Gene Exchange Common Among Sex-manipulating Bacteria

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ScienceDaily (Mar. 26, 2009) — Certain bacteria have learned to manipulate the proportion of females and males in insect populations. Now Uppsala University researchers have mapped the entire genome of a bacterium that infects a close relative of the fruit fly.

The findings, published in PNAS, reveal extremely high frequencies of gene exchange within this group of bacteria. In the future it is hoped that it will be possible to use sex-manipulating bacteria as environmentally friendly pesticides against harmful insects.
Bacteria belonging to the Wolbachia group are adapted to invertebrate animals such as insects, spiders, scorpions, and worms. These bacteria spread via the female's eggs from one generation to the next and manipulate the sex quotas among the infected animals so that more females are produced in the population. Mechanically speaking, the bacteria convert genetic males into females or kill male embryos that are then eaten by their sisters or make females lay unfertilized eggs that all become females. However, what happens most commonly is that the males cannot reproduce with non-infected females. This gives the infected females a great advantage, and the infection spreads rapidly among the population.
The studies of the whole genome have shown that these bacteria carry genes that are common among higher organisms, but rare among other bacteria. The scientists believe that the bacteria have stolen these genes from the genome in the host cell and that they now use them to manipulate the sex quotas among the insects.
"With the help of viruses, these bacteria exchange genes with each other, which leads to a rapid dissemination of genes that are thought to be important for sex manipulation," says Lisa Klasson, one of the researchers behind the study.
The researchers have shown that the genomes of these bacteria are evolutionary mosaics, with DNA pieces from many closely related bacteria. The effect is that each gene has its own evolutionary history and that the potential for variation is infinite.
"It's fascinating that bacteria, with only 1,000 genes, can control complicated developmental processes and behaviors in insects," says Siv Andersson.
By mapping how the genes in these bacteria change over time and figuring out the mechanisms behind sex manipulation, scientists will be able to lay a foundation for finding new pesticides for insects, based on nature's own principles.
Journal reference:
Klasson et al. The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans. Proceedings of the National Academy of Sciences, 2009; DOI: 10.1073/pnas.0810753106
Adapted from materials provided by Uppsala University.

When Intestinal Bacteria Go Surfing: Molecular Signal Pathway In Diarrhea Illnesses Identified

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ScienceDaily (Mar. 26, 2009) — The bacterium Escherichia coli is part of the healthy human intestinal flora. However, E. coli also has pathogenic relatives that trigger diarrhea illnesses: enterohemorrhagic E.coli bacteria. During the course of an infection they infest the intestinal mucosa, causing injury in the process, in contrast to benign bacteria.

The EHECs adhere to the surface of the mucosal cells and alter them internally: a part of the cellular supportive skeleton - the actin skeleton - is rearranged in such a manner that the cell surface beneath the bacteria forms plinth-like growths, so-called pedestals. The bacteria are securely anchored to this pedestal; the pedestals, in contrast, are mobile. This enables the bacteria, seated upon them, to surf over the cell surface and reproduce upon it, without being flushed from the intestine. But how do the bacteria bring the host cells to convert the actin skeleton? Researchers at the Helmholtz Centre for Infection Research (HZI) have now identified the signal pathway that leads to the formation of this pedestal.
"Prerequisite for this signal pathway is a special secretion system - a sort of molecular syringe, through which the bacteria insert entire proteins in the host cell," explains Theresia Stradal, head of the Signal Transduction and Motility research group at HZI. Two factors, Tir and EspFU, are brought into the host cell from the bacterium for pedestal formation. Following this, the host cell presents Tir on its surface; the bacterium recognises "its" molecule Tir and adheres to the host cell. EspFU then triggers the signal for local actin conversion.
"It has been unclear thus far how the two bacterial effectors Tir and EspFU enter into contact with one another in the host cell," says Theresia Stradal. Her research group has now found the missing link: "The molecule comes from the host cell, is called IRSp53 and gathers on the cell surface, directly beneath the bacteria sitting on it," explains cell biologist Markus Ladwein, who is also involved in the project. IRSp53, then, establishes the connection between Tir and EspFU. It ensures that actin conversion is concentrated locally. Together with the biochemist Dr. Stefanie Weiß, a former post-graduate student with the research group, Markus Ladwein also provided the counter evidence: "Cells in which IRSp53 is lacking are no longer able to form pedestals for the bacteria."
The signal pathway clarified by the Braunschweig researchers – published in the journal Cell Host & Microbe – is a good example of how pathogenic bacteria develop progressively with their host. With the aid of bacterial factors, they therefore manage to simulate signals and set in motion complex processes in the host, which they then abuse for their own purposes.
Journal reference:
Stefanie M. Weiss, Markus Ladwein, Dorothea Schmidt, Julia Ehinger, Silvia Lommel, Kai Städing, Ulrike Beutling, Andrea Disanza, Ronald Frank, Lothar Jänsch, Giorgio Scita, Florian Gunzer, Klemens Rottner, and Theresia E.B. Stradal. IRSp53 Links the Enterohemorrhagic E. coli Effectors Tir and EspFU for Actin Pedestal Formation. Cell Host & Microbe, 2009; 5 (3): 244-58 DOI: 10.1016/j.chom.2009.02.003
Adapted from materials provided by Helmholtz Association of German Research Centres.

Fast Magnetic Fix For Sepsis? Micromagnetic-microfluidic Device Could Quickly Pull Pathogens From The Bloodstream

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ScienceDaily (Mar. 26, 2009) — Sepsis, an infection of the blood, can quickly overwhelm the body's defenses and is responsible for more than 200,000 deaths per year in the U.S. alone. Premature newborns and people with weakened immune systems are especially vulnerable.

Since most existing treatments are ineffective, researchers in the Vascular Biology Program at Children's Hospital Boston have come up with a first line of defense--using magnetism to quickly pull pathogens out of the blood.
Their blood-cleansing device, developed by Chong Wing Yung, PhD, a researcher in the laboratory of Don Ingber, MD, PhD, is described in the journal Lab on a Chip. (The article is scheduled for formal online publication on April 13.)
The system they envision will work like this: The patient's blood is drawn, and tiny magnetic beads, pre-coated with antibodies against specific pathogens (such as the fungus Candida albicans) are added. The blood is then run through a microfluidic system in which two liquid flow streams run side by side without mixing -- one containing blood, the other a saline-based collection fluid. The beads bind to the pathogens, and a magnet then pulls them (along with the pathogens) into the collection fluid, which is ultimately discarded, while the cleansed blood in reintroduced into the patient.
Tested with contaminated human blood, a device with four parallel collection modules achieved over 80 percent clearance of fungi in a single pass, at a flow rate and separation efficiency that would be viable for clinical applications. Yung and Ingber estimate that a scaled-up system with hundreds of channels could cleanse the blood of an infant within several hours.
"This blood-cleansing microdevice offers a potentially new weapon to fight pathogens in septic infants and adults, that works simply by removing the source of the infection and thereby enhancing the patient's response to existing antibiotics," says Ingber.
Yung, Ingber and physicians Mark Puder, MD, PhD, and Jay Wilson, MD. from the Department of Surgery at Children's Hospital Boston, with collaborators from Draper Laboratories, recently won a $500,000 grant from the Center for Integration of Medicine and Innovative Technology (CIMIT) to further the work. The next phase will be to test the device in an animal model.
The study was funded by CIMIT, with additional resources from Harvard University's Center for Nanoscale Systems (CNS) and the National Nanotechnology Infrastructure Network (NNIN) initiative.
Journal reference:
Yung Chong Wing Yung, Jason Fiering, Andrew J. Mueller and Donald E. Ingber. Micromagnetic-microfluidic blood cleansing device. Lab on a Chip, 2009; DOI: 10.1039/b816986a
Adapted from materials provided by Children's Hospital Boston.

New Anti-cancer Drug: 200 Times More Active In Killing Tumor Cells

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ScienceDaily (Mar. 26, 2009) — A team of 24 researchers from the U.S., Europe, Taiwan and Japan and led by University of Illinois scientists has engineered a new anti-cancer agent that is about 200 times more active in killing tumor cells than similar drugs used in recent clinical trials.

The study appears this week in the Journal of the American Chemical Society.
The new agent belongs to a class of drugs called bisphosphonates. These compounds were originally developed to treat osteoporosis and other bone diseases, but were recently found to also have potent anti-cancer and immune boosting properties.
Drug developers have tried for years to design drugs to inhibit cell survival pathways in tumor cells, focusing on a protein called Ras since nearly a third of all human cancers involve a mutation in the Ras gene that causes cell signaling to go awry. These efforts have met with limited success.
Bisphosphonates act on other enzymes, called FPPS and GGPPS, which are upstream of Ras in the cell survival pathway. Inhibiting these enzymes appears to be a more effective strategy for killing cancer cells.
When used in combination with hormone therapy in a recent clinical trial, the bisphosphonate drug zoledronate significantly reduced the recurrence of breast cancer in premenopausal women with estrogen-receptor-positive breast cancer. Similar results were reported previously for hormone-refractory prostate cancer.
But zoledronate quickly binds to bone, reducing its efficacy in other tissues.
"We're trying to develop bisphosphonates that will be very active but won't bind to the bone, because if they bind to the bone they're not going to go to breast, lung or other tissues," said University of Illinois chemistry professor Eric Oldfield, who led the new study.
Oldfield's team also wanted to design a compound that would inhibit multiple enzymes in the tumor cell survival pathway, rather than just one, an approach analogous to the use of multi-kinase inhibitors in cancer therapy.
Andrew Wang, of Academia Sinica, Taipei, and Illinois chemist Rong Cao began by producing crystallographic structures of the target enzymes and drug candidates, allowing the researchers to identify those features that would enhance the drugs' ability to bind to the enzymes. Using this and other chemical data, Illinois chemistry department research scientist Yonghui Zhang engineered new bisphosphonate compounds that bound tightly to multiple enzyme targets, but not to bone.
One of the new compounds, called BPH-715, proved to be especially potent in cell culture and effectively inhibited tumor cell growth and invasiveness.
Tadahiko Kubo, of Hiroshima University, then found that BPH-715 also killed tumor cells in mice. And Socrates Papapoulos, of Leiden University, the Netherlands, showed that the compound had a very low chemical affinity for bone.
In humans, compounds like BPH-715 and zoledronate have an added benefit in fighting cancer: They spur the proliferation of immune cells called gamma delta T-cells, which aid in killing tumor cells.
"The new drugs are about 200 times more effective than the drugs used in recent clinical trials at killing tumor cells and in activating gamma delta T-cells to kill tumor cells," Oldfield said. "They also prevent tumor progression in mice much better than do existing bisphosphonate molecules."
Journal reference:
Zhang et al. Lipophilic Bisphosphonates as Dual Farnesyl/Geranylgeranyl Diphosphate Synthase Inhibitors: An X-ray and NMR Investigation. Journal of the American Chemical Society, 2009; 090323145321030 DOI: 10.1021/ja808285e
Adapted from materials provided by University of Illinois at Urbana-Champaign.

New Possibilities For Hydrogen-producing Algae

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ScienceDaily (Mar. 25, 2009) — Photosynthesis produces the food that we eat and the oxygen that we breathe ― could it also help satisfy our future energy needs by producing clean-burning hydrogen? Researchers studying a hydrogen-producing, single-celled green alga, Chlamydomonas reinhardtii, have unmasked a previously unknown fermentation pathway that may open up possibilities for increasing hydrogen production.

C. reinhartii, a common inhabitant of soils, naturally produces small quantities of hydrogen when deprived of oxygen. Like yeast and other microbes, under anaerobic conditions this alga generates its energy from fermentation. During fermentation, hydrogen is released though the action of an enzyme called hydrogenase, powered by electrons generated by either the breakdown of organic compounds or the splitting of water by photosynthesis. Normally, only a small fraction of the electrons go into generating hydrogen. However, a major research goal has been to develop ways to increase this fraction, which would raise the potential yield of hydrogen.
In the new study by Dubini et al., published in the Journal of Biological Chemistry, researchers at the Carnegie Institution's Department of Plant Biology, the National Renewable Energy Laboratory (NREL), and the Colorado School of Mines (CSM), examined metabolic processes in a mutant strain that was unable to assemble an active hydrogenase enzyme.
The researchers, who include Alexandra Dubini (NREL), Florence Mus (Carnegie), Michael Seibert (NREL), Matthew Posewitz (CSM), and Arthur Grossman (Carnegie), expected the cell's metabolism to compensate by increasing metabolite flow along other known fermentation pathways, such as those producing formate and ethanol as end products. Instead, the algae activated a pathway leading to the production of succinate, which was previously not associated with fermentation metabolism in C. reinhardtii. Notably, succinate, a widely used industrial chemical normally synthesized from petroleum, is included in the Department of Energy's list of the top 12 value added chemicals from biomass.
"We actually didn't know that this particular pathway for fermentation metabolism existed in the alga until we generated the mutant," says Carnegie's Arthur Grossman. "This finding suggests that there is significant flexibility in the ways that soil-dwelling green algae can metabolize carbon under anaerobic conditions. By blocking and modifying some of these metabolic pathways, we may be able to augment the donation of electrons to hydrogenase under anaerobic conditions and produce elevated levels of hydrogen."
Grossman points out that it makes evolutionary sense that a soil organism such as Chlamydomonas would have a variety of metabolic pathways at its disposal. Oxygen levels, nutrient availability, and levels of metals and toxins can be extremely variable in soils, over both the short and long term. "In such an environment", Grossman says, "these organisms must evolve flexible metabolic circuits; the variety of conditions to which the organisms are exposed might favor one pathway for energy metabolism over another, which would help the organism compete in the soil environment over evolutionary time."
Grossman led the effort to generate a fully sequenced Chlamydomonas genome, which has allowed researchers to identify key genes encoding proteins involved in both fermentation and hydrogen production. Grossman feels that it is of immediate importance to generate new mutant strains to help us understand how we may be able to alter fermentation metabolism and the production of hydrogen. NREL's Michael Seibert, the project's Principal Investigator, observed that "the overarching goal of the work is to gain a fundamental understanding of the total suite of metabolic processes occurring in Chlamydomonas and how they interact; this discovery effort will lead to the development of novel ways to produce renewable hydrogen and other biofuels, which will benefit all of us".
"These are really exciting times in the field," says Matthew Posewitz. "The tools developed at Carnegie and by other groups in the field are presenting unprecedented opportunities for scientists to make important advances in our understanding of the basic biology of organisms such as Chlamydomonas."
As an energy source to potentially replace fossil fuels, hydrogen would greatly reduce the emission of greenhouse gases. Proponents of algal-based hydrogen production point out that, unlike ethanol produced from crops, it would not compete with food production for agricultural land.
The Project is being supported by the US Department of Energy's GTL Program within the Office of Biological and Environmental Research.
Journal reference:
Alexandra Dubini, Florence Mus, Michael Seibert, Arthur R. Grossman and Matthew C. Posewitz. Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity. Journal of Biological Chemistry, 2008; 284 (11): 7201 DOI: 10.1074/jbc.M803917200
Adapted from materials provided by Carnegie Institution, via EurekAlert!, a service of AAAS.

Evolution Of Fins And Limbs Linked With That Of Gills

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ScienceDaily (Mar. 25, 2009) — The genetic toolkit that animals use to build fins and limbs is the same genetic toolkit that controls the development of part of the gill skeleton in sharks, according to a new study.

The research is published in Proceedings of the National Academy of Sciences on March 23, 2009, by Andrew Gillis and Neil Shubin of the University of Chicago, and Randall Dahn of Mount Desert Island Biological Laboratory.
"In fact, the skeleton of any appendage off the body of an animal is probably patterned by the developmental genetic program that we have traced back to formation of gills in sharks," said Andrew Gillis, lead author of the paper and a graduate student in the Department of Organismal Biology and Anatomy at the University of Chicago. "We have pushed back the evolutionary origin of the developmental genetic program that patterns fins and limbs."
This new finding is consistent with an old theory, often discounted in science textbooks, that fins and (later) limbs evolved from the gills of an extinct vertebrate, Gillis added. "A dearth of fossils prevents us from definitely concluding that fins evolved from gills. Nevertheless, this research shows that the genetic architecture of gills, fins and limbs is the same."
The research builds on the breakthrough discovery of the fossil Tiktaalik, a "fish with legs," by Neil Shubin and his colleagues in 2006. "This is another example of how evolution uses common developmental programs to pattern different anatomical structures," said Shubin, who is the senior author on the PNAS paper and Professor and Associate Dean of Organismal and Evolutionary Biology at the University of Chicago. "In this case, shared developmental mechanisms pattern the skeletons of vertebrate gill arches and paired fins."
The research also showed for the first time that the gill arch skeleton of embryonic skates (a living relative of sharks that has gill rays) responds to treatment with the vitamin A derivative retinoic acid in the same way a limb or fin skeleton does: by making a mirror image duplicate of the structure as the embryo develops. According to the researchers, the genetic circuitry that patterns paired appendages (arms, legs and fins) has a deep evolutionary origin that actually predates the origin of paired appendages themselves.
"These findings suggest that when paired appendages appeared, the mechanism used to pattern the skeleton was co-opted from the gills," Gillis said. "Perhaps we should think of shark gills as another type of vertebrate appendage—one that's patterned in essentially the same way as fins and limbs."
The deep structural, functional, and regulatory similarities between paired appendages and developing gill rays, as well as the antiquity of gills relative to paired appendages, suggest that the signaling network that is induced by retinoic acid had a patterning function in gills before the origin of vertebrate appendages, the research concludes. And this function has been retained in the gill rays of living cartilaginous fishes.
Journal reference:
J. Andrew Gillis, Randall D. Dahna, and Neil H. Shubin. Shared developmental mechanisms pattern the vertebrate gill arch and paired fin skeletons. Proceedings of the National Academy of Sciences, 2009; DOI: 10.1073/pnas.0810959106
Adapted from materials provided by University of Chicago Medical Center, via EurekAlert!, a service of AAAS.

'Master Regulator' Of Skin Formation Discovered

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ScienceDaily (Mar. 25, 2009) — Researchers at Oregon State University have found one gene in the human body that appears to be a master regulator for skin development, in research that could help address everything from skin diseases such as eczema or psoriasis to the wrinkling of skin as people age.

Inadequate or loss of expression of this gene, called CTIP2, may play a role in some skin disorders, scientists believe, and understanding the mechanisms of gene action could provide a solution to them.
"We found that CTIP2 is a transcriptional factor that helps control different levels of skin development, including the final phase of a protective barrier formation," said Arup Indra, an OSU assistant professor of pharmacy. "It also seems particularly important in lipid biosynthesis, which is relevant not only to certain skin diseases but also wrinkling and premature skin aging."
The findings of this research, done in collaboration with Mark Leid, OSU professor of pharmacy, were recently published in the Journal of Investigative Dermatology. This work is supported by the National Institutes of Health, which has provided $1.5 million for its continuation.
Skin is actually the largest organ in the human body, and has important functions in protecting people from infection, toxins, microbes and solar radiation. But it's not static – skin cells are constantly dying and being replaced by new cells, to the extent that human skin actually renews its surface layers every three to four weeks. Wrinkles, in fact, are a reflection of slower skin regeneration that occurs naturally with aging.
Major advances have been made in recent years in understanding how skin develops in space and time, and in recent breakthroughs scientists learned how to re-program adult skin cells into embryonic stem cells.
"When you think about therapies for skin disease or to address the effects of skin aging, basically you're trying to find ways to modulate the genetic network within cells and make sure they are doing their job," Indra said. "We now believe that CTIP2 might be the regulator that can do that. The next step will be to find ways to affect its expression."
One of the ways that some ancient botanical extracts or other compounds may accomplish their job in helping to rejuvenate skin, Indra said, is by stimulating gene expression. A more complete understanding of skin genetics might allow that process to be done more scientifically, effectively and permanently.
Journal reference:
Olga Golonzhka, Xiaobo Liang, Nadia Messaddeq, Jean-Marc Bornert, Adam L Campbell, Daniel Metzger, Pierre Chambon, Gitali Ganguli-Indra, Mark Leid and Arup K Indra. Dual Role of COUP-TF-Interacting Protein 2 in Epidermal Homeostasis and Permeability Barrier Formation. Journal of Investigative Dermatology, 2008; DOI: 10.1038/jid.2008.392
Adapted from materials provided by Oregon State University.

Genomic Fossils In Lemurs Shed Light On Origin And Evolution Of HIV And Other Primate Lentiviruses

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ScienceDaily (Mar. 24, 2009) — A retrovirus related to HIV became stably integrated into the genome of several lemurs around 4.2 million years ago, according to research led by Dr. Cédric Feschotte at the University of Texas, Arlington. The new analysis of prosimian immunodeficiency virus (pSIV) offers new insights into the evolution of lentiviruses.

During replication, retroviruses integrate within the chromosomes of their host cells. If germ cells are infected, the integrated viral DNA can be transmitted from parent to offspring and may eventually become assimilated as part of the genetic material of the host species. This 'endogenization' process has occurred repeatedly during evolution, and has involved diverse retroviruses, giving rise to a sizeable portion of the genome of many vertebrate species – for example, ~8% of the human genome. Until now, the process was believed to be extremely rare for lentiviruses, an evolutionarily elusive group of retroviruses that infect diverse mammals, including humans (in the form of human immunodeficiency virus [HIV]).
Based on 'fossil' sequences collected from different lemur species, the researchers computationally reconstructed an apparently intact and complete DNA sequence for the ancestral prosimian lentivirus. The discovery that two different species of lemurs endemic to Madagascar suffered, independently and quasi-simultaneously, multiple germline infections of pSIV provides evidence that lentiviruses have repeatedly infiltrated the germline of prosimian species.
These findings should allow future functional analysis of the extinct virus and advance our understanding of the biology of lentiviruses, including HIV. In addition, the characterization of this ancient lentivirus in lemurs raises the possibility that HIV-like retroviruses are still circulating today in the mammalian fauna of Madagascar.
Journal reference:
Gilbert et al. Parallel Germline Infiltration of a Lentivirus in Two Malagasy Lemurs. PLoS Genetics, 2009; 5 (3): e1000425 DOI: 10.1371/journal.pgen.1000425
Adapted from materials provided by Public Library of Science, via EurekAlert!, a service of AAAS.

No More Cold Sores? Scientists Find Cellular Process That Fights Herpes Virus

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ScienceDaily (Mar. 24, 2009) — Scientists have discovered a new way for our immune system to combat the elusive virus responsible for cold sores: Type 1 herpes simplex (HSV-1). As reported in the advance online edition of Nature Immunology, a group of virus hunters from the Université de Montréal, in collaboration with American colleagues, have identified a cellular process that seeks out and fights herpes.

The five-year study, partially supported by the Canadian Institutes of Health Research, was a joint project with Washington University and Pennsylvania State University.
"Once human cells are infected with Type 1 herpes simplex, the virus comes back because it hides and blocks protection from our immune system," says Luc English, the study's lead author and a doctoral student at the Université de Montréal's Department of Pathology and Cell Biology. "For the first time, our research team has indentified a combative cellular mechanism in this game of hide-and-seek."
"We've found that the nuclear membrane of an infected cell can unmask Type 1 herpes simplex and stimulate the immune system to disintegrate the virus," says English.
The team made its discovery while conducting various tests in HSV-1 infected mice cells. They replicated environments when Type 1 herpes simplex thrives, namely periods of low-grade fever between 38.5 to 39 degrees, and found that herpes-fighting mechanisms were unleashed.
The research team now plans to study how activation of the herpes-combating cellular process could be applied to other illnesses. The outcome could hasten the development of therapies to prevent other immune-evading bacteria, parasites and viruses. "Our goal is to further study the molecules implicated in this mechanism to eventually develop therapies against diseases such as HIV or even cancer," says English.
According to Dr. Michel Desjardins, senior author and a professor in the Department of Pathology and Cell Biology at the Université de Montréal, treatment options might be imaginable in a decade.
"Now that we've identified the novel mechanism in cells that activate immune response to Type 1 herpes simplex, scientists are one step closer to creating new treatments that can activate the defence against this and other viruses," says Dr. Desjardins. "While it may not be possible to completely eradicate Type 1 herpes simplex in people who are already infected, at the very least, future therapies may be able to keep the virus in its dormant state."
This study was funded by the Canadian Institutes of Health Research, the Natural Science and Engineering Research Council of Canada, the Fonds de la Recherche en Santé du Québec, the U.S. National Institutes of Health and the foundation Research to Prevent Blindness.
About Herpes
There are two types of herpes viruses: Type 1 herpes simplex causes facial cold sores and Type 2 causes genital herpes. Both types of herpes affect an estimated 80 million people in America alone and there is currently no cure for the condition.
Journal reference:
Luc English, Magali Chemali, Johanne Duron, Christiane Rondeau, Annie Laplante, Diane Gingras, Diane Alexander, David Leib, Christopher Norbury, Roger Lippé & Michel Desjardins. Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection. Nature Immunology, 2009; DOI: 10.1038/ni.1720
Adapted from materials provided by University of Montreal.

New Stem Cell Therapy May Lead To Treatment For Deafness

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ScienceDaily (Mar. 23, 2009) — 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.
Journal reference:
Wei Chen, Stuart L. Johnson, Walter Marcotti, Peter W. Andrews, Harry D. Moore, Marcelo N. Rivolta. Human Fetal Auditory Stem Cells (hFASCs) Can Be Expanded In Vitro And Differentiate Into Functional Auditory Neurons And Hair Cell-Like Cells (p N/A). Stem Cells, 2009; DOI: 10.1002/stem.62
Adapted from materials provided by Wiley - Blackwell.

Artificial Genetics: New Type Of DNA Has 12 Chemical Letters Instead Of Usual 4

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ScienceDaily (Mar. 23, 2009) — In a dramatic rewrite of the recipe for life, scientists from Florida are describing the design of a new type of DNA with 12 chemical letters instead of the usual four.

Presented in Salt Lake City, Utah, at the 237th National Meeting of the American Chemical Society (ACS), this artificial genetic system already is helping to usher in the era of personalized medicine for millions of patients with HIV, hepatitis and other diseases.
The research may also shed light on how life arose on Earth, by producing a self-sustaining molecule capable of Darwinian evolution and reproduction, much like one that many scientists suggest arose at the dawn of life on Earth nearly four billion years ago.
Led by Steven Benner, Ph.D., this team is rewriting the rulebook that Nobel laureates James Watson and Francis Crick started when they described DNA's structure in 1953. One of the crowning discoveries of 20th century science, Watson and Crick's discovery established how the four chemical "letters" of DNA — A, T, C and G — pair up.
"This is a man on the moon goal," says Steven Benner, Ph.D. "It has dragged us kicking and screaming into uncharted territory. But we've learned all sorts of reasons about how the Watson and Crick rules don't enable technology to do useful things like highly parallel amplification of DNA or highly parallel diagnosis of human diseases. These things are worth a lot of money."
These pairing rules, for instance, make it very difficult for researchers to develop multiplexed diagnostic tests for viral diseases — tests that require identification and tagging of viral DNA. Old methods used regular DNA to bind and tag foreign genetic material. But natural DNA would often bind with non-disease DNA and generate confusing false positive and false negative results.
Benner's artificial genetic system does not operate under Watson-Crick rules, so the tagging gives accurate results. Benner's artificial alphabet already has been applied commercially. It is the basis of a viral load detector, which helps personalize the health care of those 400,000 patients annually infected with hepatitis B, hepatitis C, and HIV, the cause of AIDS.
"This is a hundred million dollar product right now," Benner noted. "It's used to manage cystic fibrosis, as well. We can also use this technology to go into biological samples and extract known genes with cancer-causing mutations. We can do all of this because we have an artificial DNA system.
For patients with HIV and hepatitis, the viral load detector can mean the difference between life and death.
Modern drug cocktails for these diseases are highly effective, reducing the viral load in the bloodstream to nearly zero. But at some point, the virus mutates, enabling it to evade the drugs and repopulate. As the viral tide rises, there are no outward symptoms in the patient, so the mutated strain is often discovered long after the virus has spread again.
The viral load detector, which relies on Benner's 12 letter system to tag DNA, may change that.
"What we want to do with personalized care is to give you a cocktail, and then monitor you and discover when the virus becomes resistant to it," explains Benner. "Now we don't want to do that too soon – that would waste a lifetime of good viral inhibitors — but not too late, of course. The patient would go in once a month to get their viral load measured. At some point the virus mutates and its viral load goes up. Then you know you better change the cocktail."
Benner says that the artificial DNA system is poised to become an essential tool in genomics research. The 12 letter alphabet already underlies new work at the National Human Genome Research Institute to connect large quantities of genomic data with human medicine.
The 12 letter system might also shed light on one of most mysterious times in Earth's history — the dawn of life nearly four billion years ago. Many scientists believe that this might have occurred when DNA's ancient cousin, RNA, began to act like a living organism.
"The idea has been that life originated on earth as RNA molecules assembled randomly and spontaneously in the prebiotic soup," says Benner. "Then, one of them found the ability to make copies of itself. In doing so, it made those copies with imperfections, so that some of its 'kids' were a bit better. Most were worse, so the better ones took over more resources. That started Darwinian processes. The rest is history."
Benner's ultimate goal is to synthesize a similar life form in his lab at the Foundation for Applied Molecular Evolution. His 12 letter genetic system is capable of nearly all of the actions that define a living thing — reproduction, growth and response to its environment — all without the benefit of genes refined over billions of years of evolution.
"But it still isn't self-sustaining," Benner explains. "You need a graduate or post-doc to come in the morning and feed it. It doesn't look for its own food. No one has gotten that first step to work. If you start making estimates of how many molecules you have to look for in order to find one that does this, you're talking about 10,000,000,000,000,000,000,000,000,000,000,000 molecules."
While Benner continues to pursue a chemical system fully capable of Darwinian evolution, he emphasized the lessons already learned from the development of the 12 letter system.
"We haven't just taken things from nature, but we've actually understood something about how chemical structure is related to genetic behavior. With that, we've been able to make new versions of it," says Benner.
Adapted from materials provided by American Chemical Society, via EurekAlert!, a service of AAAS.

How Proteins Find The Right DNA Sequences

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ScienceDaily (Mar. 23, 2009) — Researchers at Uppsala University and Harvard University have collaboratively developed a new theoretical model to explain how proteins can rapidly find specific DNA sequences, even though there are many obstacles in the way on the chromosomes.

In living cells, DNA-binding proteins regulate the activity of various genes so that different cells carry out the right tasks at the right time. For this to work, the DNA-binding proteins need to find the right DNA site sufficiently quickly. The research team behind the new study has previously succeeded in determining that it takes only a few minutes for an individual protein molecule to look through the millions of nearly identical binding alternatives and find the right place to bind. This is nevertheless slower than what is predicted by the established theoretical model for how DNA-binding proteins find their way to the proper place by alternating between diffusing in the cell cytoplasm and along DNA strands.
"By also taking into consideration the fact that there are many obstacles in the way when proteins are to diffuse along DNA strands, we can now calculate more exactly how long it takes them to find their way," says Johan Elf, associate professor of molecular biotechnology at the Center for Bioinformatics.
Besides offering a more precise prediction regarding the time needed to find the right site on DNA, the new theoretical model explains why there is an optimal total concentration of DNA-binding proteins. If there were more, it would simply be impossible for them to find a binding place in a reasonable time, since the proteins would be in each other's way. If there were fewer it would go slower as well, since not enough proteins would be searching. Finally, the new model provides an explanation why so many DNA-binding proteins also bind auxiliary binding sites close to the regulatory site, thus forming DNA loops. It turns out that this can shorten the time to find the right sites.
"This more detailed understanding of gene regulation is important, since it can ultimately provide a better understanding of diseases that occur as a result of problems in the control functions of cells, such as in cancer" says Johan Elf.
The researchers behind the study are Gene-Wei Li, Otto G. Berg, and Johan Elf. The findings are being published March 16 in the scientific journal Nature Physics.
Adapted from materials provided by Uppsala University.

First Automated Carbohydrate 'Assembly Line' Opens Door To New Field Of Medicine

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ScienceDaily (Mar. 23, 2009) — Scientists from Germany have reported a major advance toward opening the doors of a carbohydrate-based medicine chest for the 21st century. Much more than just potatoes and pasta, these carbohydrates may form the basis of revolutionary new vaccines and drugs to battle malaria, HIV, and a bevy of other diseases.

Speaking at the 237th National Meeting of the American Chemical Society, Peter H. Seeberger, Ph.D., described development of an automated carbohydrate synthesizer, a device that builds these intricate molecules in a few hours — rather than the months or years required with existing technology.
"Our automated synthesizer is now the fastest method to make complex carbohydrates," says Seeberger, principal investigator for the research. "There are currently no competitive methods available. Today, if people working in biology run into a problem related to carbohydrates, they usually drop it because there are no tools available. They can't buy anything from a catalogue. It becomes a royal pain in the neck."
Scientists trying to synthesize DNA and protein-based molecules experienced a similar pain-in-the neck decades ago, until the invention of automated DNA and protein synthesizers. These devices helped kick start a revolution in genetics and proteomics. The carbohydrate synthesizer may do the same thing for the emerging fields of glycochemistry and glycobiology — named for carbohydrate sugar chains known as "glycans."
In 2001, Seeberger and colleagues reported the design of a prototype synthesizer. Revealed for the first time at the ACS National Meeting, the latest version is now fully automated, much faster, and can be operated by a non-expert, says Seeberger. Developed at the Swiss Federal Institute of Technology in Zurich, the instrument produces significant quantities of carbohydrate molecules that were nearly inaccessible until now.
Carbohydrates are tough molecules to build because of their complicated, branched structure. So instead of trying to build carbohydrates from scratch, scientists today use molecules isolated from nature, a painstaking process that could take months.
"We make things chemically that people used to isolate," explains Seeberger. "The automated synthesizer puts single sugars, the building blocks of carbohydrates, together like beads on a string."
Carbohydrates play crucial roles in the immune system, especially in the body's defenses against disease-causing viruses and bacteria. Most of these microbes have unique carbohydrate markers on their surfaces. The immune system recognizes these carbohydrates as foreign material, and creates antibodies that launch an immune response to battle the infection.
"Vaccines 'educate' the immune system to recognize a specific molecule on the surface of infectious organisms," explains Seeberger. "The synthesizer allows us to make not one but many carbohydrate structures from a particular organism and test those to see if they protect against the microbe. Synthetic carbohydrates that show promising protective qualities then may become the basis for new vaccines.
In a recent finding, the team discovered a carbohydrate on the surface of the malaria parasite P. falciparum that enables the parasite to infect human red blood cells, thus solving a long-standing mystery about how infection happens.
Seeberger's group used the carbohydrate synthesizer to develop a malaria vaccine. Clinical trials for the vaccine are scheduled for 2010 in Mozambique and Tanzania. Its unique "anti-disease" mechanism makes it the only vaccine of its kind, he says.
"To my knowledge, ours is the first attempt at an anti-disease vaccine. It doesn't actually kill the malarial parasite; it blocks its toxic action. You create antibodies against the sugar structure, and these antibodies block the carbohydrate toxin from leading to inflammation and anemia, the hallmarks of malarial infection," says Seeberger.
He explained that they will pair the carbohydrate vaccine with a traditional, protein-based one to create a "conjugate vaccine," which is best suited to immunize the most vulnerable group of potential malaria victims — children under the age of two.
The malaria vaccine is only one of almost a dozen vaccines from Seeberger's lab headed for clinical trials. Carbohydrate-based vaccines could target some of today's most serious infectious diseases, including antibiotic-resistant infections and HIV.
Seeberger is commercializing the carbohydrate synthesizer through his start-up company, Ancora Pharmaceuticals, based in Medford, Mass. Looking ahead, Seeberger discussed the other major obstacle facing carbohy¬¬¬drate research.
"In the area of glycobiology, there are two technological hurdles right now. One is to get access to molecules, which we have now addressed. The second one is sequencing. If you look at the human genome project, or genomics and proteomics, sequencing and synthesis were always the key issues," says Seeberger.
Seeberger saw firsthand the profound effect that automated DNA synthesizers had on genetics and biotechnology. His doctoral advisor, Marvin H. Caruthers, Ph.D., of the University of Colorado, helped develop the first model in 1980.
"We hope that we have the same effect on carbohydrate research," says Seeberger.
Adapted from materials provided by American Chemical Society, via EurekAlert!, a service of AAAS.

Genetic Mechanism In Mole Rats Can Be Targeted In Cancer Research

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ScienceDaily (Mar. 23, 2009) — Cellular mechanisms that subterranean mole rats have developed in order to survive the low levels of oxygen in their underground habitat are similar to the mechanisms used by tumors to survive and progress in humans. Based on a new study, the mole rat can represent the human tumor in research, and the gene targeted in mole rats can be targeted for development of anti-cancer drugs.

This landmark discovery was revealed in a new study carried out by researchers from the Institute of Evolution at the University of Haifa and the Functional Genomics Center at the University of Illinois.
"When we understand how the subterranean mole rat developed these mechanisms for survival, we may be able to understand why they are so destructive in humans," Prof. Aaron Avivi of the University of Haifa said.
The biological significance of the blind subterranean mole rat was recognized about 50 years ago by Prof. Eviatar Nevo, who also pioneered biological studies of the organism. The current study, led by Prof. Aaron Avivi from the Institute of Evolution at the University of Haifa and Dr. Mark Band from the University of Illinois, is supported by a grant from the U.S-Israel Binational Science Foundation (BSF), and has just been published in the online FASEB Journal.
Based on the new study, the mole rat can represent the human tumor in research, and the gene targeted in mole rats can be targeted for possible development of anti-cancer drugs. It is therefore predicted that understanding the survival mechanism in the blind subterranean mole rat can help in the advancement of cancer research. "When we understand how the subterranean mole rat developed these mechanisms for survival, we may be able to understand why they are so destructive in humans," Prof. Avivi of the University of Haifa pointed out.
Experiments were conducted on groups of hypoxia-tolerant mole rats and hypoxia-intolerant "regular" rats. A group of each species was exposed to normal levels of oxygen while other groups were exposed to low oxygen levels, ranging from 3 to 10 percent. The gene BNIP3, which becomes active in the regular rats to protect their bodies from low oxygen and to prevent resulting damage, was shown to be active in heart and skeletal muscles. On the other hand, in the mole rats that tolerate low levels of oxygen, the gene was less expressed and less active, suggesting and supporting previous findings by these scientists that on a physiological level their cells and tissues do not become hypoxic.
The hypoxia-regulated pattern of BNIP3 expression and the gene's activity in the mole rat echoes its behavior in cancer cells. In both cases waves of normal oxygen levels and hypoxic levels lead to changes in the regulation of hypoxia-induced genes behavior. In mole rats this fluctuation is due to rainfalls that flood their underground tunnels, limiting the availability of oxygen and, moreover, forcing them to rebuild the tunnels and exhaust the limited oxygen; in tumor cells it occurs as they divide faster than blood vessels, which supply oxygen, sprouting into new cells.
Among a growing list of hypoxia-induced genes that were studied in the mole rat by Prof. Avivi and his colleagues and collaborators, this is the third gene that shows a similar pattern of expression as in cancer. In the past it has also been revealed that VEGF (Vascular Endothelial Growth Factor - a major growth factor that regulates the growth of new blood vessels) and p53 (a "master" gene, responsible for activating a battery of other genes engaged in either programmed cell death [apoptosis] or DNA-repair in cells) exhibit a similar mode of action in mole rats and in cancer growths, which is why they are so destructive in human cancer growths, Prof. Avivi pointed out.
Adapted from materials provided by University of Haifa.

Mice With Disabled Gene That Helps Turn Carbs Into Fat Stay Lean Despite Feasting On High-carb Diet

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ScienceDaily (Mar. 23, 2009) — Researchers at the University of California, Berkeley, have identified a gene that plays a critical regulatory role in the process of converting dietary carbohydrates to fat. In a new study, they disabled this gene in mice, which consequently had lower levels of body fat than their normal counterparts, despite being fed the equivalent of an all-you-can-eat pasta buffet.

The authors of the study, to be published in the March 20 issue of the journal Cell, say the gene, called DNA-PK, could potentially play a role in the prevention of obesity related to the over-consumption of high-carbohydrate foods, such as pasta, rice, soda and sugary snacks.
DNA-PK, which stands for DNA-dependent protein kinase, has already been the subject of much research because it helps repair breaks in the DNA. Suppression of DNA-PK has been used as a technique by researchers to enhance the ability of cancer treatments to kill tumor cells. Its role in fat synthesis, then, came as a surprise to the UC Berkeley researchers.
"It turns out that DNA-PK is critical to a metabolic process we have been trying to understand for 20 years," said Hei Sook Sul, a professor in UC Berkeley's Department of Nutritional Science & Toxicology and head of the research team behind these new findings. "For the first time, we have connected DNA-PK to the signaling pathway involved in the formation of fat from carbohydrates in the liver. Identifying this signaling pathway involving DNA-PK brings us one step forward in understanding obesity resulting from a diet high in carbohydrates, and could possibly serve as a potential pharmacological target for obesity prevention."
After a meal of pizza and soda, it is known that levels of blood glucose - the digested form of carbohydrates - go up. That rise in blood glucose triggers the secretion of the hormone insulin, which helps different cells in the body use glucose for energy. Glucose in the liver that isn't burned for energy turns into fatty acids, which then circulate to other parts of the body, primarily to fat tissue.
This conversion of excess glucose into fatty acids occurs in the liver, but the exact molecular pathway involved has not been fully understood until now. Researchers have known that insulin binds to receptors on the liver cells, which activates protein phosphatase-1 (PP1), the first molecule of the insulin-signaling pathway inside the liver cell. Sul's lab had previously shown that upstream stimulatory factor (USF) is needed to activate certain genes, such as fatty acid synthase (FAS), which converts glucose to fatty acids.
The link between PP1 and USF was still a mystery until Roger H. F. Wong, a UC Berkeley graduate student in comparative biochemistry in Sul's lab, finally connected the dots through proteomic sequencing. He found that DNA-PK, which is regulated by PP1, controls the activation of USF and the subsequent conversion of glucose to fatty acids.
"The missing link was DNA-PK," said Wong. "We determined that DNA-PK acts as a signaling molecule in the chain reaction that begins when insulin binds to receptors on liver cells. This helps explain why untreated Type 1 diabetics, who cannot produce insulin, may experience significant weight loss. Without treatment, they basically have trouble making enough fat."
"This insulin-signaling pathway is also disrupted in Type 2 diabetes, in which the body still produces insulin, but the cells become resistant to its effects," said Wong.
After identifying DNA-PK, the researchers put the gene to the test in mice fed a diet containing 70 percent carbohydrates, but no fat. A typical lab mouse diet is made up of both fat and carbohydrates. Half the mice had the DNA-PK gene disabled, and the other half comprised a control group of normal mice.
"The DNA-PK disabled mice were leaner and had 40 percent less body fat compared with a control group of normal mice because of their deficiency in turning carbs into fat," said Wong. "The knockout mice were resistant to high carbohydrate-induced obesity and had lower plasma lipids, which can reduce the risk of cardiovascular disease. With all of these health benefits, this gene can serve as a potential pharmacological target for obesity prevention."
The researchers noted that although interest in low-carb diets persists, there are many sources of carbohydrates, including fruits and vegetables, legumes and whole grain breads and pastas, that have important nutritional benefits.
"The best way to control your body weight is to eat a well-balanced diet and limit your caloric intake," said Wong. "We hope that this research will one day help people eat bread, pasta and rice and not worry about getting fat."
This study is part of the larger research effort by the Sul lab to understand the molecular mechanisms underlying the synthesis of fatty acids, creation of fat cells and how fat is stored in the body. Recently, the lab published a study in the journal Cell Metabolism describing how a molecule called Pref-1 blocks the creation of fat cells. Two months ago, the discovery by Sul's lab of an enzyme called AdPLA critical to the breakdown of fat cells, was published in the journal Nature Medicine. This latest paper in Cell details the very first step of fat synthesis - making fat from carbohydrate.
Other co-authors of this study are members of Sul's lab and include several undergraduate students in the Department of Nutritional Science & Toxicology.
The National Institutes of Health helped support this study in Cell.
Journal reference:
Roger H.F. Wong, Inhwan Chang, Carolyn S.S. Hudak, Suzanne Hyun, Hiu-Yee Kwan, Hei Sook Sul. A Role of DNA-PK for the Metabolic Gene Regulation in Response to Insulin. Cell, 2009; 136 (6): 1056 DOI: 10.1016/j.cell.2008.12.040
Adapted from materials provided by University of California - Berkeley.

Plant Biologists Discover Gene That Switches On 'Essence Of Male'

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ScienceDaily (Mar. 20, 2009) — Biologists at the University of Leicester have published results of a new study into plant sex – and discovered that a particular gene switches on 'the essence of male'. The study takes to a new level understanding of the genes needed for successful plant reproduction and seed production.

Professor David Twell and colleagues in the Department of Biology at the University of Leicester reported the discovery of a gene that has a critical role in allowing precursor reproductive cells to divide to form twin sperm cells.
Their study is reported in the journal Public Library of Science Genetics (PLoS Genetics) and was funded by the Biotechnology and Biological Sciences Research Council (BBSRC).
Professor Twell said: "Flowering plants, unlike animals require not one, but two sperm cells for successful fertilisation. One sperm cell to join with the egg cell to produce the embryo and the other to join with the central cell to produce the nutrient-rich endosperm tissue inside the seed. A mystery in this 'double fertilisation' process was how each single pollen grain could produce the pair of sperm cells needed for fertility and seed production.
"We now report the discovery of a dual role for DUO1, a regulatory gene required for plant sperm cell production. We show that the DUO1 gene is required to promote the division of sperm precursor cells, while at the same time promoting their specialised function as sperm cells. It effectively switches on the essence of male.
"We show that DUO1 is required for the expression of a key protein that controls cell division and for the expression of genes that are critical for gamete differentiation and fertilisation.
"This work provides the first molecular insight into the mechanisms through which cell cycle progression and gamete differentiation are coordinated in flowering plants.
"This knowledge will be helpful in understanding the mechanisms and evolution of gamete development in flowering plants and may be useful in the control of gene flow and crossing behaviour in crop plants."
The researchers also report on the presence of genes closely related to DUO1 in a wide variety of flowering plants and even in lowly land plants such as moss, which suggests that DUO1 may be part of an ancient sperm cell regulatory network that evolved even before pollen and flowers appeared on the scene.
Interestingly, DUO1 is also related to a super class of Myb regulator proteins also present in animals that have an important role in controlling cell proliferation and that are implicated in certain human cancers such as leukemias. So like animal cell Myb proteins, DUO1 is needed for control of cell proliferation, but DUO1 is distinguished by its specific role in plant reproduction, namely its dual role in sperm cell production and switching on their ability to fertilize.
Professor Twell added that the study could help to unravel the evolutionary origins of plant sperm cells and provide new molecular tools for the manipulation of plant fertility and hybrid seed production – as well as to control gene flow in transgenic crops where the male contribution may need to be eliminated.
Background
The unique double fertilisation mechanism in flowering plants depends upon a pair of functional sperm cells. During male gametogenesis, each haploid microspore undergoes an asymmetric division to produce a large, non-germline vegetative cell and a single germ cell that divides once to produce the sperm cell pair. Despite the importance of sperm cells in plant reproduction, relatively little is known about the molecular mechanisms controlling germ cell proliferation and specification.
Adapted from materials provided by University of Leicester, via EurekAlert!, a service of AAAS.

Cellular Discovery May Lead To Targeted Treatment For Rare Form Of Anemia

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ScienceDaily (Mar. 20, 2009) — University of Cincinnati (UC) researchers have identified the specific biological mechanisms believed to lead to a rare and incurable blood disease known as Diamond Blackfan anemia (DBA). Scientists say with further investigation, their discoveries could result in drastic changes to current thinking about treatment for this disease and may lead to promising new drug therapies.

George Thomas, PhD, Stefano Fumagalli, PhD, and collaborators report their findings online ahead of print in the journal Nature Cell Biology on March 15, 2009. The research will also appear in the April print issue of the journal and is being presented at the 10th annual International Diamond Blackfan Anemia Consensus Conference in New York, which concludes Monday, March 16.
DBA is a rare blood disorder characterized by the bone marrow's failure to produce red blood cells. This failure is due to an intrinsic defect that makes the red blood cells prone to cell death before they mature. Red blood cells travel through the bloodstream to deliver oxygen to the body's tissues, which is critical to the health and proper function of all tissues.
According to the Centers for Disease Control and Prevention, approximately 25-35 new cases of DBA are diagnosed each year, with the majority of patients being identified before age 1. The most common treatments include blood transfusions and corticosteroids. The disease is characterized by extreme anemia—with a propensity to develop into leukemia—and often has no cure.
Using a preclinical laboratory model, Thomas' team was able to explain how cell death occurs in DBA and identified a specific step in the biological chain of events leading to disease onset where targeted medical intervention may effectively slow—or even stop—red blood cell death.
DBA has recently been attributed to a ribosomal protein defect that the UC team hypothesizes leads to abnormal activation of p53, causing premature death of red blood cells. P53 is a protein that normally functions to trigger "cell suicide" in response to severe cellular damage, therefore protecting the body from overgrowth of defective cells.
Previous research has attributed p53 activation to the passive diffusion of ribosomal protein L11 from the nucleolus, the part of the nucleus where ribosomes are produced to the nucleoplasm.
The UC research, however, suggests that p53 activation is not due to nucleolar breakdown, but is actually the result of an active increase in the production of L11. They suggest that in DBA, a series of L11 interactions results in cell cycle arrest and ultimately leads to cell death and anemia.
"Previous studies suggested L11 was passively coming out of the nucleolus when ribosome production was disrupted. Our study actually showed that the nucleolus stayed intact as ribosomes were still being produced, suggesting selective upregulation of L11," explains Thomas, the John and Gladys Strauss endowed professor of cancer biology at UC and scientific director at UC's Genome Research Institute. "If we can target the L11 interaction, we might be able to spare other stress pathways that mediate potential benefits of p53 induction."
Thomas believes DBA slowly evolves into cancer when this specific molecular checkpoint is lost. This results in the body being genetically reprogrammed over time, leading to the onset of additional medical problems, particularly leukemia, in DBA patients later in life.
"By understanding the chain of biological events leading to this abnormal cell death and targeting the specific molecular checkpoint that controls cell death, we may be able to develop new drugs that would interrupt or stop the process and allow the body to recover, rebuilding healthy bone marrow," adds Thomas.
This research was funded in part by the National Cancer Institute's Mouse Models in Human Cancer Consortium. In addition to Thomas and Funagalli, manuscript co-authors include Sandy Schwemberger, PhD, and George Babcock, MD, and Arti Neb-Gulati of UC; Alessandro Di Cara, PhD of Friedrich Miescher Institute for Biomedical Research in Switzerland; Francois Natt and Jonathan Hall of Novartis Institutes for Biomedical Research in Switzerland; Rosa Bernardi MD, PhD, of San Raffaele, Institute Via Olgettina in Italy; and Pier Paolo Pandolfi, MD, PhD, of Beth Israel Deaconess Medical Center in Boston.
"It is our hope that these discoveries will lead to new treatments for the disease. As anyone can imagine, in any disease where more than 90 percent of patients present before 1 year of age the families clamor for additional breakthroughs," adds Marie Arturi, executive director of the Daniella Maria Arturi Foundation. "We are deeply indebted to all who help in this effort."
Adapted from materials provided by University of Cincinnati.

Lab-grown Nerves Promote Nerve Regeneration After Injury

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ScienceDaily (Mar. 20, 2009) — Researchers at the University of Pennsylvania School of Medicine have engineered transplantable living nerve tissue that encourages and guides regeneration in an animal model. Results were published in March in the journal Tissue Engineering Part A.

About 300,000 Americans suffer peripheral nerve injuries every year, in many cases resulting in permanent loss of motor function, sensory function, or both. These injuries are a common consequence of trauma or surgery, but there are insufficient means for repair, according to neurosurgeons. In particular, surgeons need improved methods to coax nerve fibers known as axons to regrow across major nerve injuries to reconnect healthy targets, for instance muscle or skin.
“We have created a three-dimensional neural network, a living conduit in culture, which can be transplanted en masse to an injury site,” explains senior author Douglas H. Smith, MD, Professor, Department of Neurosurgery and Director of the Center for Brain Injury and Repair at Penn. Smith and colleagues have successfully grown, transplanted, and integrated axon bundles that act as ‘jumper cables’ to the host tissue in order to bridge a damaged section of nerve.
Previously, Smith and colleagues have “stretch-grown” axons by placing neurons from rat dorsal root ganglia (clusters of nerves just outside the spinal cord) on nutrient-filled plastic plates. Axons sprouted from the neurons on each plate and connected with neurons on the other plate. The plates were then slowly pulled apart over a series of days, aided by a precise computer-controlled motor system.
These nerves were elongated to over 1 cm over seven days, after which they were embedded in a protein matrix (with growth factors), rolled into a tube, and then implanted to bridge a section of nerve that was removed in a rat.
“That creates what we call a ‘nervous-tissue construct’,” says Smith. “We have designed a cylinder that looks similar to the longitudinal arrangement of the nerve axon bundles before it was damaged. The long bundles of axons span two populations of neurons, and these neurons can have axons growing in two directions - toward each other and into the host tissue at each side."
The constructs were transplanted to bridge an excised segment of the sciatic nerve in rats. Up to 16 weeks post-transplantation, the constructs still had their pre-transplant shape, with surviving transplanted neurons at the extremities of the constructs spanned by tracts of axons.
Remarkably, the host axons appeared to use the transplanted axons as a living scaffold to regenerate across the injury. The authors found host and graft axons intertwined throughout the transplant region, suggesting a new form of axon-mediated axonal regeneration. “Regenerating axons grew across the transplant bridge and became totally intertwined with the transplanted axons,” says Smith
Axons throughout the transplant region showed extensive myelination, the fatty layer surrounding axons. What’s more, graft neurons had extended axons beyond the margins of the transplanted region, penetrating deep into the host nerve. Remarkably, the constructs survived and integrated without the use of immunosuppressive drugs, challenging the conventional wisdom regarding immune tolerance in the peripheral nervous system.
The researchers suspect that the living nerve-tissue construct encourages the survival of the supporting cells left in the nerve sheath away from the injury site. These are cells that further guide regeneration and provide the overall structure of the nerve.
“This may be a new way to promote nerve regeneration where it may not have been possible before,” says co-first author D. Kacy Cullen, PhD, a post doctoral fellow in the Smith lab. “It’s a race against time - if nerve regeneration happens too slowly, as may be the case for major injuries, the support cells in the extremities can degenerate, blunting complete repair. Because our living axonal constructs actually grow into the host nerve sheath, they may ‘babysit’ these support cells to give the host more time to regenerate.”
The other co-first author is Jason Huang, MD, Assistant Professor of Neurosurgery at Rochester University, who participated in the study during his Neurosurgical residency at Penn.
This work was funded by the National Institutes of Neurological Disorders and Stroke and the Sharpe Trust.
Journal reference:
Jason H. Huang, D. Kacy Cullen, Kevin D. Browne, Robert Groff, Jun Zhang, Bryan J. Pfister, Eric L. Zager, Douglas H. Smith. Long-Term Survival and Integration of Transplanted Engineered Nervous Tissue Constructs Promotes Peripheral Nerve Regeneration. Tissue Engineering Part A, 2009; 090220122151069 DOI: 10.1089/ten.tea.2008.0294
Adapted from materials provided by University of Pennsylvania School of Medicine.

Missing Piece Of Plant Clock Found

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ScienceDaily (Mar. 19, 2009) — Biologists at the University of California, San Diego have identified a key protein that links the morning and evening components of the daily biological clock of plants.

Their discovery, detailed in the March 13 issue of Science, solves a longstanding puzzle about the underlying biochemical mechanisms that control plant clocks and could provide a new way to increase the growth and yield of agricultural crops.
The finding is the first outcome of a larger effort to assemble a complete library of all proteins called transcription factors, which regulate genes, in Arabidopsis, a plant often used as a genetic model.
Scientists previously had identified two primary feedback loops in the plant daily clock – one that detects the onset of light in the morning and another that tracks when light fades in the evening.
"The best way to construct a robust clock would be to connect the loops so that they both communicate that information to each other," said Steve Kay, dean of the Division of Biological Sciences at UC San Diego whose research team made the discovery. "Now a protein we call CHE has provided that link."
CHE, first predicted nearly a decade ago, has proved difficult to find. Multiple backup systems for many important functions in plants, including timekeeping, frustrate efforts to identify the function of an individual molecule or gene.
"In plants there are a lot of redundancies – proteins that do similar things," said Jose Pruneda-Paz, a postdoctoral fellow at UC San Diego and the first author of the study. "In the clock, on top of the redundancies, you have feedback loops that are interconnected. So it's difficult to perturb the system."
Disrupting a protein will fail to reveal its function if the system can compensate for its loss, so the team took a different approach. They sorted through proteins with the ability to bind to DNA, and therefore to regulate genes, and selected candidates mostly likely to be part of a clock: the ones that cycle between abundant and scarce.
Of those cyclical proteins, only CHE stuck specifically to the part of plant DNA that controls a critical component of the morning loop. Further experiments demonstrated that CHE also binds to an evening loop protein providing the missing link.
Pruneda-Paz and his co-authors "solve a major puzzle in our understanding of the plant clock," wrote C. Robertson McClung, professor of biology at Dartmouth College, in a commentary on the article that will appear in the same issue of Science.
Evidence increasingly points to the clock as a critical component of functions growth and the timing of flowering. A recent paper published in Nature by a group at the University of Texas, Austin reports that an altered clock contributes to hybrid vigor, suggesting that targeting clock genes may be a way to improve the growth of crops. "It's going to be a way to come up with rational design for increasing yield in the field," Kay said.
Kay expects the growing catalog of transcription factors to be completed by the end of the year with more than 2,000 entries, he said. "This is going to be a significant resource for the plant science community developed here at UC San Diego."
Grants from the National Insitutes of Health supported his team's research.
Adapted from materials provided by University of California - San Diego, via EurekAlert!, a service of AAAS.

Microscope Reveals How Soil Bacteria 'Breathe' Toxic Metals

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ScienceDaily (Mar. 19, 2009) — Researchers are studying some common soil bacteria that “inhale” toxic metals and “exhale” them in a non-toxic form. The bacteria might one day be used to clean up toxic chemicals left over from nuclear weapons production decades ago.

Using a unique combination of microscopes, researchers at Ohio State University and their colleagues were able to glimpse how the Shewanella oneidensis bacterium breaks down metal to chemically extract oxygen.
The study, published online the week of March 16 in the journal Applied and Environmental Microbiology, provides the first evidence that Shewanella maneuvers proteins within the bacterial cell into its outer membrane to contact metal directly. The proteins then bond with metal oxides, which the bacteria utilize the same way we do oxygen.
The process is called respiration, and it’s how living organisms make energy, explained Brian Lower, assistant professor in the School of Environment and Natural Resources at Ohio State. We use the oxygen we breathe to release energy from our food. But in nature, bacteria don’t always have access to oxygen.
“Whether the bacteria are buried in the soil or underwater, they can rely on metals to get the energy they need,” Lower said. “It’s an ancient form of respiration.”
“This kind of respiration is fascinating from an evolutionary standpoint, but we’re also interested in how we can use the bacteria to remediate nasty compounds such as uranium, technetium, and chromium.”
The last two are byproducts of plutonium. The United States Department of Energy is sponsoring the work in order to uncover new methods for treating waste from nuclear weapons production in the 1960s and ‘70s.
Shewanella is naturally present in the soil, and can in fact be found at nuclear waste sites such as the Hanford site in the state of Washington, Lower explained.
With better knowledge of the bacterium’s abilities, scientists might one day engineer a Shewanella that would remediate such waste more efficiently.
“For instance, if you could enhance this bacterium’s ability to reduce uranium by having it make more of these key proteins, that could perhaps be one way to clean up these sites that are contaminated,” he said.
The danger at such waste sites is that the toxic metals are soluble, and so can leak into the local water supply. But these bacteria naturally convert the metals into an insoluble form. Though the metals would remain in place, they would be stable solids instead of unstable liquids.
For this study, Lower and his colleagues used an atomic force microscope (AFM) to test how the bacterium responded to the metallic mineral hematite.
An AFM works somewhat like a miniaturized phonograph needle: a tiny tip dangles from a cantilever above a surface that’s being studied. The cantilever measures how much the tip rises and falls as it’s dragged over the surface. It can measure features smaller than a nanometer (billionth of a meter), and detect atomic forces between the probe tip and the surface material.
They combined the AFM with an optical microscope to get a precise map of the bacteria’s location on the hematite.
Though the bacteria are very small -- several hundred thousand of them could fit inside the period at the end of a sentence -- they are still thousands of times bigger than the tip of an AFM probe. So the microscope was able to slide over the surface of individual bacteria to detect protein molecules on the cell surface and in contact with the metal.
The researchers coated their probe tip with antibodies for the protein OmcA, which they suspected Shewanella would use to “breathe” the metal.
Whenever the probe slid over an OmcA protein, the antibody coating would stick to the protein. By measuring the tiny increase in force needed to pull the two apart, the researchers could tell where on the bacteria surface the proteins were located.
The microscope detected OmcA all around the edges of the bacteria, wherever the cell membrane contacted the hematite -- which suggests that the protein does indeed enable the bacteria to “breathe” hematite. The protein was even present in a gelatinous ooze that was seeping from the bacteria. This suggests that Shewanella might create the ooze in order to obtain energy from a wider portion of the metal than it can directly touch, Lower said.
In the future, he and his partners want to test their new microscope technique on other types of cells. They also want to test whether Shewanella produces OmcA on the cell surface when exposed to uranium and technetium.
Lower’s coauthors on the paper hail from Corning, Inc.; Pacific Northwest National Laboratory; Johannes Kepler University of Linz, Austria; Ecole Polytechnique Fédérale de Lausanne, Switzerland; and Umeå University, Sweden.
Adapted from materials provided by Ohio State University.

DNA Shape Is Constrained By Evolution: Structural Approach To Exploring DNA

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ScienceDaily (Mar. 19, 2009) — A team led by researchers from Boston University and the National Institutes of Health has developed a new method for uncovering functional areas of the human genome by studying DNA's three-dimensional structure -- a topographical approach that extends the more familiar analysis of the sequence of the four-letter alphabet of the DNA bases.

Unlike the well-understood genomic sequences that code for proteins and comprise about two percent of the human genome, the remaining 98 percent is the non-coding portion, which encodes many functions. However, little is known about how this functional non-coding information is specified.
In a study which appears March 12 in the online edition of Science, the researchers focused on examining the non-coding regions of the genome for areas that are likely to play a key role in human biological function.
To do this, the researchers developed a method which incorporates information about the structure of DNA to compare sequences of genomes from humans and 36 mammalian species that included the mouse, chimpanzee, elephant and rabbit.
By examining the shapes, grooves, turns and bumps of the DNA that comprises the human genome, the team discovered that 12 percent of the human genome appears to be constrained by evolution. That's double the six percent detected by simply comparing the linear order of DNA nucleotides (A, T, G, and C, the familiar letters that make up the genome). The huge increase stems from finding some DNA sequences that differ in the order of nucleotides, but have very similar topographical shapes, and so may perform similar functions.
They went on to show that the topographically-informed constrained regions correlate with functional non-coding elements better than constrained regions identified by nucleotide sequence alone.
"By considering the three-dimensional structure of DNA, you can better explain the biology of the genome," said Thomas D. Tullius, Boston University professor of chemistry who has spent more than 20 years developing ways to map the structure of the human genome. "For this achievement Stephen Parker, a Boston University graduate student, deserves much of the credit for his development of the algorithm that incorporated DNA structure into evolutionary analysis."
Bringing a molecular biologist's point of view and expertise in comparing the genomes of different species was Elliott Margulies, an investigator at NHGRI's Genomic Technology Branch. "Proteins that influence biological function by binding to DNA recognize more than just the sequence of bases," he said. "These binding proteins also see the surface of the DNA molecule and are looking for a shape that allows a lock-and-key fit."
In their Science paper the researchers also explored how small genetic changes, or variations, known as SNPs (Single Nucleotide Polymorphisms) could prompt structural changes that might lead to disease. In studying these mutations from a database of 734 non-coding SNPs associated with diseases, such as cystic fibrosis, Alzheimer's disease, and heart disease, they found that disease-associated SNPs produced larger changes in the shape of DNA than SNPs not associated with a disease.
The new research findings on evolutionary conservation of DNA structure stem from recent progress in analyzing the functional elements in a representative fraction of the human genome. That study, known as ENCODE (ENCyclopedia of DNA Elements), organized by the National Human Genome Research Institute (NHGRI), challenged the traditional view of the human genetic blueprint as a collection of independent genes. Instead, researchers found a complex network of genes, regulatory elements, and other DNA sequences that do not code for proteins.
Researchers say DNA sequence is not always a good indicator of function. They found that very similar DNA sequences may assume very different topographical shapes, which can have a major impact on their function or lack of function. On the other hand, different DNA sequences may assume very similar topographical shapes and perform very similar functions. So, in many instances, DNA structure may be a better predictor of function than DNA sequence.
The study determined, for the first time, where many types of functional elements are located, how they are organized, and how the genome is pervasively made into RNA. The current research on genome structure and function is based on some of the ENCODE findings, noted Tullius, whose work in developing the new technology was funded through the ENCODE project.
In addition to Tullius and Margulies, the other authors of the Science paper are Stephen C.J. Parker and Loren Hansen, both BU graduate students in bioinformatics, and Hatice Ozel Abaan, a technician in Margulies' laboratory.
Journal reference:
. Local DNA Topography Correlates with Functional Noncoding Regions of the Human Genome. Science, March 13, 2009
Adapted from materials provided by Boston University, via EurekAlert!, a service of AAAS.

Process That Regulates Seed Germination Identified

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ScienceDaily (Mar. 18, 2009) — Purdue University researchers have determined a process that regulates activity of genes that control seed germination and seedling development.

Mike Hasegawa, the Bruno C. Moser Distinguished Professor of Horticulture and Landscape Architecture, and Kenji Miura, a former Purdue postdoctoral researcher and now an assistant professor at Tsukuba University in Japan, discovered the step involved in keeping seeds from germinating in adverse conditions such as freezing temperatures or drought, a factor in the survival of plant species.
The work, which was published March 11 in the early online version of the Proceedings of the National Academy of Sciences, is part of ongoing research that has uncovered that similar processes affects a plant's freeze tolerance and absorption of phosphate.
"We've found the process, called sumoylation, is involved in the regulation of some major agricultural traits," Hasegawa said. "It is fundamental, basic research like this that allows us to understand how plants respond to hormones and environmental conditions."
Seeds produce a hormone called abscisic acid, or ABA, that prevents germination. When environmental factors such as temperature are not optimal for seed germination, ABA levels are high, which causes production of higher levels of a protein called ABI5. When the ABI5 protein is active, it switches on genes that prevent germination.
Hasegawa's research showed that when a SUMO peptide is attached to the ABI5 protein, the protein becomes inactive, switching off the genes that prevent germination and seedling development.
"A single stimulus such as ABA affects transcription factors, which are major controllers of genes involved in complex processes such as seed germination," Hasegawa said. "Sumoylation seems to be an important process in the control of significant plant characteristics."
Hasegawa said that the ABI5 protein can become active again, halting germination and seedling development if condition are no longer optimal. When conditions change to make plant development possible, the protein can once again be deactivated.
The National Science Foundation and the U.S. Department of Agriculture have funded the research in Hasegawa's laboratory. Hasegawa's next step is to determine how the sumoylation process leads to gene suppression and expression.
Adapted from materials provided by Purdue University.