the walls of blood vessels much more completely than they do in mice and rats, possibly playing a much more important role in keeping agents in the blood from entering the brain and in regulating blood flow.

The work was funded by the G. Harold and Leila Y. Mathers Charitable Foundation and by the National Institute of Neurological Disorders and Stroke.

Brain building: Study Shows Brain Growth Tied to Cell Division in Mouse Embryos
How your brain grows might come down to how your cells divide

In the April 6 issue of the Journal of Cell Biology (JCB), Lake and Sokol report that mouse protein Vangl2 controls the asymmetrical cell division and developmental fate of progenitor neurons.

Vangl2 (aka Strabismus in flies) is a component of the PCP (planar cell polarity) pathway that is active in a variety of tissues and organisms. Mice that lack Vangl2 have a number of neurological defects including incomplete neural tube closure and reduced brain size.

Sokol and Lake wondered how Vangl2 might influence brain development. In the cerebral cortex, neurons are born from a pool of progenitor cells, and the time of their birth determines their fate. The research duo found that Vangl2-lacking mouse embryos had large numbers of early-born neurons and few remaining progenitor cells. This hinted that Vangl2-lacking neurons were differentiating prematurely—a suspicion confirmed in vitro.

The progenitor pool is maintained by asymmetrical division—one daughter cell becomes a neuron, the other self-renews. This fate asymmetry is thought to depend on the orientation of cell division, and the authors observed an increase in the number of symmetrically dividing progenitors in the brains of Vangl2-lacking mouse embryos. Also, Vangl2-lacking cells in culture showed symmetrical distribution of a spindle-orienting factor that in normal cells distributes asymmetrically.

Such similarities between Vangl2-lacking cells in vitro and in vivo will facilitate ongoing studies of the PCP pathway in neurogenesis.

Experimental Design Could Reduce Need for Animal Tests
Accounting for environmental changes may be better than trying to control them

Researchers could cut the use of animals in their experiments by changing the way they analyze their results, according to a study by scientists based in Germany and the United States.

In a typical animal experiment, researchers will try to standardize factors such as the animals' genetic backgrounds and laboratory conditions to make it as easy as possible for other researchers to reproduce their results later. Now, a team led by Hanno Würbel at the Justus-Liebig-University in Giessen, Germany, has reanalyzed a study of mouse behaviour by taking such genetic and environmental variations into account, and they got fewer spurious results, or false positives, than the initial study.

In an article in this week's Nature Methods1, Würbel's team argues that initial chemical or drug screenings that include such natural variations in animals could help researchers cut the number of expensive secondary screenings and make their results more reproducible.

"In agricultural [and human] experiments it's absolutely recognized that there is uncontrollable variation," says study coauthor Joseph Garner of Purdue University in West Lafayette, Indiana. "It's only in laboratory animals that we have this draconian idea that we can control all variation."

To see whether a different experimental design might work better, Garner, along with Würbel and his student Helene Richter, reanalyzed data from a published multilaboratory mouse behaviour study2. They report that analyzing the data from the mice without accounting for different environmental conditions created about 10 times as many spurious results as when they directly compared such mice in what they call a "heterogeneous" experimental design.

Garner says that uncontrollable variations in the conditions from lab to lab may be throwing off the results. He blames interference from environmental factors such as the location of a mouse's cage within a lab, which might introduce additional light, noise or odours that can cause behaviour-changing anxiety.

"There has to be a gremlin, a Machiavellian gremlin, that is causing false positives in these experiments," Garner says.

"That might be a good thing to do, but I think they would need to show it experimentally," says John Crabbe of the Oregon Health & Science University in Portland.

Crabbe published a study3 in 1999 that found that certain mouse behaviours, particularly those thought to be genetically influenced, were easy to replicate from one lab to the next, but others, particularly those involving emotional responses, were more difficult to replicate.

Crabbe argues that problems with replicability also occur in other fields, including biochemistry and physics, and that in practical terms, any provocative experiment "is going to be done again anyhow ... and robust findings will persist".

In an accompanying article in Nature Methods4, neuroscientist and geneticist Richard Paylor, of Baylor College of Medicine in Houston, also says a fresh experiment designed to test Würbel's idea is the "obvious next step".

Würbel says he and his team are already working on such an experiment. "It's impractical to design every experiment as a multilab study," he concedes. "What we need to work out [next] is one or two factors that we can vary within the lab."

Computer Simulations Explain Limitations of Working Memory
Researchers at Karolinska Institutet (KI) have constructed a mathematical activity model of the brain´s frontal and parietal parts, to increase the understanding of the capacity of the working memory and of how the billions of neurons in the brain interact. One of the findings they have made with this "model brain" is a mechanism in the brain´s neuronal network that restricts the number of items we can normally store in our working memories at any one time to around two to seven

Working memory, which is our ability to retain and process information over short periods of time, is essential to most cognitive processes, such as thinking, language and planning. It has long been known that the working memory is subject to limitations, as we can only manage to "juggle" a certain number of mnemonic items at any one time. Functional magnetic resonance imagery (fMRI) has revealed that the frontal and parietal lobes are activated when a sequence of two pictures is to be retained briefly in visual working memory. However, just how the nerve cells work together to handle this task has remained a mystery.

The study, which is published in the journal PNAS, is based on a multidisciplinary project co-run by two research teams at KI led by professors Torkel Klingberg and Jesper Tegnér. Most of the work was conducted by doctors Fredrik Edin and Albert Compte, the latter of whom is currently principal investigator of the theoretical neurobiology group at IDIBAPS in Barcelona.

For their project, the researchers used techniques from different scientific fields, applying them to previously known data on how nerve cells and their synapses function biochemically and electrophysiologically. They then developed, using mathematical tools, a form of virtual or computer simulated model brain. The computations carried out with this "model brain" were tested using fMRI experiments, which allowed the researchers to confirm that the computations genuinely gave answers to the questions they asked.

"It´s like a computer programme for aircraft designers," says Fredrik Edin, PhD in computational neuroscience. "Before testing the design for real, you feed in data on material and aerodynamics and so on to get an idea of how the plan´s going to fly."

"The model predicts, for instance, that increased activation of the frontal lobes will improve working memory," continues Dr Edin. "This finding was also replicable in follow-up fMRI experiments on humans. Working memory is a bottleneck for the human brain´s capacity to process information. These results give us fresh insight into what the bottleneck consists of."

Publication:
Fredrik Edin, Torkel Klingberg, Pär Johansson, Fiona McNab, Jesper Tegnér & Albert Compte
Mechanism for top-down control of working memory capacity
PNAS, online early edition 30 March - 3 April 2009.


Distinguishing Single Cells With Nothing But Light
Researchers at the University of Rochester have developed a novel optical technique that permits rapid analysis of single human immune cells using only light

Availability of such a technique means that immunologists and other cellular researchers may soon be able to observe the responses of individual cells to various stimuli, rather than relying on aggregate statistical data from large cell populations. Until now scientists have not had a non-invasive way to see how human cells, like T cells or cancer cells, activate individually and evolve over time.

As reported today in a special biomedical issue of Applied Optics, this is the first time clear differences between two types of immune cells have been seen using a microscopy system that gathers chemical and structural information by combining two previously distinct optical techniques, according to senior author Andrew Berger, associate professor of optics at the University of Rochester.

Berger and his graduate student Zachary Smith are the first to integrate Raman and angular-scattering microscopy into a single system, which they call IRAM.
"Conceptually it's pretty straightforward—you shine a specified wavelength of light onto your sample and you get back a large number of peaks spread out like a rainbow," says Berger. "The peaks tell you how the molecules you're studying vibrate and together the vibrations give you the chemical information."

According to Smith, "Raman spectroscopy is essentially an easy way to get a fingerprint from the molecule."
Structural information is simultaneously gathered by examining the angles at which light incident on a sample is bumped off its original course. Together the chemical and structural information provide the data needed to classify and distinguish between two different, single cells. Berger and Smith verified this by looking at single granulocytes—a type of white blood cell—and peripheral blood monocytes. "One of the big plusses with our system is that it's a non-labeling approach for studying living cells," says Berger.

IRAM differs from most standard procedures where markers are inserted in, or attached to cells. If a marker sticks to one cell, and not the other, you can tell which cell is which on the basis of specific binding properties.
While markers are often adequate for studying cells at a single point in time, monitoring a cell over time as it changes is more problematic, since the marker can affect dynamic cell activities, like membrane transport. And internal markers actually involve punching holes in the membrane, damaging or killing the cell in the process. "Our method uses only light to effectively reach inside the cell," says Smith. "We can classify internal differences in the cell without opening it up, attaching anything to it, or preparing it in any special way. It's really just flipping a switch."

Despite being relatively intense, the light used with IRAM does not harm or inhibit normal cell functionality. This is because the wavelength of the light can be precisely calibrated to minimize absorption by the cells. The near-infrared spectrum has proven particularly optimal for allowing almost all of the light to pass through the cells.

With the availability of a technique where making a measurement does not alter cellular activity, scientists will be able to better observe individual cell responses to stimuli, which Berger and Smith suspect may have far reaching implications for current understandings of cell activation and development.

"In the cell sensing community it's currently a pretty hot area to figure out how to analyze activation responses on a cell-by-cell basis," says Berger. "If individual information was available on top of existing ensemble data, you'd have a richer understanding of immune responses."

Perfecting IRAM has been a stepping stone process so far. Now that individual cells can be distinguished, Berger and Smith are actively investigating activation processes more explicitly. Preliminary IRAM experiments conducted on T cells have revealed perceivable differences between the initial resting state of a T cell and its state following an encounter with an invader.

The next step will be to use IRAM to gather data continuously so that scientists can effectively watch single cells undergo activation and react to stimuli in real-time. The ability to know not only about the aggregate responses of cells, but also be able to observe the earliest changes among individual cells, may be of profound importance in time-critical areas, such as cancer research and immunology.

"There's an obvious desire among cell researchers to be able to deliver a controlled stimulant to a single cell and then study its response over time," says Berger. "The clinical insights that might arise are currently in the realm of speculation. We won't know until we can do it—and now we can."



TUESDAY March 31, 2009---------------------------News Archive

Hundreds of Natural-Selection Studies Could Be Wrong
Scientists at Penn State and the National Institute of Genetics in Japan have demonstrated that several statistical methods commonly used by biologists to detect natural selection at the molecular level tend to produce incorrect results

"Our finding means that hundreds of published studies on natural selection may have drawn incorrect conclusions," said Masatoshi Nei, Penn State Evan Pugh Professor of Biology and the team's leader. The team's results will be published in the Online Early Edition of the journal Proceedings of the National Academy of Sciences during the week ending Friday 3 April 2009 and also in the journal's print edition at a later date.

Nei said that many scientists who examine human evolution have used faulty statistical methods in their studies and, as a result, their conclusions could be wrong. For example, in one published study the scientists used a statistical method to demonstrate pervasive natural selection during human evolution. "This group documented adaptive evolution in many genes expressed in the brain, thyroid, and placenta, which are assumed to be important for human evolution," said Masafumi Nozawa, a postdoctoral fellow at Penn State and one of the paper's authors. "But if the statistical method that they used is not reliable, then their results also might not be reliable," added Nei. "Of course, we would never say that natural selection is not happening, but we are saying that these statistical methods can lead scientists to make erroneous inferences," he said.

The team examined the branch-site method and several types of site-prediction methods commonly used for statistical analyses of natural selection at the molecular level. The branch-site method enables scientists to determine whether or not natural selection has occurred within a particular gene, and the site-prediction method allows scientists to predict the exact location on a gene in which natural selection has occurred.

"Both of these methods are very popular among biologists because they appear to give valuable results about which genes have undergone natural selection," said Nei. "But neither of the methods seems to give an accurate picture of what's really going on."

Nei said that for many years he has suspected that the statistical methods were faulty. "The methods assume that when natural selection occurs the number of nucleotide substitutions that lead to changes in amino acids is significantly higher than the number of nucleotide substitutions that do not result in amino acid changes," he said. "But this assumption may be wrong. Actually, the majority of amino acid substitutions do not lead to functional changes, and the adaptive change of a protein often occurs by a rare amino acid substitution. For this reason, statistical methods may give erroneous conclusions." Nei also believes that the methods are inaccurate when the number of nucleotide substitutions observed is small.

To demonstrate the faultiness of the statistical methods, Nei's team compiled data collected by their Emory University colleague, Shozo Yokoyama, on the genes that control the abilities of fish to see light at different water depths and on the genes that control color vision in a variety of animals. The team used these data to compare statistically predicted sites of natural selection with experimentally determined sites. They found that the statistical methods rarely predicted the actual sites of natural selection, which had been identified by Yokoyama through experiments. "In some cases, statistical method completely failed to identify the true sites where natural selection occurred," said Nei. "This particular exercise demonstrated the difficulty with which statistical methods are able to detect natural selection."

To demonstrate how small sample sizes can lead to incorrect results, the team used computer simulations to examine the evolution of genes in three primates: humans, chimpanzees, and macaques. The scientists mimicked the procedures used by the authors of a 2007 paper, which applied the branch-site method to 14,000 orthologous genes -- genes that are genealogically identical among different species -- and which found that the method predicted selection in 32 of the genes. Nei and his team also studied selection using Fisher's exact test, but this test did not detect any selection. "The results indicate that the number of nucleotide substitutions that occurred were too small to detect any selection; therefore, all of the 32 cases obtained by the branch-site method must be false positives," said Nozawa.

"These statistical methods have led many scientists to believe that natural selection acted on many more genes in humans than it did in chimpanzees, and they conclude that this is the reason why humans have developed large brains and other morphological differences," said Nei. "But I believe that these scientists are wrong. The number of genes that have undergone selection should be nearly the same in humans and chimps. The differences that make us human are more likely due to mutations that were favorable to us in the particular environment into which we moved, and these mutations then accumulated through time."

Nei said that to obtain a more realistic picture of natural selection, biologists should pair experimental data with their statistical data whenever possible. Scientists usually do not use experimental data because such experiments can be difficult to conduct and because they are very time-consuming.

A third author on the study is Yoshiyuki Suzuki, a researcher at the National Institute of Genetics in Japan. This research was supported by the National Institutes of Health.

New Images of Marine Microbe Illuminate Process of Carbon and Nitrogen Fixation
Stunning new pictures of a cell's inner workings are helping scientists solve a long-standing debate over how a vital marine microbe is able to fix both carbon and nitrogen

Trichodesmium is unusual among marine microbes because it both "breathes" carbon dioxide like plants, while also taking nitrogen gas from the air and "fixing" it into a fertilizer of the seas. How Tricho does both these things has long puzzled researchers, since the two processes don't work well together: fixing carbon dioxide creates oxygen, and oxygen inhibits the enzyme that makes fixing nitrogen possible. It would be as if breathing made it hard to grow. "Everyone has been trying to figure out how they do it," said USC microbiologist Juliette Finzi-Hart. "The more we can understand how it functions, the better we can model it, and the better we can understand its role in the context of the global carbon and nitrogen cycles."

In a new study to be published Monday in the Early Edition of the Proceedings of the National Academy of Sciences, Finzi-Hart and colleagues offer new evidence that Tricho spends part of the day focusing on carbon and the other part on nitrogen.

Using advanced imaging technology rarely used in the marine sciences, the study lends support to the theory that Tricho manages to fix both by separating the processes in time. The stunning pictures also revealed specific hotspots where fixed nitrogen is temporarily stored, and offers strong evidence that Tricho fixes both nitrogen and carbon in the same cell.

As a cyanobacterium, Tricho uses sunlight to fix carbon dioxide and turn it into food, using a process like photosynthesis. In addition, it's a major nitrogen fixer, making it a "fertilizer for the open ocean." Just like fertilizer in a field, Tricho's fixed nitrogen makes the seas thrive, and along with other nitrogen fixers, they in effect control the health of entire ecosystems.

Theories about how Tricho fixes both carbon and nitrogen have largely fallen into two camps. "Time" advocates believe that Tricho, like some other microorganisms, separates carbon and nitrogen fixation by time. At peak sun, carbon fixation gets inhibited, less oxygen is formed, and a window opens for nitrogen fixation to ramp up. "Space" advocates, on the other hand, believe that Tricho physically separates the carbon and nitrogen fixation processes in a kind of "division of labor."

Tricho is a single-cell organism that lives in colonies. Each cell is stacked on top of one another in long filaments called trichomes, and these trichomes clump together. "They look like eyelashes," Finzi-Hart said. "When you collect a sample, you get a big bucket and a big scoop, and you have a table of 15 scientists literally picking out Tricho by hand."

In at least one other type of cyanobacteria, the cells of a filament form groups: some cells fix carbon, while others take care of the nitrogen. "For a lot of organisms, they either fix carbon during the day and nitrogen at night, or if they're a long chain organism, they'll have certain cells that are partitioned off," Finzi-Hart explained. The time-space debate is tricky in the case of Trichodesmium, however. "With Tricho, it looks like one individual cell can do both."

Finzi-Hart and colleagues turned to NanoSIMS, a specialized imaging technology used mainly in the planetary and medical sciences. "The idea behind this was how we can get inside a cell and see if and how a cell is fixing carbon and nitrogen at the same time." USC's Doug Capone and Ken Nealson were among the first to adopt the technique in marine microbiology, working closely with Jennifer Pett-Ridge and Peter Weber of the Lawrence Livermore National Laboratory. "You would never get images like this before," Finzi-Hart said. "We've opened a door to the insides of the cells."

NanoSIMS allows scientists to see stable isotopic forms of carbon and nitrogen as they are fixed. These isotopes light up like honing beacons in the images produced by the spectrometer. In this study, Finzi-Hart and colleagues examined the insides of Tricho cells to watch the tagged carbon and nitrogen at different points over a 24-hour period. The resulting pictures showed both where the nitrogen ended up within a cell and also how that distribution, and the quantity, changed over time. In short, the technique offered a new way to look at the debate from both the time and space points of view.

The study found strong evidence in support of the temporal separation thesis. Over the 24-hour period, the images clearly demonstrated that just after carbon fixation peaked, nitrogen fixation picked up. When it came to nitrogen, the images were startling: one could see the fixed tagged nitrogen slowly enter the cell, and after a few hours pool into concentration "hotspots," then see these hotspots subside into a more uniform distribution. (Comparing these images to others suggested that the hotspots were storage organelles called cyanophycin.)

"Basically they were taking the nitrogen, fixing it, storing it, using it, and then starting the whole process again the next day," Finzi-Hart said.

In terms of the spatial segregation theory, things were more complicated. At first, the researchers thought they uncovered clear evidence that there was no division of labor, because carbon and nitrogen tags were uniformly present across a broad sample of cells. In other words, there was no cluster of cells that fixed only nitrogen and other clusters that fixed only carbon, as was predicted by the spatial segregation theory.

However, previous studies have shown that after fixing nitrogen, Tricho cells are able to redistribute the nitrogen in as little as 90 seconds. Since the earliest NanoSIMS images were taken 15 minutes after introducing the isotopes, it is possible that there was spatial segregation that just didn't get captured. "Because they're able to redistribute their product so quickly, we can't say definitively that there's no spatial segregation," Finzi-Hart said.

Still, the evidence is pointing towards Tricho having the ability to fix both carbon and nitrogen without divvying up the work. "If there is a division of labor, it's transient," she said.

Brain building: Study Shows Brain Growth Tied to Cell Division in Mouse Embryos
How your brain grows might come down to how your cells divide

In the April 6 issue of the Journal of Cell Biology (JCB), Lake and Sokol report that mouse protein Vangl2 controls the asymmetrical cell division and developmental fate of progenitor neurons.

Vangl2 (aka Strabismus in flies) is a component of the PCP (planar cell polarity) pathway that is active in a variety of tissues and organisms. Mice that lack Vangl2 have a number of neurological defects including incomplete neural tube closure and reduced brain size.

Sokol and Lake wondered how Vangl2 might influence brain development. In the cerebral cortex, neurons are born from a pool of progenitor cells, and the time of their birth determines their fate. The research duo found that Vangl2-lacking mouse embryos had large numbers of early-born neurons and few remaining progenitor cells. This hinted that Vangl2-lacking neurons were differentiating prematurely—a suspicion confirmed in vitro.

The progenitor pool is maintained by asymmetrical division—one daughter cell becomes a neuron, the other self-renews. This fate asymmetry is thought to depend on the orientation of cell division, and the authors observed an increase in the number of symmetrically dividing progenitors in the brains of Vangl2-lacking mouse embryos. Also, Vangl2-lacking cells in culture showed symmetrical distribution of a spindle-orienting factor that in normal cells distributes asymmetrically.

Such similarities between Vangl2-lacking cells in vitro and in vivo will facilitate ongoing studies of the PCP pathway in neurogenesis.

Gene Linked to Lupus Might Explain Gender Difference in Disease Risk
In an international human genetic study, researchers at UT Southwestern Medical Center have identified a gene linked to the autoimmune disease lupus, and its location on the X chromosome might help explain why females are 10 times more susceptible to the disease than males

Identifying this gene, IRAK1, as a disease gene may also have therapeutic implications, said Dr. Chandra Mohan, professor of internal medicine and senior author of the study. “Our work also shows that blocking IRAK1 action shuts down lupus in an animal model. Though many genes may be involved in lupus, we only have very limited information on them,” he said.

The study appears online this week in the Proceedings of the National Academy of Sciences.

Locating IRAK1 on the X chromosome also represents a breakthrough in explaining why lupus seems to be sex-linked, Dr. Mohan said. For decades, researchers have focused on hormonal differences between males and females as a cause of the gender difference, he pointed out.

“This first demonstration of an X chromosome gene as a disease susceptibility factor in human lupus raises the possibility that the gender difference in rates may in part be attributed to sex chromosome genes,” Dr. Mohan said.

Systemic lupus erythematosus, or lupus for short, causes a wide range of symptoms such as rashes, fever or fatigue that make it difficult to diagnose.

The multicenter study involved 759 people who developed lupus as children, 5,337 patients who developed it as adults, and 5,317 healthy controls. Each group comprised four ethnicities: European-Americans, African-Americans, Asian-Americans and Hispanic-Americans.

In previous genetic studies, the researchers had found an association but not a definite link between lupus and IRAK1.

For the current study, the researchers studied five variations of the IRAK1 gene in the subjects, and found that three of the five variants were common in people with either childhood-onset or adult-onset lupus.

To further test the link, the researchers then took mice of a strain that normally is prone to developing lupus and engineered them to lack the IRAK1 gene. In the absence of IRAK1, the animals lacked symptoms associated with lupus, including kidney malfunction, production of autoimmune antibodies and activation of white blood cells.

“The extensive involvement of IRAK1 in the regulation of the immune response renders its association with lupus a prime candidate for careful genetic and functional analysis,” Dr. Mohan said.

Future research will investigate the role that X-linked genes, versus hormonal differences, play in the gender susceptibility rates of lupus.

The study was funded by the National Institutes of Health, the Alliance for Lupus Research and the Republic of Korea Ministry for Health.

New Radiation-free Targeted Therapy Detects and Eliminates Breast-Cancer Tumors in Mice
Caltech researcher helps develop technique that homes in on aggressive, difficult-to-treat HER2+ breast cancer cells

Combining a compound known as a gallium corrole with a protein carrier results in a targeted cancer therapy that is able to detect and eliminate tumors in mice with seemingly fewer side effects than other breast-cancer treatments, says a team of researchers from the California Institute of Technology (Caltech), the Israel Institute of Technology (Technion) and the Cedars-Sinai Medical Center.

A paper describing their work is highlighted in this week's issue of the online edition of the Proceedings of the National Academy of Sciences (PNAS).

Corroles have very similar structures to the porphyrin molecules used in a well-studied cancer treatment known as photodynamic therapy, or PDT, in which porphyrin compounds injected into the body are exposed to specific wavelengths of laser light. The light prompts the porphyrins to produce active, tumor-killing oxygen radicals.

The difference between porphyrins and corroles, says Harry Gray, Caltech's Arnold O. Beckman Professor of Chemistry and founding director of the Beckman Institute, is that some corroles don't require a laser boost to turn lethal. "The striking thing about gallium corroles is that they apparently kill cancer cells in the dark," says Gray. "We don't yet know exactly how this works, but what we've seen so far tells us that it does work."

He notes that "ongoing work in our laboratories focuses on testing our leading hypotheses for elucidating the mechanism of action."

In the experiments described in the PNAS paper, the team paired a gallium corrole with a carrier protein, then aimed it at cells that carry the human epidermal growth factor receptor 2 (HER2). The presence of a HER2 receptor is the hallmark of about 25 percent of breast cancers, and marks those tumors as particularly aggressive and difficult to treat.

In trials in mice, the targeted corrole was able to shrink tumors at doses five times lower than that of the standard chemotherapeutic agent for HER2-positive tumors, a drug called doxorubicin. In contrast with doxorubicin, the corrole was injected into the bloodstream, rather than directly into the tumor.

"We looked at three groups of mice with human tumors," explains paper coauthor Lali Medina-Kauwe, an assistant professor of medicine at the David Geffen School of Medicine at UCLA, and a faculty research scientist in the Department of Biomedical Science at Cedars-Sinai Medical Center in Los Angeles. "In one, we introduced just the protein carrier, without the corrole; tumor growth in those mice did not change. In other mice, we gave the corrole without the carrier protein; this led to some tumor suppression. But it was the last group, the ones that got the corrole with the carrier protein, that experienced the most therapeutic effect."

"The fact that we can target this compound means we can give it at very low concentrations," adds coauthor Daniel Farkas, director of Cedar-Sinai's Minimally Invasive Surgical Technologies Institute. "Using lower concentrations means less toxicity. Doxorubicin tends to have significant heart toxicity; this therapy seems likely to be much less damaging to the heart."

In addition, adds Medina-Kauwe, targeted compounds can seek out tumors wherever they may be. "One of the beauties of targeting," she says, " is that we can go after metastatic tumors that are too small to be seen."

These targeted gallium corroles are not only effective, they're also easy to study, notes Zeev Gross, the Reba May & Robert D. Blum Academic Chair at Technion, the Israeli Institute of Technology, in Haifa, and another of the paper's coauthors. "In most cases, if people want to get a closer look at a drug in vivo, they have to attach a fluorescent probe to it--and that turns it into a different molecule. But in our case, the active molecule we're tracking does the fluorescing. We get to track the original, unmodified molecule and are hence able to follow its distribution among different organs in live animals."

The difficulty in getting to this point, notes Gray, is that corroles have been challenging to synthesize. "Then Zeev came up with a powerful synthetic method to make corroles," he says. "We went from being able to make a couple of milligrams in two years to being able to make two grams in two days. It really puts corroles on the map."

Gray and Gross further add, "It is truly fulfilling to see how the close collaboration between our research groups at Caltech and the Technion, which started 10 years ago with a focus on developing the fundamental science of corroles, led to pharmaceutical utility when we joined forces with Medina-Kauwe and Farkas, who are experts in cellular biology and biomedical imaging technologies."

The work described in the paper, "Tumor detection and elimination by a targeted gallium corrole," was supported by grants from the National Science Foundation, the National Institutes of Health, the U.S. Department of Defense, Susan G. Komen for the Cure, the Donna and Jesse Garber Award, the Gurwin Foundation, the United States-Israel Binational Science Foundation, and by the U.S. Navy Bureau of Medicine and Surgery.

Colon Cancer and The Microbes in Your Gut
A typical Western diet, rich in meat and fats and low in complex carbohydrates, is a recipe for colon cancer, Professor Stephen O'Keefe from the University of Pittsburgh, USA, told the Society for General Microbiology meeting at Harrogate today (Tuesday 31 March)

He described an expanding body of evidence to show that the composition of the diet directly influences the diversity of the microbes in the gut, providing the link between diet, colonic disease and colon cancer.

People eating a healthy diet containing high levels of complex carbohydrate had significant populations of micro-organisms in their gut called Firmicutes. These bacteria use the undigested residues of starch and proteins in the colon to manufacture short-chain fatty acids and vitamins such as folate and biotin that maintain colonic health. One of these fatty acids, butyrate, not only provides most of the energy to maintain a healthy gut wall, but it also regulates cell growth and differentiation. Both experimental and human studies support its role in reducing colon cancer risk.

However, gut microbes may also make toxic products from food residues. Diets high in meat will produce sulphur - this decreases the activity of 'good' bacteria that use methane and increases the production of hydrogen sulphide and other possible carcinogens by sulphur-reducing bacteria.

"Colon cancer is the second leading cause of cancer-related deaths in adults in Westernized communities." said Professor O'Keefe, "Our results suggest that a diet that maintains the health of the colon wall is also one that maintains general body health and reduces heart disease."

"A diet rich in fibre and resistant starch encourages the growth of good bacteria and increases production of short chain fatty acids which lessen the risk of cancer, while a high meat and fat diet reduces the numbers of these good bacteria." Professor O'Keefe went on. "Our investigations to date have focused on a small number of bacterial species and have therefore revealed but the tip of the iceberg, our colons harbour over 800 bacterial species and 7,000 different strains. The characterization of their properties and metabolism can be expected to provide the key to colonic health and disease."



MONDAY March 30, 2009---------------------------News Archive

A Splice of Life
In a new study this week in Nature, researchers at Brandeis University and the MRC Laboratory of Molecular Biology (Cambridge, U.K.) for the first time shed light on a crucial step in the complex process by which human genetic information is transmitted to action in the human cell and frequently at which point genetic disease develops in humans

The scientists report that they were able to crystallize a very large complex of a macromolecular "machine" in the human cell and determine its structure or what it actually looks like, thereby zeroing in on the process of genetic encoding. Importantly, 15 to 20 percent of all human genetic disorders, including muscular dystrophy, are caused by defects in this genetic encoding process known as RNA splicing.

Using x-ray crystallography, the scientists for the first time were able to create a three-dimensional structure of an integral complex of the human spliceosome, which consists of specialized RNA and protein subunits. The spliceosome's job is to modify the message relayed from our genetic material—DNA—by clipping, or splicing, genetic bits in such a manner that they are acceptable for translation into protein. Importantly, the spliceosome also rearranges the genetic bits of the message in such a way that it can generate multiple and varied proteins which can and do have dramatic effects on human development, said lead author and Brandeis biochemist Daniel Pomeranz Krummel.

"The process of RNA splicing is vital to human cell development and survival," said Pomeranz Krummel. "In this process, the regions of our DNA encoding for protein are removed from non-encoding regions and brought together—quite often in alternative arrangements. Defects in this process can have disasterous repercussions in the form of genetic disorders," said Pomeranz Krummel, adding that neuronal development can be particularly affected when things go awry. Indeed, defects in this process have recently been implicated in various human neurological disorders, including epilepsy.

Specifically, this macromolecular machine clips, or splices, gene sequences transcribed as part of a precursor to the mRNA, removing them before the final mRNA product is translated into protein. The spliceosome must clip these sequences, known as introns, at the right place in the precursor mRNA.

"In human cells one gene can be made into a variety of proteins, so if the process just goes slightly wrong, the genetic alteration can lead to incredible disaster; yet on the other hand, this incredible complexity has led to our amazing evolutionary progress," said Pomeranz Krummel. "The human genome is not terribly different from the earthworm's with regards to its size, but the process of RNA splicing that occurs in our cells is different. The fundamental difference between us and the earthworm is that our cells have evolved to utilize this process of RNA splicing to generate a whole other dimension to the transmission of genetic information."

Pomeranz Krummel's lab will next focus on understanding how this complex interacts with other macromolecular machines in the human cell. The study was funded by the Medical Research Council (U.K.) and the Human Frontier Science Program.

Skin Cancer Study Uncovers New Tumor Suppressor Gene
National Institutes of Health (NIH) researchers have identified a gene that suppresses tumor growth in melanoma, the deadliest form of skin cancer

The finding is reported today in the journal Nature Genetics as part of a systematic genetic analysis of a group of enzymes implicated in skin cancer and many other types of cancer.

The NIH analysis found that one-quarter of human melanoma tumors had changes, or mutations, in genes that code for matrix metalloproteinase (MMP) enzymes. The findings lay the foundation for more individualized cancer treatment strategies where MMP and other key enzymes play a functional role in tumor growth and spread of the disease.

Tumor suppressor genes encode proteins that normally serve as a brake on cell growth. When such genes are mutated, the brake may be lifted, resulting in the runaway cell growth known as cancer. In contrast, oncogenes are genes that encode proteins involved in normal cell growth. When such genes are mutated, they also may cause cancer, but they do so by activating growth-promoting signals. Cancer therapies that target oncogenes usually seek to block or reduce their action, while those aimed at tumor suppressor genes seek to restore or increase their action.

The new study may help to explain the disappointing performance of drugs designed to treat cancer by blocking MMP enzymes. Because members of the MMP gene family were thought to be oncogenes and many tumors express high levels of MMP enzymes, researchers have spent decades pursuing MMPs as promising targets for cancer therapies. However, when MMP inhibitors were tested in people with a wide range of cancers, the drugs failed to slow — and in some cases even sped up — tumor growth.

Now, it turns out that one of the most often mutated MMP genes in melanoma is not an oncogene at all. In its study, the team led by researchers from the National Human Genome Research Institute (NHGRI) found that MMP-8 actually serves as a tumor suppressor gene in melanoma. Consequently, in the estimated 6 percent of melanoma patients whose tumors harbor a mutated MMP-8 gene or related tumor suppressor(s), it may not be wise to block all MMPs. The study suggests that a better approach may be to look for drugs that restore or increase MMP-8 function or for drugs that block only those MMPs that are truly oncogenes.

"This research is an illustrative proof of concept that shows the value of genomic strategies for understanding cancer and possible therapies," said NHGRI Scientific Director Eric Green, M.D., Ph.D. "It is gratifying to see that genomic technologies are guiding scientific discovery, advancing cancer research, especially melanoma research."

Melanoma is the most serious form of skin cancer. In the United States and many other nations, melanoma is becoming more common every year. A major cause is thought to be overexposure to the sun. The ultraviolet radiation in sunlight can damage DNA and lead to cancer-causing genetic changes within skin cells.

MMP enzymes help the body to break down and recycle proteins, playing a crucial role in the process of remodeling skin after sunburns, cuts or other injuries. The MMP gene family has been associated with tumor growth in a variety of cancers, including breast, colon and melanoma.

To explore the role of MMP genes in melanoma, the NHGRI researchers studied a bank of tumor and blood samples collected from 79 patients with aggressive melanoma by collaborator Steven Rosenberg, M.D., Ph.D., chief of surgery at the National Cancer Institute (NCI). Specifically, they compared the sequence of MMP genes in tumors and normal DNA from the same patients, looking for mutations in all 23 members of the MMP gene family.

The researchers identified 28 different mutations in eight MMP genes in the melanoma tumors studied. These mutations were found to be distributed in different frequencies and patterns among the tumor samples. Nearly one-quarter of the tumors analyzed had at least one MMP gene mutation. Some mutations were found in as few as 3 percent of tumors, while more than 6 percent of tumors had mutations in MMP-8 and more than 7 percent had mutations in MMP-27, which codes for an enzyme very similar to MMP-8.

"We often talk about cancer as though it is one disease, and cancers do have many common denominators. But when we look at the DNA level, we see that different cancers have different genetic profiles, and so do different patients who have the same cancer," said the study's senior author, Yardena Samuels, Ph.D., an investigator in the Cancer Genetics Branch of the NHGRI's Division of Intramural Research.

Dr. Samuels and her collaborators followed up their DNA sequencing work with cell and animal studies to see whether MMP-8 mutations affect enzyme function. Strikingly, the researchers showed that five of the mutations reduced activity of the MMP-8 enzyme. The researchers next studied whether MMP-8 mutations promote activities related to cancer. Indeed, cells with MMP-8 mutations showed increased ability to multiply outside the constraints of normal cells, a hallmark of cancer development known as anchorage-independent growth. Likewise, cells with MMP-8 mutations had a greater ability to migrate — a key aspect of cancer metastasis — than normal cells.

The researchers found that mice injected with cells expressing normal MMP-8 did not develop skin ulcers, which are one of the most important measures of cancer aggression in melanoma. In contrast, mice injected with cells expressing mutated MMP-8 went on to develop ulcerations and metastases in their lungs.

In addition to Dr. Samuels's and Dr. Rosenberg's laboratories, the NIH team included researchers from the National Institute on Aging and the National Institute of Dental and Craniofacial Research, who helped with the mouse studies; and NHGRI's Genome Technology Branch, Bioinformatics and Scientific Programming Core, and Office of Laboratory Animal Medicine. Other collaborators included researchers from the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins in Baltimore, and the University of South Carolina School of Medicine in Columbia.

For a high resolution micrograph of metastatic melanoma, go to www.genome.gov/pressDisplay.cfm?photoID=20152 and www.genome.gov/pressDisplay.cfm?photoID=20153.

NHGRI is one of the 27 institutes and centers at the NIH, an agency of the Department of Health and Human Services. The NHGRI Division of Intramural Research develops and implements technology to understand, diagnose and treat genomic and genetic diseases. Additional information about NHGRI can be found at its Web site, www.genome.gov.

The National Institutes of Health — "The Nation's Medical Research Agency" — includes 27 institutes and centers, and is a component of the U.S. Department of Health and Human Services. It is the primary federal agency for conducting and supporting basic, clinical and translational medical research, and it investigates the causes, treatments and cures for both common and rare diseases. For more, visit www.nih.gov.

Fireflies and Jellyfish Illuminate Quest for Cause of Infertility
Genes taken from fireflies and jellyfish are literally shedding light on possible causes of infertility and autoimmune diseases in humans

Genes taken from fireflies and jellyfish are literally shedding light on possible causes of infertility and autoimmune diseases in humans.

Scientists are using the luminescent and florescent genes to illuminate cells that produce a hormone linked to conditions, which include rheumatoid arthritis and lupus.

The technique will help scientists track the production of the hormone prolactin, which is crucial in ensuring supplies of breast milk in nursing mothers but can be overproduced by some pituitary tumours, causing infertility.

Prolactin has been linked to more than 300 biological functions. It is believed to play a role in autoimmune diseases, such as lupus and rheumatoid arthritis, as well as in the inflammation of cells and tissues.

Scientists from the Universities of Edinburgh, Manchester and Liverpool harnessed firefly and jellyfish genes, which enable these creatures to emit light, and used them to create a chemical reaction to light up cells expressing prolactin in rats.

The technique means that scientists can identify when and where prolactin is expressed to look at how the hormone works in real time.

Sabrina Semprini, whose study is published in the journal Molecular Endocrinology, said: "The lighting up of cells expressing this hormone will help us to understand its role within the body and could help research looking for treatments for conditions in which prolactin is involved."

The research, funded by the Wellcome Trust, identified cells producing prolactin throughout the body. This included the pituitary gland, the thymus – an organ in the chest which helps protect against autoimmunity – the spleen and inflammatory cells in the abdominal cavity.

Scientists Excise Vector, Exotic Genes from Induced Stem Cells
Two University of Rhode Island scientists have revealed how a cancer causing protein is regulated by reactive oxygen species (ROS) - a type of stress signal

A team of scientists from the Morgridge Institute for Research at the University of Wisconsin-Madison reports that it has created induced human pluripotent stem (iPS) cells completely free of viral vectors and exotic genes.

By reprogramming skin cells to an embryonic state using a plasmid rather than a virus to ferry reprogramming genes into adult cells, the Wisconsin group's work removes a key safety concern about the potential use of iPS cells in therapeutic settings.

The new method, which is reported in March 26th issue online of the journal Science, also removes the exotic reprogramming genes from the iPS equation, as the plasmid and the genes it carries do not integrate into an induced cell's genome and can be screened out of subsequent generations of cells. Thus, cells made using the new method are completely free of any genetic artifacts that could compromise therapeutic safety or skew research results, according to the Science report.

The new work was conducted in the laboratory of James Thomson, the UW-Madison scientist who was the first to successfully culture human embryonic stem cells in 1998 and, in 2007, co-discovered a way to make human-induced pluripotent stem cells. Thomson, a professor in the UW-Madison School of Medicine and Public Health, is also the director of regenerative biology for the Morgridge Institute for Research, the private, nonprofit side of the new Wisconsin Institutes for Discovery at UW-Madison.

"We believe this is the first time human-induced pluripotent stem cells have been created that are completely free of vector and transgene sequences," says Thomson.

The new study was led by geneticist Junying Yu, the Wisconsin researcher who, with Thomson, co-discovered a method for reprogramming adult skin cells to behave like embryonic stem cells, the master cells that arise at the earliest stages of development and that ultimately develop into all 220 cell types in the human body.

While the methods first devised for reprogramming adult cells yielded embryonic-like cells, the process resulted in the permanent integration of both viral genes and the genes used for reprogramming into the genomes of the newly induced cells. Such genetic baggage posed safety concerns for potential therapies like cell transplants, and confounded work in the lab, as the introduced genes sometimes spurred mutations that interfered with the normal function of induced cells.

The new work was accomplished using a plasmid, a circle of DNA, and cells from the foreskins of newborns. "The plasmids carry all the needed transgenes, but don't integrate into the host DNA, they just float around as episomes" in the cell, Thomson says.

The plasmids replicate, but they do so somewhat inefficiently, Thomson explains, so that after they perform the job of reprogramming, they can subsequently be weeded out, leaving the induced cells free of any exotic genetic material. "Once the transgenes have done their job and are no longer needed, one can merely recover induced pluripotent stem cells that have lost their episomes."

The resulting cells, says Thomson, are remarkably similar to embryonic stem cells and show the same capacity to proliferate indefinitely in culture and diversify into all the cell types of the human body.

"The recent discovery that adult cells could be reprogrammed to iPS cells that resemble embryonic stem cells opened up tremendous potential for regenerative medicine," says Marion Zatz of the National Institute of Health's National Institute of General Medical Sciences, which partially funded the new work. "However, the early methods posed significant risks in using iPS cells in a clinical setting. This latest discovery by Thomson's group of a new method for generating iPS cells without inserting viral vectors into the cells' genetic material is a major advance toward safely reprogramming cells for clinical use."

Thomson notes that researchers have developed other promising approaches using mouse cells, and previously had removed most of the vector and exogenous gene sequences from human-induced pluripotent stem cells. However, those efforts did not succeed in removing all of the genetic artifacts of reprogramming, which could still result in mutations in induced cells.

"Given the rapid pace of the field, it won't be surprising if there are several alternative methods for producing vector and transgene free cells very soon," says Thomson. "However, it will be essential to determine which of these methods most consistently produces induced pluripotent stem cells with the fewest genetic abnormalities. Any problems would impact research, drug development and possible transplantation therapies."

The new viral vector-free iPS cells will be available to researchers almost immediately through the International Stem Cell Bank at the WiCell Research Institute, which also carries the four original iPS cell lines developed by the Thomson lab in 2007.

Targeting Oxidized Cysteine Through Diet Could Reduce Inflammation and Lower Disease Risk
A team of scientists at Emory University School of Medicine has identified a direct link between oxidative stress and inflammatory signals in the blood

The finding could lead to improved strategies for preventing several diseases by including antioxidants in the diet and for reducing the impact of inflammation in critically ill patients by adding cysteine to intravenous or tube feeding.

Many normal metabolic functions produce reactive forms of oxygen that can damage cells. Oxidative stress, a disruption of the body's ability to control reactive forms of oxygen, has been connected with heart disease, diabetes and several neurodegenerative diseases.

However, scientists are still learning about the best ways to measure and reduce oxidative stress, says Dean P. Jones, PhD, professor of medicine and director of the Clinical Biomarkers Laboratory at Emory University School of Medicine. For example, large-scale clinical trials have shown little benefit in supplementing the diet with antioxidants such as vitamins C and E.

Jones and his colleagues, including Thomas R. Ziegler, MD of the Emory Department of Medicine, have been concentrating on a measure of oxidative stress in the blood: cysteine, an amino acid found in most proteins in the body. Cysteine can exist in two forms: oxidized and reduced. The higher the level of oxidative stress outside the cell, the more oxidized cysteine there is. Other indicators such as glutathione are more important inside cells.

Several studies have shown that levels of oxidized cysteine in the blood tend to rise as people age. Smoking and alcohol consumption are also linked with higher levels of oxidized cysteine. In addition, Jones and Ziegler have found that critical illness and malnutrition are associated with oxidative stress and oxidized cysteine in the blood.

Working with Jones, graduate student Smita Iyer and immunologist Mauricio Rojas, MD, found that a high level of oxidized cysteine drives white blood cells to send out inflammatory messages in the form of the protein IL-1 beta.

The researchers used a mouse model of sepsis to test the effects of dietary cysteine on reducing inflammation. They treated the mice with LPS, which mimics the inflammatory effect of bacteria on the human immune system and causes an increase in the level of IL-1 beta. When they supplemented the diet of the mice with cysteine, however, IL-1 beta levels dropped, thus blunting the impact of a sepsis-like inflammation.

In a subsequent study of healthy, but overweight adult volunteers with an average age of 62, IL-1 beta levels also rose and fell in association with the amount of dietary cysteine.

"Our research shows a direct mechanistic link between the oxidative stress biomarker (cysteine redox potential) and pro-inflammatory cytokines, which have been linked to multiple age-related and chronic diseases," says Jones. "Our group and others have already established that cysteine redox potential is oxidized with aging and with a number of health risk factors. This suggests that one could target cysteine redox potential as a means to decrease chronic proinflammatory signaling as an intervention for age-related diseases and for the acute inflammation of sepsis or lung injury."

The researchers plan to continue studying the relationship between cysteine and markers of inflammation in different age groups, in overweight and normal weight individuals and in critically ill patients requiring intravenous feeding.

The research was funded by the National Institutes of Health.

Reference: "Cysteine Redox Potential Determines Pro-Inflammatory IL-1b Levels." PLoS One, published online March 27, 2009.

Difference in Fat Storage May Explain Lower Rate of Liver Disease in African-Americans
Where different ethnic groups store fat in their bodies may account for differences in the likelihood they’ll develop insulin resistance and non-alcoholic fatty liver disease, researchers at UT Southwestern Medical Center have found

According to research reported in the online edition and the March issue of Hepatology, African-Americans with insulin resistance might harbor factors that protect them from this form of metabolic liver disease.

Despite similarly high rates of associated risk factors such as insulin resistance, obesity and diabetes among African-Americans and Hispanics, African-Americans are less likely than Hispanics to develop non-alcoholic fatty liver disease, or NAFLD. The disease is characterized by high levels of triglycerides in the liver and affects as many as one-third of American adults.

“If we can identify the factors that protect African-Americans from this liver disease, we may be able to extrapolate those to other populations and perhaps develop targeted therapies to help populations prone to NAFLD,” said Dr. Jeffrey Browning, assistant professor of internal medicine in the UT Southwestern Advanced Imaging Research Center and the study’s senior author.

Previous research has shown that when African-Americans do develop NAFLD, they’re less likely to reach the later stages of liver disease. Prior work by Dr. Browning and other UT Southwestern scientists has revealed that NAFLD is more prevalent among Hispanics than African-Americans or Caucasians. For the current study, Dr. Browning and his colleagues analyzed data gathered in the multi-ethnic, population-based Dallas Heart Study. Starting in the year 2000, more than 2,100 participants provided blood samples and underwent multiple body scans with magnetic resonance imaging and computed tomography to examine the liver, heart and other organs. Body composition, including fat distribution, also was scrutinized.

The study found that African-Americans and Hispanics both have obesity rates of about 48 percent among their respective populations, as well as diabetes rates of about 21 percent. Only 23 percent of African-Americans, however, have NAFLD, compared with 45 percent of Hispanics. Similarly, African-Americans are less likely to have high levels of triglycerides and abdominal fat – both characteristics of insulin resistance – when compared with Hispanics or Caucasians, even though overall rates of insulin resistance among all groups are the same, researchers found. “This presents something of a paradox,” Dr. Browning said. The explanation might lie in where different ethnic groups typically store fat.

Obese Hispanics tend to deposit fat in the liver and visceral adipose tissue — the area around the belly. Obese African-Americans deposit fat predominantly in subcutaneous adipose tissues — the area around the hips and thighs, Dr. Browning said. “This may be protective,” Dr. Browning said. “In animal studies, if subcutaneous fat is increased as opposed to visceral fat, you can actually reverse fatty liver disease.” Scientists aren’t sure why the location of fat storage matters. “This seems to argue that there is a fundamental difference in the lipid metabolism between African-Americans and Hispanics or Caucasians, and this difference is maintained even when insulin resistance is present,” Dr. Browning said.

Differences in liver-fat content in Caucasians seem to be based on gender. Caucasian males are at the highest risk for NAFLD, on par with the risk faced by Hispanics in general. Caucasian females are on par with the African-American population, at about 23 percent. Caucasian females, like African-Americans, might benefit from the greater predilection to store fat in lower extremities.

“Research studies traditionally have been based on examining Caucasian males, but this information suggests that there are sometimes ethnic and gender differences that need to be studied individually to determine if there are important clues we’re missing because we’re lumping everybody together,” Dr. Browning said. Researchers next will study how differences in metabolism affect fatty liver disease.

Other researchers from UT Southwestern involved in the study were lead author Dr. Richard Guerrero, a postdoctoral trainee clinician in internal medicine; Dr. Gloria Vega, professor of clinical nutrition; and Dr. Scott M. Grundy, director of the Center for Human Nutrition.

The study was funded by the Donald W. Reynolds Foundation and the National Institutes of Health.
Visit http://www.utsouthwestern.org/digestive to learn more about UT Southwestern’s clinical services in digestive disorders, including liver disease.

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