RxPG News : Microbiology

Predatory bacteria attack in 'military-style' waves

Sun, 23 Nov 2008 10:54:06 PST

( from http://www.rxpgnews.com ) Washington, Oct 30 - A soil bacteria like M. xanthus executes a wave-like 'military-style' attack in a swarm against their prey, before gobbling them up and moving on.

Despite its deadly role, M. xanthus is harmless to humans and might be used to destroy harmful bacteria on surfaces or in human infections, said John Kirby, associate professor of microbiology at the University of Iowa - Carver College of Medicine.

'When an M. xanthus aggregate is placed inside a colony of E. coli bacteria,' it 'proceeds to eat the colony from the inside out and creates a rippling pattern as the swarm moves through the prey cells,' Kirby said.

'We now know that this rippling pattern is the highly organised behaviour of thousands of cells working in concert to digest the prey.'

'It may be that we can modify this predator-prey relationship or apply it to medically relevant situations,' Kirby said. 'It would be amazing if we could adapt its predatory ability to get rid of harmful bacteria that reside in places we don't want them, including in hospitals or on medical implants.'

The U-I team also showed that the ripple wavelength is adaptable. At high prey density, M. xanthus forms ripples with shorter wavelengths. As prey density decreases, the ripple wavelength gets longer. Eventually, when there is no more prey, the rippling behaviour dissipates.

M. xanthus lives in a multi-cellular unit that can change its structure and behaviour in response to changing availability of prey. This adaptive ability to control movement in response to an environmental stimulus is called chemotaxis, and the research team coined the term predataxis to describe its behaviour in response to prey, said a U-I press release.

These findings were published online in the Proceedings of the National Academy of Sciences - early edition.

In earlier studies, Kirby and James Berleman, postdoctoral fellow in Kirby's lab, showed that the presence of prey causes M. xanthus to form parallel rippling waves that move toward and through prey bacteria.

Now, how the bacteria organise to form these travelling waves in response to the presence of prey is the subject of the study.

The Strange Case of the Radiation-Resistant Bacteria

Mon, 26 Mar 2007 10:54:42 PST

( from http://www.rxpgnews.com ) Fifty years ago, scientists experimenting with gamma radiation to sterilize canned foods were surprised to find spoiled meat in cans zapped with what they thought were lethal levels of ionizing radiation (IR). Inside the bulging cans, they discovered a strain of bacteria now called Deinococcus radiodurans. This extremely resilient microbe can endure 100 times the IR levels that kill other bacteria and levels 2,000 times higher than the lethal human dose.

Researchers investigating the nature of radiation toxicity long ago settled on DNA as its principal target. Within this framework, efforts to understand D. radioduransâs resistance have focused on the mechanisms of DNA repair, with each study revealing seemingly greater levels of efficiency. Surprisingly, this extremophile relies on a set of apparently universal DNA repair proteins, raising an even bigger paradox: DNA repair and synthesis depends on proteins, but these proteins suffer radiation damage, too. And no matter how efficient DNA repair enzymes might be under normal conditions, itâs not clear how they manage to resurrect a radiation-shattered genome if they are also damaged.

Over the past few years, several observations have challenged the DNA-centered view of IR toxicity. For one thing, the D. radiodurans genome, sequenced in 1999, revealed nothing clearly unusual about its DNA repair components. And it appears that bacteria at the opposite ends of resistance sustain about the same amount of DNA damage from a given IR dose, with many bacterial species succumbing to IR doses that cause very little DNA damage. Shewanella oneidensis, for example, cannot survive doses causing less than one double-strand DNA break per genome although it encodes DNA repair systems that appear more complex than those in D. radiodurans, which can weather the 100 double-strand breaks per genome caused by much higher doses just fine.

Might the hypothetical genes identified in the D. radiodurans genome encode proteins with novel repair functions? Or perhaps resistant bacteria can use standard DNA repair equipment in ways other organisms cannot. Or maybe thereâs something special in the way the microbe packages its chromosomes.

A 2004 study by Michael Daly et al. found that IR-resistant and IR-sensitive cells had significantly different mineral concentrations, lending support to a role of manganese and iron in recovery. The researchers showed that the most resistant cells contained about 300 times more manganese and three times less iron than the most sensitive cells. In a new study investigating the functional consequences of this disparity, Daly et al. show that high cytosolic manganese and low iron concentrations facilitate resistance by protecting proteins, but not DNA, from IR-induced oxidative damage. Their findings offer a novel perspective on the long-cryptic nature of D. radiodurans resistance, shifting the focus of toxicity and resistance away from DNA damage and repair toward a potent form of protein protection.

Image overlay of transmission electron microscopy, light microscopy, and X-ray fluorescence microprobe analyses of D. radiodurans. Average abundance of manganese (blue, green, and pink) and iron (red) are shown within a single D. radiodurans diplococcus.

Exposing cells to IR generates a range of potentially harmful molecules called reactive oxygen species (ROS). When ROS accumulate faster than cellular scavengers can neutralize them, they cause oxidative stress and can kill cells. Hydroxyl radicals, one of the primary ROS products of irradiated water (the major component of cells), are particularly toxic to DNA, and can generate other ROS, including hydrogen peroxide and superoxide (a simple peroxyl radical).

High intracellular concentrations of manganese ions are known to alleviate oxidative stress in several bacterial species; these ions can interact with different ROS depending on their oxidation state and their binding with different molecules. Daly et al. reasoned manganese might affect ROS generation during irradiation. They first tested manganeseâs ability to scavenge hydroxyl and superoxide radicals to determine whether its activity protects DNA or proteins. Whereas hydroxyl radicals target both DNA and proteins, superoxide radicals selectively damage proteins. The researchers irradiated DNA and a DNA-modifying enzyme and found that, although manganese ions failed to protect DNA from hydroxyl radicals generated during irradiation, the ions did prevent enzyme damage and preserved enzyme activity.

To understand the nature of manganese protection in cells, the researchers then irradiated IR-sensitive and IR-resistant bacteria and compared their levels of oxidative protein damage. The sensitive cells with the lowest manganese to iron concentration ratios, they found, sustained high levels of protein oxidation; the resistant cells with the highest ratios had no detectable protein oxidation. They showed that proteins purified from D. radiodurans are not inherently oxidation-resistant, and when cells were depleted of manganese, cells were rendered sensitive to IR and protein oxidation. This suggests that the microbe actively offsets the effects of IR by protecting proteins using manganese, specifically with divalent manganese (Mn(II)) ions.

Resistant bacteria, the researchers suspected, might use Mn(II) to transform superoxide radicals, which canât easily cross the cell membrane, into hydrogen peroxide, which can. And thatâs what they found: irradiated D. radiodurans and a second resistant bacteria with high manganese concentrations (Lactobacillus plantarum) released hydrogen peroxide (likely as a product of the âredoxâ reactions that neutralize superoxide radicals), while sensitive and non-irradiated resistant bacteria did not. The researchers went on to show that the resistance of normal D. radiodurans can be controlled externally by inhibiting manganese redox recycling.

Electron micrograph of a cross-section of a D. radiodurans tetracoccus (cluster of four cells).

In the context of previous studies, these results suggest that D. radiodurans relies not on a highly specialized DNA repair machinery, but on a detoxifying mechanism associated with the microbeâs unusual intracellular environment. Most organisms contain near-millimolar concentrations of iron, which under IR will contribute to the formation of hydroxyl radicals and superoxide radicals. In resistant bacteria, millimolar Mn(II) concentrations appear to protect proteins from oxidative damage by eliminating superoxide and its derivatives. This oxidative protection may in turn shield proteins involved in DNA repair, and subsequently allow them to quickly heal DNA lesions, which in sensitive bacteria turn lethal because their repair proteins are ravaged by radiation.

This new model of radiation toxicity opens up novel avenues for radioprotection in diverse settings. Individuals exposed to chronic or acute doses of radiation could potentially benefit from treatments that deliver purified D. radiodurans Mn complexes into their cells. Similarly, the toxic effects of radiation therapy in cancer patients might be ameliorated by antioxidant drugs based on such a protection paradigm. And given that many bacteria, such as S. oneidensis, with favorable bioremediation functions are extremely sensitive to radiation, the new insight on how D. radiodurans survives radiation might prove useful in efforts to contain the toxic runoff from the immense radioactive- and heavy-metal-contaminated waste sites left over from the Cold War.

Evolution of typhoid bacteria

Wed, 29 Nov 2006 10:47:32 PST

( from http://www.rxpgnews.com ) Typhoid fever remains a major health problem in the developing world and continues to cause disease in Europe and on the american continent. The evolutionary history and population structure of Typhi were poorly understood, partly because these bacteria show little genetic diversity. Now a team led by Mark Achtman and Philippe Roumagnac from the Max Planck Institute for Infection Biology, Berlin, has applied population genetic experience from prior work with Yersinia pestis, Escherichia coli, Helicobacter pylori and Neisseria meningitidis to provide novel insights into the evolution of this pathogen.

The team combined its resources to assemble for the first time a globally representative collection of 105 strains of Typhi and investigated the sequence diversity within 90,000 base pairs per strain. Eighty-eight informative sequence differences were detected, showing that the population structure has evolved over the last 10,000 to 43,000 years. Amazingly, the ancestral strain continues to exist today, as do many of its direct descendents, indicating a neutral population structure, whereas normally selective forces lead to extinction of intermediate genotypes. Furthermore, these bacteria are distributed globally, demonstrating that Typhi has spread inter-continentally on multiple occasions.

The authors propose that the unusual population structure of Typhi reflects long-term carriage by asymptomatic carriers, who reached public notoriety at the beginning of the 20th century with "Mr. N the milker" in England and Typhoid Mary (Mary Mallon) in the U.S.A. These individuals infected 100s of people over the decades while they worked in the food production industry. Healthy carriers may have allowed Typhi to survive in hunter-gatherer populations prior to the Neolithic expansion of city states and facilitated its intercontinental spread. Healthy carriers are also consistent with the observation that individual genotypes of Typhi persist for many decades within each country.

Increasing resistance to antibiotics in recent decades has hampered efforts of clinicians to cure typhoid fever. The indiscriminate use of fluoroquinolones, which is a cost-effective, standard treatment for typhoid fever, has been accompanied by a frightening increase in the numbers of resistant Typhi. Investigations of a large strain collection from southern Asia revealed that many different genotypes independently acquired resistance to nalidixic acid, a quinolone. One of these genotypes, H58, has become predominant throughout southern Asia and has even spread to Africa. In Vietnam, up to 95% of Typhi are now resistant to nalidixic acid and many other antibiotics. Although these cases can still be treated with newer antibiotics, those antibiotics are much more expensive than standard fluoroquinolones, which raises the cost of medical treatment. Furthermore, it is likely that Typhi will develop resistance to these antibiotics as well.

The combination of these investigations raises problems for public health measures. Indiscriminate antibiotic usage results in real-time evolution of bacteria that resist treatment. Furthermore, the healthy carrier state provides a safe reservoir for these bacteria which allows them to evade short-term antibiotic treatment and vaccination, indicating that typhoid fever will remain a major health problem for the foreseeable future.

New Treatment Using Human Antibodies to Target Harmful Toxins May Protect Against C. Difficile

Sun, 19 Nov 2006 04:22:43 PST

( from http://www.rxpgnews.com ) A new therapeutic method using human antibodies to neutralize toxins was found to prevent Clostridium difficile-induced death in hamsters say researchers from New Jersey and Massachusetts. They report their findings in the November 2006 issue of the journal Infection and Immunity.

C. difficile is the leading cause of nosocomial antibiotic-associated diarrhea, often resulting from the administration of antibiotics such as clindamycin, ampicillin, or cephalosporins. C. difficile associated diarrhea (CDAD) effects approximately 300,000 patients per year in the U.S. alone. Treatment available to date includes discontinuation of the antibiotic causing the illness as well as administration of medication such as metronidazole or vancomycin. Although both methods offer initial relief, there is currently a 10 to 20% relapse rate among patients. Due to the recent emergence of more virulent C. difficile strains, in addition to increasing vancomycin resistance, researchers are focusing on new treatments and relapse prevention therapy.

In the study mice were used to isolate human monoclonal antibodies (HuMAbs) capable of neutralizing C. difficile toxins A and B. Researchers then tested anti-toxin A HuMAb CDA1 alone and in conjunction with anti-toxin B HUMAb MDX-1388 for the ability to protect hamsters from C.difficle-induced death and relapse prevention. Results showed that combination therapy reduced mortality from 100% to 45% in the primary disease model and from 78% to 32% in the relapse model.

"These human and animal studies, taken together, demonstrate the relevance of toxin-reactive antibodies in disease outcomes," say the researchers. "Here we describe the characterization of a panel of neutralizing, fully human monoclonal antibodies (HuMAbs) directed against either toxin A or toxin B. HuMAb CDA1 (against toxin A) alone could protect hamsters from mortality, but significantly enhanced protection was observed when the antibodies were administered as a combination therapy."

Guinea Pig Aerosol Challenge Presents New Model for Q Fever Research in Humans

Sun, 19 Nov 2006 04:20:32 PST

( from http://www.rxpgnews.com ) Clinical signs and pathological changes in guinea pigs following an aerosol challenge with acute Q fever were similar to those seen in human acute Q fever indicating an effective animal model of human disease say researchers from Texas A&M University. They report their findings in the November issue of the journal Infection and Immunity.

Q fever, caused by the bacterium Coxiella burnetti, generally infects humans through inhalation with as few as 10 organisms capable of causing disease. C. burnetti has a high degree of resistance to treatment agents and can remain infectious in contaminated soils for years. Due to its highly infectious nature, the Centers for Disease Control and Prevention has listed C. burnetti as a potential weapon of mass destruction reinforcing the need for a safe and effective vaccine. There is currently no licensed vaccine available in the U.S.

In the study select guinea pigs received a killed whole-cell Q fever vaccine after which all were infected with C. burnetti through inhalation of small-particle aerosols and evaluated 28 days postinfection. Noted clinical signs included fever, weight loss, respiratory difficulty and death with the degree and duration of response correlating with the dose of organism delivered. Those guinea pigs vaccinated prior to challenge with the highest dose of C. burnetti did not develop fever and were protected against lethal infection.

"The guinea pig aerosol challenge model presented here mimics both the clinical and pathologic changes seen in human acute Q fever and Q fever pneumonia cases and will provide an accurate and valuable tool for the study of the general pathogenesis of C. burnetti infection, for vaccine assessment, and for evaluations of host immune responses," say the researchers.

Gut Bacteria Cospeciating with Plataspid stinkbug

Wed, 11 Oct 2006 04:56:37 PST

( from http://www.rxpgnews.com ) With some 1 million species and counting, insects may be the most abundant class of animals living today. Their protective exoskeleton, prolific reproductive rate, and wings help their cause, as do the symbiotic bacteria that inhabit their cells, gut, or body cavity. Endocellular symbionts live inside specialized insect cells and provide essential nutrients for their hosts, which in turn provide suitable habitat for the bacteria. Insect mothers transmit endocellular symbionts to their offspring during egg or embryo development, preserving an intimate bond between host and symbiont that is evident in both species' genomes.

Studies that use genome analysis to infer evolutionary relationships (called phylogenetics) show that the history of insect host genes (or phylogeny) often mirrors that of their endocellular symbiontindicating a shared evolutionary history, or cospeciation. Unlike endocellular symbionts, gut or body cavity symbionts are vulnerable to displacement or attack by other microbes and appear to have less-exclusive relationships with their hosts, based on reports that hostsymbiont phylogenies for termites and alydid stinkbugs do not match. But a new study suggests that not all gut symbionts go for the promiscuous lifestyle. Takahiro Hosokawa, Takema Fukatsu, and colleagues provide the first evidence of cospeciation between a group of gut symbionts and their insect hosts, plataspid stinkbugs. Not only do their phylogenies mirror each other, but the gut symbionts share many of the unique genetic traits typical of endocellular symbionts.

A mating pair of the Japanese common plataspid stinkbug Megacopta punctatissima (Image: PLoS Biology)

Plataspid stinkbug symbionts live in the bugs' posterior midgut and are vertically transmitted by the mother in symbiont capsules. When the female lays eggs, small, brown symbiont-filled capsules always appear under the egg mass. Nymph hatchlings ingest symbionts from the capsule.

Hosokawa et al. collected 12 populations of stinkbugs, representing three genera and seven species, from several locations in Japan. (Four species were used in the experiments.) All females had the same three-compartment midgut, which had been previously described in two other species: one section contains the symbionts (called the thin crypt-bearing midgut, or TCM), another secretes webbing that embeds the symbionts into the capsules, and a third produces the shell that encases the capsule. All the females also codeposited capsules and egg masses. (Males have only the TCM.)

After removing the TCM from adult females, the researchers analyzed the DNA of the resident bacteriafocusing on a ribosomal RNA gene called 16S rRNA often used to identify bacteriaand found that each bacterial species was associated with a different stinkbug species. Using the 16S rRNA sequences to infer the bacteria's evolutionary origins, they discovered that the sequences didn't match any other bacterial sequences in the databasesthey fell into their own class of Proteobacteria. Interestingly, however, the symbionts did form a sister groupindicating evolutionary kinshipwith the well-characterized obligate endosymbiont (Buchnera aphidocola) of aphids.

Given the phylogenetic similarity between the stinkbug symbionts and Buchnera, the researchers wondered whether their biology might be similar as well. They divided egg masses into two groups and deprived one group of capsules to generate sibling populations with and without gut symbionts. Adults lacking symbionts showed developmental delays, grew smaller, failed to copulate or reproduce, and died prematurely. Like aphids depend on their endosymbionts, plataspid stinkbugs depend on their gut symbionts to survivehow they do this, however, will be interesting to discover. Like Buchnera, the gut endosymbionts also appear to have co-evolved with their host. The phylogenetic tree of the stinkbugs, the researchers found, perfectly agreed with the phylogenetic relationships of the gut symbionts. Maternal transmission of the symbiont capsule provides a means of stable transmission, but other factors such as physiological compatibility may come into play.

The symbiotic lifestyle appears to have shaped the genome evolution of endocellular symbionts, which have a small genome, a high percentage of A and T nucleotides in their DNA, and accelerated molecular evolution. Whether these genetic traits arose from population genetic forcesfor example, small population size and bottlenecksor from some aspect of the endocellular environment has been a matter of dispute. Hosokawa et al. found the same peculiar genetic patterns in the gut symbionts, lending support to the population genetic hypothesis. They named these gut symbionts Candidatus Ishikawaella capsulata, in honor of Hajime Ishikawa, a pioneer in the molecular study of symbiosis, who recently passed away.

How the symbiont capsule evolved remains an open, and intriguing, question. With some 530 species and 56 genera in the Plataspidae family, researchers have their work cut out for them as they survey the lineages for a stinkbug without a capsule. But with this unique plataspid stinkbug system, they will be well equipped to study insect symbiosis and its influence on genome evolution.

How West Nile virus evades immune defenses

Thu, 05 Oct 2006 01:04:37 PST

( from http://www.rxpgnews.com ) West Nile virus evades the body's immune defenses by blocking immune signaling by a protein receptor, a finding that could pave the way for a vaccine to protect against North American strains of the virus, UT Southwestern Medical Center researchers report.

Researchers discovered the receptor's key role in controlling West Nile infection by conducting a study, described in October's Journal of Virology, that compares the genetics of an illness-causing Texas strain of the virus to a harmless African strain.

The Texas strain can inflict illness because it blocks the signaling activity of a protein receptor called the interferon alpha/beta receptor, or IFNAR, disrupting a cell's ability to direct the immune system to fight off the virus.

The African strain does not block IFNAR activity, so the immune system renders it harmless. The strain is harmful, however, in mice with dysfunctional receptors.

"We now hope to harness the African strain as the basis for West Nile vaccine studies. The virus has spread across the country and infected more than 2,100 U.S. residents 180 in Texas this year alone, so we have to learn how to deal with it," said Dr. Michael Gale, associate professor of microbiology at UT Southwestern and director of the study. Brian Keller, a student in the Medical Scientist Training Program at UT Southwestern, is the first author of the study.

West Nile virus, which is transmitted by mosquito bite, arrived in the United States in 1999 and has become an epidemic that flares up in the summer and lasts into fall.

Infection causes mild flu-like symptoms in most people, but about one in every 150 develop serious illness, that can include high fever, coma, seizures and encephalitis and meningitis. Children, the elderly or people with weak immune systems are most at risk.

There is no vaccine. Doctors can only treat symptoms of the disease.

Searching for clues that might allow development of a vaccine, Dr. Gale and his research team compared one strain from each of West Nile's two basic categories: the harmful strains associated with outbreaks of encephalitis and meningitis in North America, and non-harmful strains from Madagascar and Cyprus.

They studied a harmful strain isolated from an infected grackle from Hall County, Texas, in 2002, and a harmless strain isolated from an infected parrot from Madagascar in 1978.

They mapped the genetic makeup of each strain, and then tested the viruses in mice.

West Nile infection triggers production of interferon, a group of proteins that are crucial in immune defense. Interferon, which binds to IFNAR, subsequently signals the JAK-STAT molecular pathway, a series of biochemical reactions essential for turning on immune-defense genes, allowing the body to clear out the virus. This process occurs normally in the African strain.

Infection by the Texas strain, however, blocked IFNAR signaling activity, allowing the virus to replicate and spread.

This highlights the integral role of interferon and IFNAR signaling in innate immunity.

Dr. Gale said the mechanisms at work in the African strain could be used as a basis for a vaccine, perhaps mutating North American strains so they no longer disrupt immune signaling. The remaining key is figuring out the exact mechanics of how the strains block signaling, a project Dr. Gale's team is already at work on.

Fortunately, North American strains are extremely similar in fact, the one that appeared in the United States in 1999 and the Texas strain used in this study are 99 percent identical. One vaccine could, in theory, prevent illness from many of the harmful strains, Dr. Gale said.

"We feel a vaccine could be highly effective in preventing infection," said Dr. Gale.

An infectious agent of deception, exposed through proteomics

Sun, 01 Oct 2006 22:55:37 PST

( from http://www.rxpgnews.com ) Salmonella bacteria, infamous for food poisoning that kills hundreds of thousands worldwide, infect by stealth. They slip unnoticed into and multiply inside macrophages, the very immune system cells the body relies on to seek and destroy invading microbes.

Just how Salmonella escapes detection by macrophages, turning predator cells to prey complicit in promoting infection, has seemed impossibly complicated, a needle-in-a-haystack proposition involving thousands of proteins, the building blocks that carry out cells' vital functions.

Applying the high-volume sorting and analytical power of proteomics--a detailed survey of microbial proteins present in the 24 hours that follow mouse-macrophage infection--a team led by Liang Shi of the Department of Energy's Pacific Northwest National Laboratory has turned up a suspect protein.

PNNL scientists have identified a protein in Salmonella bacteria that enables it to infect immune cells called macrophages. Seen here: Salmonella, isolated from infected macrophrages. (Mildly color-enhanced. Photo credit: Pacific Northwest National Laboratory.)

The discovery of the protein, dubbed STM3117, is detailed today (Sept. 29) in The Journal of Biological Chemistry. Knocking out the gene that codes for STM3117, the researchers subsequently crippled the microbe's ability to multiply inside macrophages. Shi and colleagues say the protein and two closely related proteins discovered in the study are similar in genetic sequence to those known to make and modify chemicals in the microbe's cell wall called peptidoglycan.

Drug and vaccine designers armed with this mouse-model information can target chemicals or immune responses that disrupt peptidoglycan synthesis and other processes linked to Salmonella's colonization of macrophages in humans, said Joshua Adkins, a co-author on Shi's paper and lead author of a related study in Molecular & Cellular Proteomics last month. A quick identification of these proteins, Adkins added, could help physicians assess the virulence of a given strain.

The candidate proteins were winnowed from among 315 possibilities that emerged through a combination of techniques, culminating in measurements by Fourier-transform mass spectrometry, or FT-MS. A suite of FT-MS instruments customized by co-author and PNNL-based Battelle Fellow Richard D. Smith enabled the team to rapidly separate and identify many proteins at once even as macrophages were being infected.

Most of the initial candidates were designated "house-keeping" proteins, or those whose numbers relative to other proteins remained more or less constant during the course of infection. But 39 proteins shot up in number during bacterial colonization of macrophages, and of those, a handful or so--including STM3117--responded specifically to a macrophage protein associated with resistance to microbial infection. A standard assay called Western blot confirmed the abundance increases of that small group of proteins during infection.

Gram positive bacterial membrane mystery solved

Fri, 01 Sep 2006 17:55:37 PST

( from http://www.rxpgnews.com ) A 25-year quest to identify the first biochemical step that many disease-causing bacteria use to build their membranes has led to a discovery that holds promise for effective, new antibiotics against these bacteria, according to investigators at St. Jude Children's Research Hospital. The finding is significant because the biochemical step the antibiotic would block is not used by humans. Therefore, such a drug would not cause dangerous side effects.

A report on this finding appears in the September 1 issue of Molecular Cell.

The discovery also demonstrated that current textbooks use the wrong type of bacterium as a model to explain a critical biochemical step that most disease-causing bacteria use to make their membranes, according to Charles Rock, Ph.D., a member of the St. Jude Department of Infectious Diseases and senior author of the paper. As bacteria grow in size or divide, they must make additional membrane using a series of biochemical reactions. The first step in this process is the transfer of a fatty acid to a molecule called G3P. Bacteria then convert this molecule into a variety of other molecules called phospholipids, which are the building blocks of membranes.

"We identified a biochemical process that uses a previously unrecognized molecule as a raw material to make phospholipid," Rock said. "That discovery solved a mystery that has puzzled researchers for 25 years."

Scientists have used E. coli bacteria for many years as a model to understand how disease-causing bacteria make membrane phospholipids, but E. coli is an unsuitable model for most pathogens (disease-causing bacteria), according to Rock.

First, E. coli is a so-called gram-negative bacterium, while many of the pathogens researchers are interested in are gram-positive, Rock noted. Among those gram-positive organisms are Staphylococcus aureus, which causes skin infections and serious blood infections, and Streptococcus pneumoniae, which causes pneumonia. The terms "gram-positive" and "gram-negative" refer to the response of bacteria to a standard laboratory process by which they are stained as a first step in identification.

Laboratory strains of E. coli do not cause disease; and the enzyme E. coli uses during the first step in making membranes does not exist in most other bacteria, including gram-positive pathogens. Therefore, the way gram-positive bacteria make phospholipid building blocks remained a mystery for over more than two decades. Now, however, the St. Jude team reports that the gram-positive pathogens use two enzymes, called PlsX and PlsY, to kick off phospholipid synthesis.

"In fact, the biochemical pathway that uses PlsX and PlsY is the most widely distributed bacterial pathway for initiating the production of phospholipids," explained the study's first author, Ying-Jie Lu, Ph.D., of the St. Jude Department of Infectious Diseases. "It turns out that E. coli is more of an oddball rather than in the mainstream when it comes to how it makes membranes."

E. coli fuses a molecule called G3P with a fatty acid in a single step. Rock's team showed that gram-positive pathogens first use PlsX to synthesize a compound called fatty acyl-phosphate, then use PlsY to transfer the fatty acid to G3P. These steps initiate membrane phospholipid formation required for cell growth.

"Our discovery of PlsX and PlsY not only solved a troublesome mystery," Rock said. "It's also important because identifying the essential components required for disease-causing bacteria to grow and multiply is a key part of developing new strategies for controlling infections."

E.Coli uses 'shock absorbers' to combat adverse conditions

Tue, 29 Aug 2006 20:53:37 PST

( from http://www.rxpgnews.com ) Bacteria have hair-like protrusions with a sticky protein on the tip that lets them cling to surfaces. The coiled, bungee cord-like structure of the protrusions helps the bacteria hang on tightly, even under rough fluid flow inside the body, researchers report in the journal PLoS Biology.

A group of researchers at the University of Washington in Seattle and ETH Zurich in Switzerland have been studying how the bacterium E. coli attaches to mucous membranes in the body. In their previous research, they explained how the protrusions, known as fimbriae, have an adhesive protein called FimH at their tip that binds in an unusual way to a sugar molecule on a surface.

The FimH-sugar combination makes a "catch bond" that acts like a finger trap, and actually gets stronger as drag force is exerted on a bacterium. Rather than being swept away by fluids moving through the human body, the bacterium grips even more tightly, helping it stick around and form an infection, like those seen in the urinary tract, for instance. The catch bonds release their grip when there is little or no force on the bacteria.

In new research, the scientists have learned that the mechanical properties of the bungee-like fimbriae also play a key role in the tenacity of E. coli clinging to mucousal surfaces. The tiny fiber-like protrusions are made up of interlocking protein segments in a tightly coiled helix shape, like a seven-nanometer-wide Slinky toy. The researchers found that under force, the fimbriae stretch to many times their original length as the protein segments uncoil one by one. If the force on them drops, the fimbriae coils are compressed, keeping tension on the bond between the bacterium and the mucous membrane.

"The fimbriae uncoil and recoil to dampen sudden changes in forces caused by rough and rapidly changing flow conditions," explained study co-author Dr. Viola Vogel, professor in the Department of Materials at ETH Zurich. This process maintains an optimal force required to keep the finger trap-like FimH anchor from breaking loose.

"This system is similar to a set of shock absorbers on your car that dampen turbulent forces caused by bumpy road conditions," added study co-author Dr. Wendy Thomas, assistant professor of bioengineering at the UW.

The researchers found that fimbrial uncoiling and recoiling events balance each other at an intermediate force level that corresponds to the force at which the sticky protein tip forms the most stable bond with the surface. Thus, the mechanical and adhesive features of the system evolved together to help the bacteria persist in tough environments inside a host animal or person.

"Research on these fimbriae uncovers something that's essentially a mechanical nanotechnological device created by nature, and gives us the opportunity to adapt such a system for biotechnological and even other technical uses," said study co-author Dr. Evgeni Sokurenko, associate professor of microbiology at the UW and a principal investigator at the NIH Bioengineering Research Partnership that primarily funded the project. "It also improves our understanding of how to fight bacteria that persist in turbulent environments, like the human urinary tract or intestines."

Innovative method for creating a human cytomegalovirus vaccine outlined

Wed, 02 Aug 2006 11:42:37 PST

( from http://www.rxpgnews.com ) Each year, about 40,000 children are born infected with human cytomegalovirus, or CMV, and about 8,000 of these children suffer permanent disabilities due to the virus almost one an hour. These disabilities can include hearing loss, vision loss, mental disability, a lack of coordination, and seizures. According to the Centers for Disease Control and Prevention, CMV is as common a cause of serious disability as Down syndrome, fetal alcohol syndrome, or neural tube defects.

Because of the dangers posed by the virus to infants, the Institute of Medicine has declared that development of a CMV vaccine should be one of the highest priorities for vaccine makers. Now, in a new study in the August 1 issue of The Journal of Virology, researchers at The Wistar Institute outline an innovative approach that could be used to create such a vaccine.

The Wistar scientists began with the observation that mice harbor a species-specific form of CMV that is unable to sustain an infection in humans and is completely harmless to them. They then asked whether, using recombinant technologies, there might not be a way to shift the mouse-specific virus closer to the human-specific virus to generate a version of the virus able to elicit a protective immune response but not a dangerous infection in humans.

With this goal, the researchers began to systematically introduce selected genes from human CMV into the genome of mouse CMV in the laboratory. The result was a novel form of CMV virus that infected human cells well enough that it might trigger an immune response but not well enough to sustain an infection.

"It should be possible to develop a safe and effective CMV vaccine using the method we've described in our study," says Gerd G. Maul, Ph.D., a professor in the Gene Expression and Regulation Program at Wistar and senior author on the new study. "Success will depend on achieving a certain balance between the immune-stimulating genes from the human virus and the basic safety of the mouse virus."

Over the years, a number of scientists have worked to create a vaccine against human CMV. Among these is former Wistar researcher Stanley A. Plotkin, M.D., who during his career at the Institute between 1960 and 1991 developed the rubella vaccine that eradicated the disease in the U.S. and co-developed a new rotavirus vaccine approved in the U.S. in 2006. Plotkin's approach to developing a CMV vaccine was to attenuate, or weaken, human CMV, as he had done to create the rubella vaccine. In contrast, the rotavirus vaccine was developed using co-infection techniques to incorporate selected elements from several human rotavirus strains into a bovine rotavirus backbone. The vaccine that resulted takes advantage of the immunological characteristics of both the human and bovine viruses. This approach is conceptually similar to Maul's strategy for developing a CMV vaccine, although the recombinant technologies available today should help to streamline the task.

CMV infection is widespread in the U.S., according to the Centers for Disease Control and Prevention. Between 50 and 80 percent of adults are infected with CMV by the time they reach 40 years of age, but the virus generally poses little threat to them. The exceptions are AIDS patients, transplant recipients, and others with compromised immune systems are at risk for complications. The virus is a member of the herpes virus family, which includes the herpes simplex viruses, the viruses that cause chicken pox (varicella-zoster virus), and infectious mononucleosis (Epstein-Barr virus.) Infectious transmission is via body fluids.

Cracking Virus Protection Shield

Mon, 19 Jun 2006 02:17:37 PST

( from http://www.rxpgnews.com ) Ebola, measles and rabies are serious threats to public health in developing countries. Despite different symptoms all of the diseases are caused by the same class of viruses that unlike most other living beings carry their genetic information on a single RNA molecule instead of a double strand of DNA. Now researchers from the Institut de Virologie Moléculaire et Structurale [IVMS] and the Outstation of the European Molecular Biology Laboratory [EMBL] in Grenoble have obtained a detailed structural picture of a protein that allows the rabies virus to withstand the human immune response and survive and replicate in our cells. The study that is published in this week's online edition of Science suggests new potential drug targets in rabies and sheds light on how similar approaches can help fighting other viral diseases.

When the rabies virus enters a human cell through the membrane, the RNA molecule that carries its genes is transported into the centre of the cell. Here it redirects the cellular machinery of the host to produce many new copies of the virus that go on to infect more cells. One molecule that is crucial in this process is a viral protein called nucleoprotein. The protein ensures that on its way through the cell the virus RNA is not destroyed by the immune response of the host.

"Nucleoprotein is vital for the rabies virus," says Rob Ruigrok, Head of the IVMS. "It is one of the few proteins that the virus brings into the host cell and it wraps around the RNA like a protection shield. Without this shield the RNA would be degraded by the enzymes of the human immune system that try to eliminate the invader."

To investigate how exactly this protection shield works, Aurélie Albertini from Ruigrok's team obtained crystals of nucleoprotein bound to RNA. Examining the crystals with high-intensity X-ray sources at the European Synchrotron Radiation Facility [ESRF], Amy Wernimont from Winfried Weissenhorn's group at EMBL Grenoble produced a high-resolution image of the protein.

"Nucleoprotein acts like a clamp," says Weissenhorn. "It consists of two domains that like two jaws clasp around the RNA strand. Many nucleoproteins bind side-by-side along the length of an RNA molecule and make it inaccessible for degrading enzymes but also for the machinery needed to replicate the virus. This means that the protection shield must be flexible and able to distinguish between different types of enzymes trying to gain access."

The detailed structural picture suggests that upon a signal a part of the protein located between the two main domains might act as a hinge that moves the upper jaw out of the way when time for replication has come.

"This dynamic mechanism makes nucleoproteins an excellent drug target," says Ruigrok, "Small agents that bind to the protein in such a way to block its flexibility and keep it in the closed state, would prevent replication of the virus and would stop it from spreading."

Rabies virus shares this protection strategy with other viruses of its class; in Ebola, measles and Borna virus similar complexes of RNA and nucleoproteins have been found.

"This means that our results do not only have implications for the design of new drugs against rabies, but they suggest new therapeutic approaches in a variety of diseases, some of which are much more threatening than rabies. On a different note, the conservation of the nucleoprotein system also leaves room for evolutionary speculations about common ancestors and primordial infectious units of RNA viruses," Weissenhorn concludes.

Viruses trade-off between survival and reproduction

Thu, 15 Jun 2006 12:08:37 PST

( from http://www.rxpgnews.com ) Living is an energy-intensive exercise that inevitably involves trade-offs. As many a mother may tell you, expending the energy necessary to raise a clutch of kids can shave years off one's life. Trade-offs between reproductive success and survival have been demonstrated for a wide variety of organisms and, in keeping with life history theory, should arise in any organism striving to maximize fitness under the constraints of finite resources.

In a new study, Marianne De Paepe and François Taddei asked whether these trade-offs extend to viruses. Though not universally considered alive because they can't replicate without the help of their host's molecular machinery, viruses pass through distinct life cycle stages, mutate, and evolve in response to selection pressures from their host. Viruses also have life history traits, such as multiplication rate in a host, survival outside the host, and mode of transmission.

Working with viruses that infect bacteria, called bacteriophages (or in this case, coliphages, which infect Escherichia coli), De Paepe and Taddei predicted that the phage, just like a full-fledged cellular organism, would display trade-offs between survival and reproduction. They discovered that, although coliphages don't wither and die like real organisms, they do experience life history trade-offs, with rapid reproducers suffering higher casualties outside the host. And, by investigating several physical properties of the coliphages, they found that two physical parameters account for most of the observed variation in survival.

During infection, phages rapidly reproduce until the bacterial cell ruptures (called lysis) and releases the virions (viral particles, which consist of little more than the viral genome encased in a capsid protein shell). A phage typically meets its end after encountering harsh conditions, such as heat or osmotic shock, that rupture the capsid, releasing its genetic contents. How well the phage can resist such stresses depends in part on the strength of the capsid, which must withstand the extreme pressure exerted by strong repulsive forces between charged DNA strands and bending of the tightly packed DNA.

To investigate the presence of life history trade-offs in phages, De Paepe and Taddei measured the decay rate (mortality) and multiplication rate of 16 coliphage strains. They determined the kinetics of mortality within and across the phage populations, by measuring the number of phage particles that completed an infectious cycle in E. coli cultures, at different times and temperatures. Although all the phages died at a constant rate over time, different strains showed considerable variation in these rates. The constant rate (meaning that the probability of death did not increase over time) suggests that the phages succumbed to a single eventindicating that they don't agerather than to a series of events, which would be consistent with aging. Decay rates increase exponentially with the inverse of the temperature, which is a characteristic of very simple chemical reactions.

To determine the factors underlying this variation, the researchers investigated the correlation between several physical parameters and decay rate. Among the parameters measured were two factors governing capsid stabilitydensity of the packaged genome and surfacic mass of the capsid, an indicator of capsid thicknessand two indicators of replication rateburst size (number of particles released during an infection cycle) and latency period (time between infection and host lysis). They found that decay rate increases significantly with the density of the packaged DNA, linking higher internal pressure with higher mortality. By contrast, decay rate decreases as surfacic mass increases, supporting the notion that a stronger capsid increases phage stability. The highest correlation was between decay rate and multiplication rate in the bacterial host. (Multiplication rate was determined by dividing burst size by latency period.) A model based on these three variablespackaged DNA density, surfacic mass, and multiplication rateaccounts for over 90% of the observed variability in phage mortality rates.

Even though they don't have their own metabolism, viruses experience the same sorts of trade-offs between survival and reproduction seen in a wide range of species. This finding suggests that models of virulence evolution, which assume that transmission rates increase along with virulence, may not be valid, since transmission depends not just on parasite multiplication rate but also on survivalwhich, they show, are negatively correlated. The fact that this trade-off is present in this very simple biological situation, the researchers write, suggests that it might be a fundamental property of evolving entities produced under constraints. If this is true, the nonliving phages that opened the door to some of the most important discoveries in molecular biology may well provide a similar service for a wide range of evolutionary phenomena.

Smart Petri Dish could rapidly screen new drugs for toxic interactions

Thu, 15 Jun 2006 11:40:37 PST

( from http://www.rxpgnews.com ) Researchers at the University of California, San Diego have developed what they call a Smart Petri Dish that could be used to rapidly screen new drugs for toxic interactions or identify cells in the early stages of cancer circulating through a patients blood.

Their invention, described in the June 20 issue of Langmuir, a physical chemistry journal published by the American Chemical Society, uses porous silicon crystals filled with polystyrene to detect subtle changes in the sizes and shapes of the cells.

One of the big concerns with any potential new drug is its toxicity, says Michael Sailor, a professor of chemistry at biochemistry at UCSD who headed the research team. Since the liver is the organ that cleans up the blood, liver cells are particularly susceptible when a toxin is introduced to the body. Pharmaceutical companies want to know early on the effect a drug has on the liver. But its very expensive to screen every potential candidate on living animals, typically rats. So if you can use just a few cells from the liver rather than the entire animal, you can perform many more thorough tests.

You could also in principle use this to identify metastatic cancer cells circulating in a patient's blood, Sailor adds, by putting blood samples from a patient onto the crystal and comparing them to normal blood samples.

In addition, says Michael Schwartz, a postdoctoral scholar in Sailor's laboratory and the first author of the paper: The potential of our technique for fundamental studies of cell toxicity is exciting, Since we can monitor cells in real time without removing them from their natural environment, the observed changes provide a time course for performing more detailed tests to find out why drugs are toxic.

The scientists constructed their Smart Petri Dish by first fabricating silicon crystals with nanometer-sized holes. This enabled them to produce a photonic crystal, capable of controlling light within the structure analogous to the way that semiconductors transmit electricity through computer chips. By attaching rat liver cells to the polystyrene within the crystals and measuring the scattering of light with a sensitive spectrometer, they were able to detect small changes in the shapes of the cells as they reacted to toxic doses of cadmium chloride and acetaminophen.

As these cells shrivel up in response to a toxin, they scatter light better, much like fog on a car windshield, allowing us to quicklydetect which drugs may have adverse side effects when taken in combination with another, says Sailor. Youre not supposed to drink alcohol when taking acetaminophen, because the combination of the two is much more toxic to your liver than either drug individually. This is known as an adverse drug-drug interaction and it is very expensive and time-consuming to screen a new drug candidate with all the possible combinations of drugs that a patient may be taking. The Smart Petri Dish allows us to perform a large number of such toxicity assays simultaneously, in order to provide an early indication of the particular physiological or pharmacological conditions that need more in-depth study.

Although we performed these experiments on rat cells, this technology can be easily extended to human cells, says Sangeeta Bhatia, a professor of bioengineering at UCSD now at MIT, who also participated in the study. This is important because we know that the enzymes that metabolize drugsthe P450 familyare very different in animal and humans. This is one of the reasons many drugs clear animal testing but end up toxic in patients. This type of sensor could help us predict human liver responses without patient exposure.

Because the Smart Petri Dish gives a continuous readout of cell damage, she adds, this type of sensor could also be very useful for understanding more about the way environmental toxicants such as water contaminants or viruses like hepatitis cause long-term liver damage.

Light scattering off liver cells on photonic crystal allows scientists to determine small changes in shapes of cells. Credit: Michael Schwartz, UCSD

Others involved in the development include Sara Alvarez, a graduate student in Sailors laboratory, and Austin Derfus, a graduate student of engineering in Bhatias former UCSD laboratory. UCSD has filed several patent applications on the device, which is now in the process of being commercialized by the Hitachi Chemical Research Center in Irvine, Ca.

The design of the new device builds on a previous development in the UCSD laboratories of Sailor and Bhatia that allowed the scientists to maintain fully functioning liver cells in culture. While many cell types can be easily grown in culture dishes, normal liver cells are much more discriminating and quickly die when removed from the body.

But by designing a porous silicon chip with miniature wells similar to those in muffin tins, the UCSD researchers were able to mimic the extracellular matrix of the liver and keep the liver cells alive. On this chip, individual cells are contained within well-like structures, 2 to 1,500 nanometers in diameter, or no wider than a human hair, that promote the flow of nutrients and chemicals through the cell culture and filter out larger particles such as bacteria and viruses. This design effectively persuades the cells to behave collectively the way they do in a fully functioning liver.

The scientists write in their paper that in their experiments the Smart Petri Dish was able to detect changes in the cells exposed to toxins before traditional assays are able to detect a decrease in viability, demonstrating the potential of the technique as a complementary tool for cell viability studies. In addition, they add, their method is noninvasive and can be performed in real time, representing a significant advantage compared to other techniques for in vitro monitoring of cell morphology, that is, for monitoring cells in the laboratory, outside of humans or animals.

Master key to yeasts' pathogenic lifestyles discovered

Fri, 28 Apr 2006 13:44:37 PST

( from http://www.rxpgnews.com ) For some microbes, the transformation from a benign lifestyle in the soil to that of a potentially deadly human pathogen is just a breath away.

Inhaled into the lungs of a mammal, spores from a class of six related soil molds found around the world encounter a new, warmer environment. And as soon as they do, they rapidly shift gears and assume the guise of pathogenic yeast, causing such serious and sometimes deadly afflictions as blastomycosis and histoplasmosis.

But how these usually bucolic fungi undergo such a transformation to become serious pathogens has always been a puzzle. Now, however, a team of scientists from the University of Wisconsin School of Medicine and Public Health reports the discovery of a master molecular sensor embedded in the spores of the fungi that triggers the transformation. The finding is reported in the April 28 edition of the journal Science.

The discovery could lead to new treatments, and possibly vaccines for the diseases caused by these Jekyll and Hyde microbes, says Bruce Klein, a UW-Madison professor of pediatrics, internal medicine and medical microbiology and immunology, and the senior author of the new study.

"These microbes have to undergo an extreme makeover to survive in a host," says Klein, an authority on fungal diseases. "The million dollar question is was what controls this change? "

Klein and colleagues Julie C. Nemecek and Marcel Wuthrich identified a molecular sensor that is conserved in these six related dimorphic fungi found worldwide. The sensor, says Klein, is like an antenna situated in the membrane of the fungi's spores. It senses temperature, and when a spore finds itself at a comfortable 37 degrees Celsius, the body temperature of a human or other animal, it kick starts a genetic program that transforms the fungi into pathogenic yeasts.

"This is a global regulator that sends signals down a molecular chain of command and governs a series of vital genetic programs," Klein explains. "It leads to changes in the organism's metabolism, cell shape, cell wall composition, and changes in virulence gene expressions."

These changes, according to Klein, are really a survival program for the microbe, conferring resistance to the host's immune responses.

The diseases caused by the fungi can be especially serious for immune compromised individuals, and some human populations seem to be more at risk for acquiring the infections. For example, U.S. soldiers who train in the American Southwest tend to be susceptible to coccidiomycosis because the organism that causes it is endemic to the region. One in three of those who train there acquire the disease, considered to be the second most common fungal infection in the United States. Of those infected, 25 percent contract pneumonia.

Histoplasmosis, a disease caused by the fungus Histoplasma capsulatum, infects as much as 80 percent of the population where the organism is endemic, including much of the eastern and central United States. It is also widespread in South America and Africa. In most instances, the infection prompts only mild symptoms. Untreated, however, it can be fatal. What's more, the microbe can lay dormant in an infected host for years.

"All of these organisms exhibit this property of latency," says Klein. "They can remain dormant until immune defenses are lowered. It's a significant medical problem in endemic regions."

The discovery of the switch that governs dimorphism and virulence in this prevalent class of fungi provides a target for new therapeutic agents and might even help underpin a vaccine able to thwart infection entirely, according to Klein.

"This could lead to therapeutics, better treatment for this class of diseases," Klein explains. "And with this finding, vaccines might now be possible. That's a strategy with promise."

The discovery of a master switch in related but diverse and geographically widespread class of fungi is an indication that it was acquired from a common ancestor deep in evolutionary history. The feature is a common mechanism used by the different organisms to adapt to a new environment: the lungs of animals.

"It is a story of how organisms are challenged in a new environment," says Klein. "They have to make themselves over so they can survive."

New hybrid virus provides targeted molecular imaging of cancer

Sat, 22 Apr 2006 19:31:37 PST

( from http://www.rxpgnews.com ) Researchers at The University of Texas M. D. Anderson Cancer Center have created a new class of hybrid virus and demonstrated its ability to find, highlight, and deliver genes to tumors in mice.

Researchers say the advance, reported in the journal Cell, is potentially an important step in making human cancer both more visible and accessible to treatment; it may also allow prediction and monitoring of how specific anti-cancer agents are actually working.

"In tumor-bearing mice, we show that this hybrid virus can target tumors systemically to deliver an imaging or therapeutic gene," says the co-leader of the study, Renata Pasqualini, Ph.D., professor of Medicine and Cancer Biology. "The signal is specific only to tumors, so one can monitor drug effectiveness at the molecular level."

The team created and characterized the hybrid viruses by combining genetic elements and biological attributes of an animal virus (adeno-associated virus, or AAV) with those of a bacterial virus (phage). Unlike animal viruses that infect mammalian cells, bacterial viruses have evolved to infect only bacterial hosts. The paper shows how particles of the hybrid virus, called AAV phage or AAVP, can serve as a vehicle for targeted delivery of genes to experimental tumors in mice and to the tumors' blood vessel supply, providing a strategy for finding tumors and genetically marking them for imaging on a clinic-ready body scanner.

The AAVP hybrid combines the ability of the bacterial virus to target specific tissues with the capability of the mammalian virus to actually deliver genes to cells. The crucial vehicles, or vectors, in the AAVP hybrid retained the properties of their respective parental viruses, which the researchers called a surprising outcome.

"This is only a proof-of-concept, and although we have yet to translate these hybrid viruses for use in humans, we hope that this new system will have future clinical applications," says Wadih Arap, M.D., the co-leader of the study and professor of Medicine and Cancer Biology. "In addition to the obvious biological interest, when the vector is refined for patient use, it could perhaps help us diagnose, monitor and treat human tumors more accurately."

The finding is the latest in a series of studies by Pasqualini and Arap that are built around their discovery that the human vasculature system contains unique molecular addresses. Organs and specialized tissues also have specific "zip codes" on their blood vessels, as do tumor blood vessels. Knowing this, Pasqualini and Arap designed, constructed, evaluated, and validated the targeted AAVP system over the past several years. Amin Hajitou, Ph.D., a post-doctoral fellow in the Arap/Pasqualini laboratory and first author of the Cell study says, "we were pleased by the strong effects of gene transfer in mouse models of common diseases such as breast and prostate cancer."

Their next step was to work closely with the team of M. D. Anderson researcher Juri Gelovani, M.D., Ph.D., chair of the Department of Experimental Diagnostic Imaging, a pioneer in development of molecular-genetic imaging tools.

"We could see by using positron emission tomography that the reporter and therapeutic genes were being expressed throughout the tumors in the animals," Gelovani says. "This is an example of the so-called "theragnostic" approach, a combination of the words therapeutic and diagnostic."

Next, the international collaborative research team plans to evaluate the safety and efficacy of other hybrid vectors in animal models. The ultimate goal is to adapt and optimize the AAVP-based targeting prototype for use in patients.

Artificial Illumination Using White or Green Light May Prevent Biofilm Formation on Artwork

Sat, 15 Apr 2006 18:40:37 PST

( from http://www.rxpgnews.com ) Using white or green light to artificially illuminate artwork may prevent biofilm formation and surface deterioration, say researchers from Spain. They report their findings in the April 2006 issue of the journal Applied and Environmental Microbiology.

Inappropriate artificial illumination of archeological remains and interior works of art can result in the development of uncontrolled photosynthetic microorganisms which form biofilms and contribute to surface biodeterioration. Biofilms are best described as a cluster of microorganisms attached to either an inert or living surface. Current control efforts include cleaning damaged surfaces and chemical treatments, both of which have had little success at biofilm prevention.

Spectral ambient light can cause variations in pigment distribution enabling an abundance of cyanobacteria and microalgae. Researchers selected green light for testing as it has previously shown to slow growth and affect pigment composition. It also represents the maximum absorbance of human vision. In the study researchers exposed artificial biofilms formed by Gloeothece membranacea and Chlorella sorokiniana to green and white light and evaluated their potential for preventing biofilm growth. Observations made suggest that green light could prevent the growth of biofilms with the exception of those capable of modifying accessory pigments.

"Although laboratory data cannot be extrapolated to natural environments, our results have prompted studies of the application of green light to artificially illuminated works of art," say the researchers.

Mass spectrometry to detect norovirus particles

Mon, 10 Apr 2006 14:07:37 PST

( from http://www.rxpgnews.com ) Scientists have used mass spectrometry for decades to determine the chemical composition of samples but rarely has it been used to identify viruses, and never in complex environmental samples. Researchers at the Johns Hopkins Bloomberg School of Public Health recently demonstrated that proteomic mass spectrometry has the potential to be applied for this purpose. Using a two-step process, researchers successfully separated, purified and concentrated a norovirus surrogate from a clinical sample within a few hours. Nanospray mass spectrometry was used to demonstrate the feasibility of detecting norovirus particles in the purified concentrates.

Human norovirus is responsible for an estimated 23 million cases of gastrointestinal illness in the United States each year. This pathogen is a particular problem aboard cruise ships. The researchers believe that their mass spectrometric method could potentially be used for biodefense and public health preparedness as a tool for rapidly detecting norovirus--a category B bioterrorism agent--and other viral public health threats. The study is published in the April 2006 edition of Applied and Environmental Microbiology.

In simplified terms, mass spectrometry is essentially a scale for weighing molecules. A laser turns a sample into ionized particles, which are then accelerated in a vacuum toward a detector. The time lapsed prior to registering on the detector helps researchers determine the mass--or weight--of the particles. By targeting characteristic particles, or peptides, belonging to the viral coat protein, the virus can be positively identified by matching the results to entries in genetic databases.

In the Hopkins study, the researchers analyzed a stool sample treated with virus-like particles, which closely resemble norovirus but are noninfectious. Using mass spectrometry, the researchers were able to detect the norovirus capsid protein down to levels typically found in clinical specimens from sick individuals.

"This is the first report of the use of mass spectrometry for the detection of norovirus," said David R. Colquhoun, lead author of the study and research fellow with the Johns Hopkins Center for a Livable Future. "This is a significant step towards using mass spectrometry as an environmental surveillance tool for the detection of pathogenic human viruses in complex environmental samples such as human and animal waste."

Typically, bacteria and viruses are identified by cultivation on selective media and cell lines. However, this process does not work for human norovirus, which cannot be cultured outside the human body.

Rolf Halden, PhD, assistant professor in the Department of Environmental Health Sciences and senior author of the study, pointed out that proteomic mass spectrometry is appealing because it has the potential to identify different types and strains of viruses regardless of whether their presence is suspected or not. "Unlike other processes, we do not need to know what we are looking for in advance. Any pathogen whose genetic information is contained in online genetic databases represents a suitable potential target. This makes the technique ideal for situations where you have an emerging infectious agent or pathogenic strain, such as in a potential terrorist attack," said Halden.

xCT molecule is a major gateway for KSHV to enter human cells

Fri, 07 Apr 2006 13:55:37 PST

( from http://www.rxpgnews.com ) Researchers at the National Institute of Allergy and Infectious Disease (NIAID), a component of the National Institutes of Health (NIH), have identified a critical human cell surface molecule involved in infection by Kaposi's sarcoma herpesvirus (KSHV), the virus that causes Kaposi's sarcoma and certain forms of lymphoma. Kaposi's sarcoma is a major cancer associated with HIV/AIDS, and it typically manifests as multiple purple-hued skin lesions.

In the March 31, 2006 issue of Science, NIAID research fellow Johnan Kaleeba, Ph.D. and senior investigator Edward A. Berger; Ph.D., describe how the molecule xCT is a major gateway that KSHV uses to enter human cells. The molecule may also play a role in the development of Kaposi's sarcoma and other syndromes associated with the virus.

The natural function of xCT in the body is to transport molecules necessary for protecting against stress into cells. When cells are stressed, they express more xCT on their surfaces. Of note, this sort of stress can be caused by KSHV itself. This suggests that the virus may facilitate its own infectivity and dissemination in the body by inducing a physiological state that results in increased numbers of its own receptor.

"The advancement of knowledge achieved in this study highlights the outstanding intramural research that takes place here on the NIH campus," says Elias A. Zerhouni, M.D., NIH director.

"Understanding the mechanisms of cell entry of Kaposi's sarcoma herpesvirus is a landmark achievement in and of itself," says NIAID director Anthony S. Fauci, M.D. "But the connection between the virus and expression of its own receptor on a cell is even more provocative because it might change the way we think about KSHV-associated diseases and their treatment."

Although less common in the United States now than early in the AIDS pandemic, Kaposi's sarcoma is still the most common cancer associated with HIV infection. Prior to the AIDS pandemic, it was an obscure disease. First identified as a multi-pigmented skin disease by a Hungarian doctor named Moritz Kaposi in 1872, it was considered to be quite rare--a medical curiosity usually found in particular populations such as older Italian men, transplant patients and young men in certain parts of sub-Saharan Africa. But then at the dawn of the AIDS pandemic in the early 1980s, the small purplish Kaposi's sarcoma skin lesions began appearing on the bodies of young American men, many of whom went on to develop opportunistic infections.

Dr. Berger became interested in KSHV because of his interest in how viruses enter cells. A decade ago, his research team was the first to identify CXCR4 as one of the coreceptors that allows HIV to gain entry into cells of the immune system. This discovery quickly led to the identification by Dr. Berger's group and several other research teams of CCR5 as the other HIV coreceptor.

By applying the same technology used to identify CXCR4, Drs. Kaleeba and Berger ultimately identified the protein xCT as the receptor that can make cells permissive for KSHV fusion.

The NIAID discovery may lead to new avenues for treating KSHV, says Dr. Berger. Moreover, their finding should enable scientists to determine whether levels of xCT determine disease severity. It also will allow researchers to study whether the expression of xCT on cells varies among different groups of people and whether these variations are genetic or environmental. This research may ultimately explain why certain groups are more at risk for Kaposi's sarcoma.

"Our finding provides a new perspective on the disease," says Dr. Kaleeba, who is originally from Uganda where Kaposi's sarcoma accounts for at least 10 percent of known tumors. "Hopefully this will be the beginning of exciting new directions in this field, as it is likely to provide a useful framework for integration of the cell biology and epidemiology of this clinically important virus."

Surprising discovery about the inner workings of vesicular stomatitis virus (VSV)

Fri, 07 Apr 2006 13:52:37 PST

( from http://www.rxpgnews.com ) Biochemists at Wake Forest University School of Medicine have made a surprising discovery about the inner workings of a powerful virus a discovery that they hope could one day lead to better vaccines or anti-virus medications.

Reporting in the April issue of the Journal of Virology, the researchers have identified a protein that plays an important role in the ability of the vesicular stomatitis virus (VSV) to invade healthy cells and reproduce itself. The finding could play a role in vaccine development and also help scientists develop anti-viral agents to stop similar viruses in their tracks.

Although VSV infects animals, it is not a human pathogen. Nevertheless, scientists study it because of its similarity to viruses such as Ebola and Marburg hemorrhagic fever viruses, as well as rabies virus. "VSV is a good model of a variety of other viruses," said John Connor, Ph.D., a research assistant professor of biochemistry. "Our research has given us a better understanding of how viruses like these are able to do the nasty things they do."

The scientists set out to study the role of a protein known as "matrix," which is produced by VSV. They suspected matrix was important in how VSV is assembled, but unexpectedly discovered the matrix protein is critical in how the virus reproduces and spreads. When they altered the matrix protein, they weakened the virus' ability to reproduce. The finding has several important implications, Connor said.

Normally, VSV is extremely powerful, with the ability to shut down a cell's system for making proteins. VSV then takes over the cell's protein-making machinery and makes its own proteins so it can replicate and spread. The scientists were able to weaken this power by altering the matrix protein, so that VSV cannot make as much protein and does not reproduce as well.

Weakened viruses such as this are often used to make vaccines because they are less likely to be harmful. Currently, another weakened form of VSV is being used for a HIV vaccine that is being tested in humans. To make the vaccine, scientists started with the weakened VSV virus and added a protein from the HIV virus so that VSV "expresses" or makes a fragment of the HIV virus. In theory, when people are inoculated with the vaccine, they will develop antibodies to the HIV protein, and if they are exposed to the actual HIV virus, their bodies will neutralize it and kill it before it infects them.

In all, several weakened forms of VSV have been developed and at least two are currently being tested in HIV vaccines. If they don't prove effective, vaccine developers can turn to one of the others, including the mutant VSV virus developed by Connor and colleagues.

"Right now, there's no way of knowing which way of weakening the virus will make the best vaccine," Connor said.

In addition to its potential for vaccine development, the new finding about VSV also provides basic information about how the virus shuts downs a cell's protein making-abilities and dominates the process.

"We always knew this happened, but the process was like a black box," said Connor. "Now, we know that the matrix protein is involved and is incredibly important in virus reproduction. This pushes forward our knowledge of how this virus is so effective at replicating."

Could the finding about matrix be used to weaken other types of viruses? The scientists aren't sure, yet. "It's a strong possibility that every virus will have an Achilles' heel like this, where they need the function of a viral protein to make lots of virus," said Connor.

Salmonella bacteria use RNA to assess and adjust magnesium levels

Fri, 07 Apr 2006 03:46:37 PST

( from http://www.rxpgnews.com ) Researchers at Washington University School of Medicine in St. Louis have added a gene in the bacterium Salmonella to the short list of genes regulated by a new mechanism known as the riboswitch.

The Salmonella riboswitch is the first to sense and respond to a metal ion, substantially expanding the types of molecules that riboswitches can detect to help cells assess and react to their environment.

First identified in 2002, riboswitches sense when a protein is needed and stop the creation of the protein if it isn't. That in itself isn't remarkable--scientists have been aware for decades of sensors in the cell that can cause molecules to bind to DNA to turn protein production on and off.

A riboswitch, however, doesn't rely on anything binding to DNA; instead, the switch is incorporated into messages for construction of proteins. These messages are protein-building instructions copied from DNA into strands of RNA. The riboswitch is a sensor within the RNA that can twist it into different configurations that block or facilitate the production of the protein encoded in the message.

Previously identified riboswitches respond to organic compounds such as nucleotides and sugars. The Salmonella riboswitch, reported in the April 7 issue of the journal Cell, responds to magnesium ions, key elements in the stability of cell membranes and reactants in an energy-making process that fuels most cells.

"Magnesium ions are essential to the stability of several different critical processes and structures in the cell, so there has to be a fairly intricate set of regulators to maintain consistent levels of it," says senior investigator Eduardo A. Groisman, Ph.D., professor of molecular microbiology. "To approach such a complex system, we study it in a simpler organism, the Salmonella bacterium."

Groisman and his colleagues uncovered the magnesium riboswitch while they were investigating the MgtA gene, which is controlled by the major regulator of Salmonella virulence, the phoP/phoQ system. The MgtA gene codes for a protein that can transport magnesium across the bacterium's cell membrane. Groisman's group showed 10 years ago that the phoP/phoQ system controls when Salmonella makes MgtA.

When Salmonella experiences a low-magnesium environment, phoQ chemically modifies phoP. The changed phoP binds to DNA, increasing the number of times instructions for making MgtA and over 100 other proteins are copied from DNA. But when Salmonella encounters a high-magnesium environment, phoQ deactivates phoP, and fewer copies of the instructions for making MgtA are made.

When Groisman and his colleagues created a mutant strain lacking the phoQ gene, though, they were surprised to find that production of the instructions to make the MgtA protein could still somehow respond to magnesium, producing less of its protein at high magnesium levels.

Researchers used a computer program to determine how RNA copied from the MgtA gene might be folding up. The program predicted RNA copied from the gene could have two significantly different configurations. Because of the significant differences between these configurations, Groisman, who is also a Howard Hughes Medical Institute investigator, became interested in a region at the beginning of the RNA strand that contains no protein-building instructions. He theorized that it might be a riboswitch that responded to high magnesium levels by twisting the RNA into a configuration where its protein-building instructions somehow could not be used or were invalidated.

"One of our tests to see if this was something more than a computer fantasy was to take this segment that contains no protein-building instructions off the MgtA gene and paste it into another genetic configuration," Groisman says. "We wanted to see if it conferred sensitivity to magnesium levels, which it did."

In addition, Groisman's group showed that one RNA configuration was common in low magnesium levels while another was common in high magnesium levels.

They also searched the genomes of other bacteria with MgtA genes to see if their DNA included a sequence similar to the riboswitch in Salmonella. In six other bacteria, a similar sequence precedes the MgtA gene and can twist RNA copied from it into different configurations.

"Normally you would expect to find that a DNA sequence that is conserved among different species is encoding part of a protein," Groisman says. "But here we're talking about a part of a message that does not encode a protein. So why would it be conserved? There must be some important role that the sequence is fulfilling that is leading to its conservation, such as giving the cell expanded ability to sense and respond to magnesium levels."

Follow-up inquiries are already underway to locate the riboswitch's "brain"--the section of the RNA strand that responds to magnesium; and to learn how the high-magnesium configuration of the RNA disrupts final production of the protein.

New human retrovirus - Xenotropic MuLV-related virus (XMRV)

Sat, 01 Apr 2006 19:26:37 PST

( from http://www.rxpgnews.com ) Howard Hughes Medical Institute researchers and their colleagues have discovered a new retrovirus in humans that is closely related to a cancer-causing virus found in mice. Their findings describe the first documented cases of human infection with a retrovirus that is native to rodents.

The researchers discovered the virus in patients with a rare type of prostate cancer. The patients in the study have a genetic mutation that compromised some of their natural defenses against viral infection. Thus, the researchers said their discovery raises the possibility that increased susceptibility to viral infection may play a role in development of some cancers. However, they emphasized that their findings by no means implicate the virus, dubbed XMRV, in causing prostate cancer. The virus may well have flourished as a result of the failure of the defense mechanism; and other factors such as chronic inflammation may play a more direct role in the cancer.

The discovery of the new virus was made by an interdisciplinary research team led by Robert Silverman of Cleveland Clinic and HHMI investigators Joseph DeRisi and Don Ganem, both at the University of California at San Francisco. A paper describing the findings was published on March 31, 2006, in the journal, Public Library of Science Pathogens.

The search for the new virus began when Silverman and his colleagues provided samples of a rare familial prostate cancer in which the viral-defense gene, RNASEL, had been mutated in a specific way. This mutation compromised the function of the enzyme produced by RNASEL, which normally shreds viral genetic material. Infected cells carrying the shredded viral genetic material are usually targeted for destruction by the immune system. While some scientists believe that such vulnerability to viral infection is connected to prostate cancer in these rare cases, others have presented evidence contesting that theory.

To screen for viruses in the prostate tissue samples, DeRisi and Ganem used the Virochip, which was invented by DeRisi and his colleagues. The Virochip consists of a microarray of some 20,000 characteristic gene sequences -- called oligonucleotides -- representing a vast array of known viruses. The oligonucleotides are deposited as tiny spots on a small glass chip.

To detect viruses from tissue samples, the researchers isolated genetic material from each sample and tagged the genetic material with a fluorescent tracer. They then applied the fluorescently tagged genetic material to the microarray chip. Since genes tended to adhere to those with a complementary genetic sequence, any viral gene sequences in the sample would attach themselves to corresponding viral sequences on the chip. The telltale fluorescence on spots on the chip signaled the presence of viral genetic material in the sample.

Although the Virochip contains only sequences from known viruses, DeRisi said it can also detect new viruses because they invariably contain sequences that have been conserved in their evolution from related viruses.

The initial screen of the RNASEL-mutant prostate cancers revealed the presence of a genetic sequence that closely resembled that of a mouse virus called murine leukemia virus (MuLV). Murine leukemia virus is known as an endogenous virus because it normally exists as an integrated part of the mouse genome, rather than as independent, infective particle. MuLV is also a retrovirus, meaning its genetic material is in the form of RNA. The RNA is then reverse transcribed into DNA that is integrated into the DNA of the host cell the virus is infecting.

When the researchers isolated and sequenced the genome of the virus, they found that it was a xenotropic virus one that can only grow in foreign cells other than mouse cells. Thus, they named the virus, Xenotropic MuLV-related virus, or XMRV.

"This finding was a big surprise because most of these endogenous viral genomes have undergone such mutation and deletion that they are incapable of giving rise to viruses any more," said Ganem. "And while some of these viruses had been induced to grow in human cells in culture, the major question is whether such infection could ever happen in nature.

"So, one of the things that is important about our study from a virologic point of view, is that this is the first really solid example of an authentic xenotropic retroviral infection in a human being," said Ganem.

According to DeRisi, the Virochip made it possible to analyze these samples without preconceived biases about what viruses might be present. "Since the chip represents every known virus in one assay, it is agnostic as to what might be found," he said. "We would never have looked for this class of virus if it wasn't for the virus chip."

Importantly, the researchers found that prostate cancers in which both copies of the RNASEL gene were crippled by mutation showed much more frequent XMRV infection than did those cancers that still had one normal copy of the RNASEL gene.

"This link between the virus and RNASEL is the second finding that is important and is firmly established in this study," noted Ganem. "We don't see the infection in people who don't have the RNASEL mutation, which suggests strongly RNASEL is an important part of the defense against retroviral infection. This is the first evidence in humans of findings that were previously made only in vitro."

DeRisi pointed out that detailed comparison of samples of the virus between people found that although all were XMRV they showed tiny genetic variations. "So, while it is the same virus in each patient, the viruses are different enough to say that they are most likely independently acquired and are not the result of some contamination of the samples," he said.

Ganem cautioned that any link between XMRV and prostate cancer is tenuous at best. "First, the genetic variant we studied occurs in familial clusters that constitute only a very small sliver of prostate cancers," he said. "And secondly, there are many reasons to believe that the virus might not relate to prostate cancer."

For example, he pointed out, analysis of prostate tissue by Silverman and his colleagues indicated that the virus appears only in a small percentage of connective tissue cells, called stromal cells, rather than in the tumors themselves. "So, one interpretation could be that the infection is entirely incidental to prostate cancer," said Ganem. "The patients with RNASEL mutations may be more likely to get the infection or perhaps less likely to clear it. Clearly XMRV is not a classic oncogenic virus."

Nevertheless, said Ganem, an indirect link to cancer cannot be ruled out, since "in cancer research these days, there is a lot of interest in the stroma as the soil in which cancer arises." He added that the chronic inflammation from infection of stromal tissues may play a role in triggering such cancers.

DeRisi observed that "it may be that men who are so-called RNASEL-mutant are just more susceptible to viruses in general, and this susceptibility has little to do with their cancer. Nevertheless, the fact that this virus is found in tumor tissue and that it is a new virus and the first of its kind ever documented in humans is an intriguing finding that demands to be followed up. This initial finding raises many questions. For example, what is the route of transmission? How is the virus passed from person to person? And are people the natural reservoir of this virus, or is it some other organism?"

DeRisi and Ganem said they are planning studies to explore whether XMRV is restricted to prostate cancers or whether it is more widespread in the body and in other segments of the human population. To answer such questions, the researchers are developing a blood test that can be used in epidemiological studies.

Genomics Sheds Light on Metabolism of Cryptic Marine Microbes

Wed, 22 Mar 2006 12:01:37 PST

( from http://www.rxpgnews.com ) In 1977 Carl Woese and George Fox expanded our appreciation of microbial diversity by analyzing the genetic sequence of a molecule (ribosomal RNA) found in all cells. They discovered that species previously classified as bacteria, called methanogenic bacteria, possessed unique enzymes and an unusual metabolism based on reducing carbon dioxide to methane. These traits were foreign to both uber domains of life, Eurkaryota and Bacteria, prompting Woese to create a new category, which he called Archaebacteria (archae means ancient in Greek), acknowledging a metabolism that would have suited the putative conditions on earth over 3 billion years ago.

Archaeal groups have been found in a wide array of habitatsfrom boiling sulfur pits, salt marshes, and hydrothermal vents to frosty Antarctic surface waters, mud flats, and freshwater habitatsyet less than 0.1% of expected species have been characterized.

In a new study, genetic analysis offers clues to the fundamentals of archaeal life and some insight into how these organisms can exist in such diverse environments. Steven Hallam, Edward DeLong, and their colleagues enlist genomics techniques to identify the pathways used by the marine sponge symbiont Cenarchaeum symbiosum to accomplish lifes most essential processes: energy metabolism and carbon assimilation. And by comparing the C. symbiosum genome sequence with sequences extracted from environmental samples collected from diverse ocean habitats, they show that planktonic Crenarchaeota share many of the same genetic components.

Many archaeal species can use inorganic compounds (rather than sunlight, like plants) as an energy source for carbon synthesis, earning them the unwieldy name of chemolithoautotroph. Several lines of evidence suggest that planktonic Crenarchaeota, significant components of the marine ecosystem, assimilate carbon in this way and that they might use ammonia (NH3) as an energy source, since they inhabit ammonia-rich Antarctic waters and are associated with high nitrite concentrations. (Nitrite is a by-product of ammonia oxidation.)

To search for genetic clues to carbon and energy metabolism in Crenarchaeota, the researchers extracted C. symbiosum DNA from its host sponge and constructed a DNA library for sequencing the symbionts genome. Hallam et al. then searched for representative genes linked to pathways associated with autotrophic carbon assimilation. They found many components of two pathways: the 3-hydroxypropionate cycle and the reductive tricarboxylic acid (citric acid) pathway (TCA). Both cycles involve a multistep series of chemical reactions that convert inorganic compoundsin this case, carbon dioxideinto organic carbon molecules. Though some components of the 3-hydroxypropionate cycle were missing in C. symbiosum, enough elements (including core proteins) were found to support a modified version of this pathway for carbon assimilation, using carbon dioxide.

In eukaryotes, the TCA cycle links the oxidative breakdown of carbon compounds with biosynthesis and energy metabolism. In prokaryotes, the process is reversed, with the oxidation of inorganic compounds (such as carbon dioxide) providing the means for carbon assimilation. Again, though some TCA components were missing, Hallam et al. found evidence suggesting that C. symbiosum could use partial TCA reactions to produce biosynthetic precursors. Its possible that other genes take the place of the missing components or that the TCA and 3-hydroxypropionate pathways overlap.

The researchers next searched for genes that might play a role in generating energy from ammonia oxidation (also called nitrification because ammonia is converted to nitrite). The C. symbiosum genome contains many genes associated with nitrification in bacteria, including genes that encode the subunits of ammonia monooxygenase, which catalyzes the first step in converting ammonia to nitrite. The researchers could not find evidence for several proteins that function downstream in this pathway, however, suggesting that the symbiont uses alternative mechanisms to effect nitrification. Supporting this possibility, the researchers found candidate genes that might take the place of some of these missing elements, as well as others that could protect the cell from the toxic nitrites generated by ammonia oxidation.

How did the genes identified here compare with planktonic Crenarchaeota gene sequences? To find out, Hallam et al. searched GenBank (the National Institutes of Health genetic sequence database) and an environmental database containing gene sequences collected from the Sargasso Sea and other ocean waters for similar sequences. Many components of both the 3-hydroxypropionate and the TCA cycle were found in the environmental database. And each of the C. symbiosum genes studied here were most closely related to sequences from planktonic Crenarchaeotasuggesting that even though these archaeal lineages evolved under different selective pressures, they rely on similar metabolic strategies.

Overall, Hallam et al. argue that, though the forms show significant divergence at the nucleotide level, C. symbiosum and planktonic Crenarchaeota share striking similarities in the identity and organization of their genes. And with gene sequences linked to fundamental processes like carbon assimilation and energy metabolism in C. symbiosum, researchers can probe parallel processes in marine Crenarchaeotaan endeavor that will likely reveal the vital role these once-enigmatic organisms play in the carbon and nitrogen cycles of marine ecosystems.

How deadly toxin botulinum neurotoxin A hijacks cells

Fri, 17 Mar 2006 14:01:37 PST

( from http://www.rxpgnews.com ) Scientists have pinpointed exactly how botulinum neurotoxin A - a potential agent of biological warfare and one of the most lethal toxins known to man - is able to sneak into cells.

The finding is crucial for the development of new treatments against botulism, a paralytic illness caused by the toxin more commonly known as botox. As small amounts of botox are also known to alleviate many medical problems, the recent work could help to quell any risks associated with the toxin's clinical use.

Writing in the current online edition of Science, a team of researchers at the University of Wisconsin-Madison and the University of Texas report that botox latches onto a protein known as SV2 to gain entry into neurons.

"Our work shows that botox is really smart and clever," says senior author Edwin Chapman, a UW-Madison professor of physiology and an investigator of the Howard Hughes Medical Institute. "It uses SV2 to sneak into nerves like a Trojan horse."

"Botulinum neurotoxins are among the six most dangerous bioterrorism threats," adds lead author Min Dong, a UW-Madison postdoctoral researcher in the department of physiology. "Knowing the protein receptor for [botulinum toxins] can pave the way for developing anti-toxin reagents which may block the entry of toxins into cells."

The botulinum toxins, of which there are seven types, are made by a bacterium commonly found in soil, known as Clostridium botulinum. Of the seven-identified by the letters A through G--botox A lasts a particularly long time in neurons. While that feature makes it especially useful in the clinic, it also means that botox A may pose a particularly dangerous threat as a biological weapon.

The toxin enters neurons by binding to nerve endings and preventing the release of crucial chemical messengers, known as neurotransmitters, that communicate with muscles. When enough nerve endings are invaded, botox can lead to paralysis and death.

By capitalizing on the ability of botox to act on a localized group of muscles, doctors have strategically used the toxin to treat an array of medical troubles, including migraine headaches, chronic inflammation and even stuttering. "I don't think there's a neuromuscular junction that hasn't been inhibited by injecting with botox A," says Chapman.

Chapman and his team located the exact molecular gateway through which botox penetrates cells by gathering clues from earlier research that pointed to the potential importance of tiny neural storage bins known as "synaptic vesicles." Situated at nerve endings, synaptic vesicles continually work to store and release neurotransmitters.

Dozens of proteins, including SV2, work to ensure that vesicles function properly. With standard screening experiments known as "entry assays," the scientists were able to zero in on SV2. To confirm that result, they acquired mice that were genetically engineered to carry reduced amounts of SV2. Without that protein around, the researchers found that botox was unable to wreak havoc.

String Test: Effective and Inexpensive Method for Detecting Helicobacter pylori

Sat, 11 Mar 2006 20:36:37 PST

( from http://www.rxpgnews.com ) Swallowing a string may offer a simple and effective alternative to costly and invasive techniques used for detecting Helicobacter pylori in patients say researchers from the U.S. and abroad. They report their findings in the March 2006 issue of the Journal of Clinical Microbiology.

Helicobacter pylori is a gram negative bacterium known for causing chronic gastric distress in individuals worldwide and can lead to the development of peptic ulcers and early onset of gastric cancer. Current methods for detecting H. pylori infection do provide highly sensitive and specific results, but they can be costly, invasive, and uncomfortable.

In the study 35 patients with gastric complaints were administered the string test (or Entero test) which involves swallowing a capsule with a protruding absorbent string whose end is held outside the mouth. The ingested string is then retrieved and microbes from the gastrointestinal tract are recovered and studied. H. pylori was cultured from 80% of the strings of those patients who had also undergone extensive biopsy procedures and received positive results. No organisms were found on strings taken from patients whose biopsy results were negative.

"Our study shows that the string test, which is minimally invasive, inexpensive, and not dependent on sophisticated or costly equipment or radioactivity, allows culture of H. pylori from infected persons about 80% as efficiently as endoscopic gastric biopsies," say the researchers. "We suggest that the H. pylori string test assays will be of increasing importance in a public health context."

Multivariate Linear Regression May Assist in Determining Virulence Factors for Microbes

Sat, 11 Mar 2006 20:36:37 PST

( from http://www.rxpgnews.com ) A well established statistical tool known as multivariate linear regression may offer a new approach in determining contributions of multiple virulence factors to the overall virulence of pathogenic microbes say researchers from Albert Einstein College of Medicine, Bronx, New York and Westminster College, Salt Lake City, Utah. Their findings appear in the March 2006 issue of the journal Infection and Immunity.

Virulence is defined as the capacity of a microbe to cause damage to its host. Although there is much literature discussing the contribution of virulence factors to microbial virulence, there is no designated methodology for determining the impact of individual virulence factors on overall microbial virulence. Identification of such a method could greatly contribute to vaccine development and antimicrobial strategies.

Multivariate linear regression is a statistical tool used to analyze the relative contributions of different parameters. Out of the three types of multivariate linear regression researchers identified hierarchical regression as the type most applicable to this study. It is described as entering variables in different blocks in a specific order with the order of entry resulting from theoretical or logical importance. This approach was applied to Cryptococcus neoformans and Bacillus anthracis and results showed the method to be useful in determining the relative contributions of virulence factors in pathogenesis.

"Multivariate linear regression can be used to identify the relative levels of importance of virulence factors in virulence studies, and this information can be used to prioritize antigen identification for vaccine development and the design of antimicrobial strategies that target virulence mechanisms," say the researchers.

Scientists develop biosensor to detect E. Coli bacteria

Sat, 25 Feb 2006 10:02:37 PST

( from http://www.rxpgnews.com ) Scientists have developed a fast working biosensor that can accurately and rapidly detect an infectious agent that causes food borne illness, including the dangerous E. Coli bacteria.

The unique technology developed by the University of Rochester Medical Centre uses a protein from the suspect bacteria as part of the sensing system that includes a silicon chip and a digital camera.

"Traditional methods of detection of bacteria can take days but the biosensor developed by them could take as much time as it takes for a snapshot," said lead researcher Benjamin Miller.

The Rochester team called the technology "arrayed imaging reflectometry".

The system utilises a silicon chip that is made in a manner so that laser light reflected off the chip is invisible unless the target bacteria are present.

A protein from the bacteria, Translocated Intimin Receptor or Tir, is placed on the chip. The Tir can be seen as a "molecular harpoon", Miller said.

The E. Coli sends out the harpoon into a cell. Once it is in, the Tir binds with an E. Coli protein called Intimin. A similar process occurs between the Tir placed on the chip and any E. Coli in the sample.

The binding of the probed sample and the bacteria alters the surface of the chip. A digital camera image of the chip captures the changes for analysis and confirmation of detection.

Describing the new technology as inexpensive, Miller said that a physician some day could use it in his office to confirm a streptococcal infection in a patient with a sore throat.

Found - bacteria with strange magnetic personality

Fri, 24 Feb 2006 02:25:37 PST

( from http://www.rxpgnews.com ) Researchers have reported the discovery of a bacterium with strange magnetic properties - it tends to swim towards south magnetic pole while being in the northern hemisphere.

While 'Magnetotactic bacteria' are known to swim toward geomagnetic north in the northern hemisphere and geomagnetic south in the southern hemisphere, researchers from the Massachusetts Institute of Technology (MIT), the Woods Hole Oceanographic Institution (WHOI) and Iowa State University have found a bacterium in New England that does just the opposite: a northern hemisphere creature that swims south.

Because this behaviour doesn't make sense in the natural environment of the bacteria, where swimming south would take them away from areas with their preferred oxygen level, the researchers believe there must be other explanations for why some magnetotactic bacteria swim in particular directions, notes an MIT release.

The team dubbed the bacterium the barbell for its appearance. In a study reported in a recent issue of Science, they describe how they used genetic sequencing and other laboratory techniques to identify the barbell, which was found coexisting with other previously described magnetotactic bacteria in Salt Pond on Cape Cod.

Magnetotactic bacteria are found throughout the world in chemically stratified marine and freshwater environments, said lead author Sheri Simmons, a graduate student of the MIT.

Simmons and colleagues studied the bacterium under laboratory conditions and say the behaviour in natural environment could be different from its laboratory behaviour. Their results, however, suggest new models are needed to explain how these magnetotactic bacteria behave in the environment.

Student discovers protein in yoghurt that fights E. coli

Fri, 24 Feb 2006 02:22:37 PST

( from http://www.rxpgnews.com ) A high school student in the US has discovered a protein in yoghurt that has the potential to fight E.coli, the leading cause of diarrhoea in the world.

The yet to be named protein was discovered by 16-year-old Serena Fasano, a junior at Glenelg High School, after three years of research at the University Of Maryland School Of Medicine. Her father is director of the Mucosal Biology Research Center.

Fasano has been awarded a patent for the protein, although it is officially owned by the University of Maryland, reported the online edition of Baltimore Sun.

The student happened to notice an unusual ingredient lactobacillus in yoghurt. She obtained - through her father - specimens of E.coli 042, added varying amounts of yoghurt to it and chronicled the results.

The dish with the most yoghurt had the least E. coli, so Fasano was able to say that yoghurt kills E.coli, which kills six million people annually in the world, mostly children in Third World nations.

Viruses can be forced to evolve as better delivery vehicles for gene therapy

Wed, 08 Feb 2006 11:33:37 PST

( from http://www.rxpgnews.com ) Viruses and humans have evolved together over millions of years in a game of one-upmanship that, often as not, left humans sick or worse.

Now, a University of California, Berkeley, researcher has shown that viruses - in this case, a benign one - can be forced to evolve in ways to benefit humans.

The adeno-associated virus, or AAV, is a common, though innocuous, resident of the body that has received a lot of attention in recent years as a possible carrier for genes in gene therapy. Because as many as 90 percent of people already have the virus, however, their immune systems are primed with antibodies to quickly tackle and neutralize it, thwarting any attempt at gene therapy.

UC Berkeley's David Schaffer, associate professor of chemical engineering and a member of the Helen Wills Neuroscience Institute, with colleagues Narendra Maheshri, James T. Koerber and Brian Kaspar, decided to speed up the process of viral evolution and direct the change in a way that would allow the virus to slip past the body's immune defenses, making it a more viable vehicle for gene therapy. In essentially two generations of accelerated evolution, requiring about two months of lab work, they succeeded.

"Directed evolution has mainly been done to change the activity of an enzyme - to make it more effective toward a new substrate or better able to catalyze a reaction, for example - or to make antibodies better binders against specific targets," said Schaffer, who also is an affiliate of the UC Berkeley wing of the California Institute for Quantitative Biomedical Research (QB3). "In the viral realm, this approach is essentially untapped."

This technique could be used to improve many other characteristics of AAV to make it a better delivery vector for genes.

"We think there are a huge variety of new problems we could address as well, such as targeting the virus to cells it is ordinarily not good at getting into, or speeding its transport through the body," he said.

Though Schaffer acknowledges that the technique could be used to help pathogenic viruses evade the human immune system, potentially making them more virulent, he said that other and easier techniques already allow this frightening possibility.

AAV consists of two genes enclosed within a ball, or capsid, of proteins. The capsid proteins are what antibodies recognize, and as a result were the target of directed evolution. To provide the raw material for evolution - the genetic variation from which nature selects the best-adapted organism - the researchers created mutant viruses by introducing small variations in the genes through an error-prone polymerase chain reaction (PCR) coupled with a test tube recombination technique. After reassembling the mutant viruses inside their capsids, they introduced them to blood serum pooled from rabbits immunized against AAV, and thus containing many types of antibodies to AAV. Only the mutant viruses good at evading antibodies to AAV survived the serum.

After passing the viruses three times through increasingly more potent serum, they isolated the survivors and subjected them to another round of PCR that introduced more mutations. After passing this second generation through serum three times, they isolated viruses that could survive AAV antibodies much better than the original strain of AAV. One strain of virus was 96 times more effective than the wild AAV, and two evolved strains survived injection into mice with nearly 1,000 times the level of antibodies required to neutralize the wild virus.

By sequencing the survivor strains, the researchers discovered that the capsid proteins of the survivors differed from those of the original strain by only seven amino acid building blocks, two of which were responsible for most of the altered interaction with antibodies.

"Starting from scratch, just trying to rationally decide which two amino acid changes to make on the virus, there is no way you would have guessed those two," Schaffer said. "Using the same algorithm as nature came up with - evolution - to solve the problem, is the best way to do it."

Since each generation takes about a month, Schaffer predicted that many types of new and improved strains could be created in a few months' time, and certainly in less than a year. He is pursuing experiments now using pooled human blood serum.

"This virus is kind of a gift from nature, a very safe and efficient virus, but nature never evolved it to be a human therapeutic. So, in a sense, we have to re-evolve it for that purpose," he said.

Slugs May Spread E. coli to Salad Vegetables

Fri, 20 Jan 2006 14:00:37 PST

( from http://www.rxpgnews.com ) A new study suggests that slugs have the potential to transmit E. coli to salad vegetables. Researchers from the University of Aberdeen, United Kingdom, report their findings in the January 2006 issue of the journal Applied and Environmental Microbiology.

Escherichia coli O157, an emerging zoonoses in many countries including the U.S. and U.K., has a 3 to 5 percent mortality rate in humans. Farm animals such as cattle and sheep have been previously identified as major reservoirs of this strain of E. coli by passing it through manure which is then used to fertilize crops. Slugs are widespread agricultural pests that continuously ingest bacteria from the soil and their environment. Their tendency to contaminate leafy vegetables often targeted for human consumption identifies them as likely source for E. coli transmission.

Laboratory testing found E. coli O157 in 0.21 % of field slugs on a sheep farm in the UK. Further studies revealed that the slug species, Deroceras reticulatum, could maintain viable E. coli on its external surface for 14 days and slugs that were fed E. coli shed viable bacteria in their feces persisting for up to 3 weeks.

"This study provides evidence that slugs can act as vectors of E. coli O157 from an environmental source to fruit or vegetables," say the researchers. "The research demonstrates that E. coli in D. reticulatum has a relatively long external and internal survival time and also shows that ability of E. coli to persist at length in excreted slug feces."

Epstein-Barr Virus Found in Breast Cancer Tissue May Impact Efficiency of Treatment

Fri, 20 Jan 2006 14:00:37 PST

( from http://www.rxpgnews.com ) Epstein-Barr virus has been detected in breast cancer tissue and tumor cells and may impact the efficiency of chemotherapeutic drug treatment say researchers from France and Japan. They report their findings in the January 2006 issue of the Journal of Virology.

A ubiquitous human herpesvirus, the Epstein-Barr virus (EBV), has been previously linked to skin and gastric cancer, as well as cancer of the salivary glands and thymus. New studies have detected EBV in breast cancer specimens and have prompted researchers to examine the effect of infection with EBV on anticancer drug treatment.

In the study biopsy specimens of breast cancer tissue and tumor cells were tested for the EBV genome. The genome was identified in about half of the specimens, however the viral load was highly variable from tumor to tumor. These findings indicate that although EBV isn't likely to cause breast cancer, it may contribute to tumor progression. In addition, researchers studied the EBV infected cells in vitro and found that the virus may contribute to the resistance of paclitaxel (taxol), chemotherapy commonly used in the treatment breast cancer, and cause overexpression of the multidrug resistance gene (MDRI).

"Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance," say the researchers.

Honeybees May Transmit Viruses to Their Offspring

Fri, 20 Jan 2006 13:55:37 PST

( from http://www.rxpgnews.com ) Researchers from the U.S. Department of Agriculture report what may be the first evidence of queen honeybees transmitting viruses to their offspring. They report their findings in the January 2006 issue of the journal Applied and Environmental Microbiology.

Honeybees contribute greatly to the annual 15 billion dollar agriculture market by assisting in the pollination of a wide variety of crops. The health of honeybee colonies is continuously threatened by various pathogens, with viruses posing the greatest risk due to lack of information concerning transmission and outbreaks.

In the study feces and tissue (including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body) of individual queen bees were tested for viral presence. All tissue forms but one, as well as feces, were found to carry viral infections. Once the viruses in the queen bees were identified, their offspring (including eggs, larvae and adult workers) were tested and found to carry the same viruses.

"The present study, using the sensitive RT-PCR method, demonstrated the vertical transmission of multiple viruses from mother queens to their offspring by two findings: first, the presence of viruses in queen excretion and queen tissues, particularly in the tissue of ovaries; and second, detection of the same viruses in queens' eggs and young larvae that are not normally associated with V. destructor, which is an important vector of bee viruses," say the researchers.

Escherichia coli doesnt gamble with its metabolism

Sat, 17 Dec 2005 15:57:38 PST

( from http://www.rxpgnews.com ) The ubiquitous and usually harmless E. coli bacterium, which has one-seventh the number of genes as a human, has more than 1,000 of them involved in metabolism and metabolic regulation. Activation of random combinations of these genes would theoretically be capable of generating a huge variety of internal states; however, researchers at UCSD will report in the Dec. 27 issue of Proceedings of the National Academy of Sciences (PNAS) that Escherichia coli doesnt gamble with its metabolism. In a surprise about E. coli that may offer clues about how human cells operate, the PNAS paper reports that only a handful of dominant metabolic states are found in E. coli when it is grown in 15,580 different environments in computer simulations.

When it comes to genomes, a great deal of complexity boils down to just a few simple themes, said Bernhard Palsson, a professor of bioengineering at UCSDs Jacobs School of Engineering and co-author of the study, which was made available online Dec. 15. Researchers have confirmed the complexity of individual parts of biochemical networks in E. coli and other model organisms, but our large-scale reconstruction of regulatory and metabolic networks involving hundreds of these parts has shown that all this genetic complexity yields surprisingly few physiological functions. This is possibly a general principal in many, if not all, species.

Palsson and his colleagues at UCSD, postdoctoral fellows Christian L. Barrett and Christopher D. Herring, and Ph.D. candidate Jennifer L. Reed, created a computer model of an E. coli cell based on the experimental results of thousands of previous experiments, some of which were completed decades ago. The goal of this study was to comprehensively simulate all the possible molecular interactions in a well studied strain of E. coli to gain a global view of the range of functional network states, said Barrett. Complex cellular networks can potentially generate lots of different behaviors, but we find that cells utilize only a few of them.

Barrett, Palsson, Herring, and Reed simulated the behavior of 1,010 of E. colis 4,200 genes. This particular subset of the bacteriums genome is tightly organized into interacting networks involved in metabolism or regulation of gene activation, or transcription. These linked networks are devoted to sensing, ingesting, and degrading potential food in the form of sugars and other energy-rich organic molecules.

E. coli must also have an efficient way to eliminate waste products. It, like all living things, generates energy in a process that involves the removal of electrons from food molecules and attaching them to acceptor molecules. For aerobic organisms, the final electron acceptor is usually oxygen, which is converted into water in the process.

E. coli can grow with or without oxygen, using nitrate or other molecules as its final electron acceptor. We found that the type of terminal electron acceptor in the growth environment and the presence or absence of glucose is very important to E. coli, said Barrett. Our simulations show that these two factors are key determinants of how the bacterium organizes itself.

This statistical projection of E. coli's computation-based activity profiles permits researchers to visualize the "space" of transcriptional regulation of genes involved in metablism and metabolic regulation. The clusters' positions are a function of the available electron acceptor, indicated by the ellipses, the carbon "food" source, and to a lesser degree by the source of nitrogen. (The number in parenthesis by each of the 13 clusters is the numbers of different activity profiles in the cluster.) (Image Courtesy: UCSD)

Barrett, Palsson, and their colleagues simulated the functional states of E. colis metabolic and transcriptional regulatory networks in the 15,580 environments of food sources and electron acceptors. To their surprise, no matter what carbon source it ingests or electron acceptor used, E. coli exhibits only six distinct functional states.

This study gives a systems biology view of how a phenotype, or a network state advantageous to a microorganism is comprised of a tiny subset of a much larger universe of possibilities as provided for in the genome, said Palsson. On a high level we can say that E. coli is obsessed with how it breathes and whether or not glucose is available to eat. All of its genetic complexity basically enables it to generate a nice steady state for itself regardless of what it has to live on.

Higher organisms have larger genomes and much more complexity, but Palsson noted that several theoretical studies predict that even eukaryotic cells will exhibit a relatively small number of functional states. When we uncover the regulatory networks in eukaryotes, including human, we will most likely be able to use computer simulations to uncover the different possible cell types in a manner similar to what was done in our work with E. coli, said Palsson.

Understanding how Rickettsia conorii interacts with host cells

Sat, 17 Dec 2005 15:40:38 PST

( from http://www.rxpgnews.com ) New research by a team of scientists in France and the United States has identified both the bacterial and host receptor proteins that enable Rickettsia conorii, the Mediterranean spotted fever pathogen to enter cells. Understanding how this bacterium interacts with the cells of its host could lead to new therapeutic strategies for diseases caused by related pathogens, including Rocky Mountain spotted fever and typhus.

Pascale Cossart, an HHMI international research scholar at the Pasteur Institute in Paris, together with her postdoctoral fellow Juan Martinez and collaborators in Paris and at Case Western Reserve University in Cleveland, Ohio, has identified the first receptor for a Rickettsial bacterium. Their findings will be reported in the December 16, 2005, issue of the journal Cell.

Rickettsial bacteria are transmitted by fleas, ticks, and lice to humans and other mammals, where they can cause dangerous and sometimes fatal infections. There are two types of Rickettsial pathogensthe spotted fever group, which includes the Rickettsia conorii bacteria studied by Cossart and her colleagues, and the typhus group. Both must live inside cells to survive. Rickettsia have been classified by the National Institute of Allergy and Infectious Diseases (NIAID) as agents with potential for use as tools for bioterrorism.

Mediterranean spotted fever is transmitted by a dog tick. The symptoms are generally mild and respond to antibiotics that shorten the course of the disease. But serious complications occur as much as 10 percent of the time, usually in patients who are elderly or who have some other underlying disease. Left untreated, Mediterranean spotted fever can be deadly.

Cossart and her team demonstrated that the Ku70 protein on the surface of host cells is critical for R. conorii to enter the cell, making it the first Rickettsial receptor ever identified. This receptor is a subunit of a protein complex present mainly in the nucleus, but also in the cell cytoplasm and at the cell membrane, said Cossart. We have thus used several approaches to establish our findings definitively. Ku70 is probably not the only receptor involved in bacterial entry, she noted.

The researchers found that R. conorii specifically binds to Ku70, and that binding and recruitment of Ku70 at the surface of the host cell are important events in R. conorii's invasion of mammalian cells. In addition, since Ku70 has previously been shown to control cell death, the new findings suggest that Rickettsia, whichlike several other intracellular parasitesprevent cell death in order to multiply inside living cells, may also use this property of their receptor for a succesful infection.

We found that Ku70 interacts with a bacterial protein called rOmpB, which is present on the surface of Rickettsia bacteria, Cossart said. The mechanism underlying this interaction remains unclear, so we are now investigating how rOmpB, expressed by R. conorii, interacts with Ku70 and allows bacterial entry.

Her team has already shown that Ku70 has to be present in certain well-organized regions of the cell membrane called rafts, and that the protein modifier called ubiquitin modifies Ku70 as soon as the bacteria interact with it. This step is critical for cell entry. Whether other Rickettsia and other pathogens use Ku70 as a receptor is still unknown, Cossart said.

CO2 sensing proves critical for fungal pathogens

Mon, 28 Nov 2005 01:33:38 PST

( from http://www.rxpgnews.com ) By using pathogenic fungi as model systems for understanding fungal diseases, two groups of researchers are reporting new work that offers insight into how carbon dioxide (CO2) governs the morphogenic changes that allow pathogenic fungi to survive in different environments and invade the human body, and they provide new evidence for how CO2 sensing and metabolism utilize evolutionarily conserved enzymes to control the growth and sexual reproduction of pathogenic microbes.

The two studies are reported by Joseph Heitman and colleagues at Duke University Medical Center and by Fritz Mühlschlegel of the University of Kent, Jochen Buck at Cornell University, and their collaborators.

How organisms sense and respond to CO2 at the cellular level is not fully understood, but it is of great importance in understanding the biology of microbes, plants, and animals alike. For example, CO2 levels govern the detection of prey by female mosquitoes, control respiration in mammals, and of course play a critical regulatory role in photosynthesis in algae and plants; in addition, CO2 sensing and transport are involved in many cellular processes and virulence attributes of diverse pathogenic bacteria and fungi--in both B. anthracis, which causes Anthrax, and C. neoformans, which causes meningitis, CO2 induces the production of sugar-based capsules that surround and protect the invading cell from the host during infection.

In their new work, Mühlschlagel and colleagues studied the function of CO2 sensing in two major human fungal pathogens, C. albicans and C. neoformans. Both cause life-threatening, invasive infections in immunocompromised patients--for example, those infected with HIV or undergoing bone-marrow transplantation. The two fungi, which are distantly related in evolution, have different attributes governing their virulence in humans. For C. albicans, a transition between different morphological forms ("yeast" and "filamentous" forms) plays a major role, whereas for C. neoformans, synthesis of a polysaccharide capsule is key.

In the bodies of mammals, the CO2 concentration is more than 150-fold higher (5%) than it is in atmospheric air (0.033%). Consequently, C. albicans and C. neoformans are exposed to dramatically elevated CO2 concentrations when causing systemic disease. In their research, the authors identify CO2 as a physiological signal that induces the pathogenic filamentous transition in C. albicans; they also demonstrate that an ancient group of enzymes called adenylyl cyclases are the so-called chemosensors mediating both the CO2 -dependent filamentation in C. albicans and the capsule biosynthesis in C. neoformans. The authors go on to show that CO2 sensing in C. albicans is essential for superficial (skin) infections, in which yeast must be able to grow despite significantly lowered CO2 levels present at the skin surface. Based on their findings, the authors conclude that CO2 sensing is a vital mediator of fungal virulence in different host environments--for example, at different sites within the body.

In a related paper, Joseph Heitman and colleagues, also using C. neoformans as a model system for understanding fungal disease, provide new evidence that CO2 sensing and metabolism govern growth, sexual reproduction, and virulence of this pathogenic microbe.

In this work, the researchers investigated the CO2-sensing mechanism of C. neoformans. This pathogen normally infects the human host (a high-CO2 environment) from the air (low CO2) in the course of causing deadly fungal meningitis. The authors found that an enzyme called carbonic anhydrase (CA) plays a critical role in the yeast's growth in ambient air; the CA enzyme accomplishes this by providing bicarbonate substrates for fatty-acid biosynthesis and other cellular processes. In contrast, the CA enzyme is dispensible for the yeast's survival in the high- CO2 environment of the host. Another major finding of the work is that for C. neoformans, high CO2 blocks sexual differentiation, which is known to be a key process for generating infectious spores, and the CA enzyme plays a central role in the process. Together with the accompanying work by Mühlschlegel and colleagues, this study implicates a key role for CO2 in growth and differentiation in the fungal kingdom.

The identification of a link between CO2 sensing and fungal virulence may have broad implications for the study of microbial pathogens and the role of adenylyl cyclase in virulence trait regulation by CO2. Furthermore, it may in time facilitate the development of a new class of antimicrobial agents.

Human Papillomavirus Could Spread Through Blood - Study

Thu, 17 Nov 2005 16:39:38 PST

( from http://www.rxpgnews.com ) Potentially transmissible human papillomavirus DNA has been identified in human blood cells suggesting that the virus, traditionally thought to be sexually transmitted, could also be spread through blood products. Researchers from the National Institutes of Health report their findings in the November 2005 issue of the Journal of Clinical Microbiology.

Human papillomavirus (HPV) is most commonly recognized as a sexually transmitted disease that can cause genital warts as well as cervical cancer. It had been widely accepted that HPVs could not be disseminated through blood. However, the successful experimental transmission of bovine papillomavirus through blood several years ago suggested that might not be the case.

In the study researchers examined HPV DNA in banked, frozen blood cells from pediatric HIV patients and fresh blood cells from healthy donors. Results showed that eight HIV patient samples (seven of which acquired HIV through blood transfusions) and three healthy donor samples were positive for two subgroups of the HPV type 16 genome and that the DNA could exist in a transmissible form.

"Peripheral blood mononuclear cells (PBMCs) might serve as a source of HPV in the infection of epithelial cells and contribute to their nonsexual spread," say the researchers. "However, additional work is needed to confirm this as a possible mode of HPV transmission."

Gene Identified in Epstein-Barr Virus that May Contribute to Cancer

Thu, 17 Nov 2005 16:36:38 PST

( from http://www.rxpgnews.com ) Researchers have identified a gene in the Epstein-Barr virus that may contribute to the development of lymphoproliferative disease (LPD) in humans. Their findings appear in the November 2005 issue of the Journal of Virology.

Epstein-Barr virus (EBV) is a form of human herpes virus that is the causative agent of mononucleosis. It is often associated with various types of human cancers, specifically lymphoproliferative disease (leukemia and hodgkins/non-hodgkins lymphoma), in immunosupressed patients. In the study immunodeficient mice infected with an EBV mutant missing a gene that controls cell lysis (the rupturing of the infected cell to release new viruses) did not develop LPD, however, when mice were challenged with EBV containing the lytic gene, development of LPD was enhanced. These results indicate that lytic gene expression contributes to EBV-associated LPD.

"Our results suggest that the decreased ability of immunosuppressed hosts to control the lytic form of EBV may promote the development of LPD not only by allowing enhanced horizontal transmission of the virus but also by increasing the number of lytically infected tumor cells," say the researchers.

New Gene Identified for Antiviral Activity

Thu, 17 Nov 2005 16:36:38 PST

( from http://www.rxpgnews.com ) Researchers have identified a gene in mice capable of producing an innate antiviral response to infection. Their findings appear in the November 2005 issue of the Journal of Virology.

The innate immune response, largely composed of the alpha/beta interferon system, is the first defense against controlling viral infections. These interferons are produced in response to viral infection and stimulate specific genes to produce antiviral compounds. These interferon-stimulated genes (ISGs) are responsible for many of the bodies' innate antiviral activities, but there are still some effects yet to be explained by the genes already identified.

In the study researchers used a modified Sindbis virus to express selected ISG responses in mice and looked for an attenuated infection. Through this approach they identified the interferon-stimulated gene 15 (ISG15) as having antiviral activity, protecting mice against mortality and decreasing viral replication in multiple organs.

"We show that expression of ISG15 in INF-á/âR mice attenuates Sindbis virus infection, providing in vivo evidence that ISG15 can function as an antiviral molecule," say the researchers.

Rapid tests for meningitis and MRSA are being developed

Mon, 17 Oct 2005 19:20:38 PST

( from http://www.rxpgnews.com ) Rapid tests for serious disease such as meningitis, chlamydia and the hospital superbug MRSA are being developed by a new company, Atlas Genetics Ltd, which is being launched using £500,000 funding and the expertise of academics at the University of Bath.

Current hospital tests take up to 72 hours, by which time the patients may have become seriously ill and may have spread the disease.

Patents have been filed in relation to Atlass key technology and it is expected that it will enable hospitals and eventually GPs to perform tests on the spot and make decisions about treatment within 20 minutes. The potential market for Atlass products is valued at over $3 billion (£1.7 billion) and is growing rapidly.

The company is working on a product that will analyse a clinical sample of blood, urine or saliva using a test cartridge inserted into a small, portable instrument.

The basis of this sensitive and specific test is an electronic tag developed by the company that automatically indicates the presence of DNA from the bacteria causing the disease.

This innovative technology has been developed over the last three years with a team of leading scientists from the Department of Chemistry at the University of Bath, including Professor Laurie Peter, Dr Toby Jenkins, Dr Chris Frost and Dr Stephen Flower. This was in collaboration with companies now acquired by Osmetech Plc, which is also a party to the current joint venture.

The commercial and technical management team of Dr John Clarkson, Dr Gordon Forrest, Dr Russ Keay, Alison Kibble and Karen Yates is highly experienced and have held senior positions in medical diagnostics, health care marketing, product development and finance.

The £500,000 already raised came in part from the Sulis Seedcorn Fund, which provides support for the new businesses set up using research carried out by the Universities of Bath, Bristol and Southampton. The Sulis institutional investment matched a £250,000 investment from a private investor who was introduced by the South West Angel and Investor Network (SWAIN), which brings together investors and companies seeking equity funding in the South West region.

Atlas was set up with the guidance and support of the University's Research and Innovation Services, which markets the University's resources of people, facilities and intellectual property to generate funds in support of research and teaching.

Getting Closer to a Vaccine for Hookworm

Fri, 07 Oct 2005 15:11:38 PST

( from http://www.rxpgnews.com ) Hookworms are intestinal parasites of mammals, including humans, dogs, and cats; in humans, these infections are a leading cause of intestinal blood loss and iron-deficiency anemia. These infections occur mostly in tropical and subtropical climates, and are estimated to infect about 1 billion people worldwideâabout one-fifth of the world's population. People who have direct contact with soil that contains human feces in areas where hookworm is common are at high risk of infection; because children play in dirt and often go barefoot, they are at highest risk.

However, since transmission of hookworm infection requires development of the larvae in soil, hookworm cannot be spread person to person. Anthelminthic chemotherapy with benzimidazole drugs is effective at eliminating existing adult parasites. But since reinfection occurs rapidly after treatment, making a vaccine against hookworm disease is a public health priority. Previous animal vaccine studies have had mixed results. Dogs have been successfully vaccinated against infection with the dog hookworm Ancylostoma caninum by immunization with attenuated third-stage infective larvae (L3). Varying levels of efficacy have been reported for vaccination against the major antigens secreted by the same larval stage in hamsters and dogs. However, only partial reductions in parasite load have been reported. In addition, protective antigens from the larval stage are only expressed in larvae, not in adult worms; hence, antibodies against L3 secretions are useless against adult stage parasites in the gut.

In this month's PLoS Medicine, Alex Loukas and colleagues suggest that the ideal hookworm vaccine would be a mixture of two recombinant proteins, targeting both the infective larva and the blood-feeding adult stage of the parasite. Such a vaccine would limit the amount of blood loss caused by feeding worms and maintain normal levels of hemoglobin, said the authors. This outcome is particularly important in young children and women of childbearing age, where menstrual and, particularly, fetal hemoglobin demands are high.

Of the different proteins expressed by blood-feeding parasitic helminths, proteolytic enzymes have shown promise as intervention targets for vaccine development. A previous study in which dogs were vaccinated with a catalytically active recombinant cysteine hemoglobinase, Ac-CP-2, induced antibodies that neutralized proteolytic activity, and provided partial protection to vaccinated dogs by reducing egg output and worm size, but there were not significant reductions of adult worm burdens or blood loss.

In the present study, the researchers found that vaccination of dogs with recombinant Ac-APR-1, an aspartic hemoglobinase that initiates the hemoglobin digestion cascade in hookworms, induced antibody and cellular responses, and resulted in significantly reduced hookworm burdens and fecal egg counts in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss and most did not develop anemia, the major pathologic sequelae of hookworm disease.

The authors went on to show that IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro, and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interfered with the parasite's ability to digest blood.

This result of vaccination against APR-1 shows the best efficacy so far reported for a recombinant vaccine aimed at reducing hookworm egg counts, intestinal worm burdens, and hookworm-induced blood loss, say the authors. They suggest that vaccination with APR-1 damaged the parasite's intestine and resulted in decreased blood intake by feeding worms, and, hence, reduced blood loss from the dogs.

The authors go on to suggest that the optimal hookworm vaccine would combine two elements: one to prevent L3 from developing into adult blood-feeding hookworms, and one to block the establishment, survival, and fecundity of the adult parasites in the intestine. Achieving both goals would require a vaccine comprised of an L3 antigen, such as ASP-2, which is now under clinical development, and an adult gut protease, such as APR-1.

These results have implications for human hookworm vaccine development; the authors finish by saying that there is now enough evidence to conclude that the counterpart vaccine for the major human hookworm Necator americanus (Na-APR-1) should be developed and entered into human clinical trials.

E.colis Defense Mechanism Uncovered

Thu, 29 Sep 2005 21:27:38 PST

( from http://www.rxpgnews.com ) Researchers at the Georgia Institute of Technology and the John Innes Centre in the United Kingdom have uncovered a mechanism with which disease-causing bacteria may thwart the bodys natural defense responses. The findings, which could ultimately lead to the development of more effective antibiotics, appear in the September 29, 2005 issue of the journal Nature.

Nitric oxide is produced by the body to fight infections. We discovered a mechanism that allows bacterial cells to detect nitric oxide and turn it into something thats harmless to the cell, said Stephen Spiro, associate professor in the School of Biology at Georgia Tech.

Spiro, along with colleagues Benoît D'Autréauz, Nicholas Tucker and Ray Dixon from the John Innes Centre, studied a non-pathogenic strain of Escherichia coli, which is very closely related to salmonella bacteria.

The pathogenic forms of E. coli and salmonella are usually transmitted to humans through undercooked meat, unwashed vegetables and cross contamination from surfaces on which these foods were prepared. Infections from either of these organisms can cause diarrhea, abdominal cramps and sometimes more serious illnesses that require hospitalization. E.coli doesnt respond well to antibiotics, while salmonella has developed several drug-resistant strains. Learning how the bacteria handle the bodys immune response is the first step in developing more effective medicines.

Spiro and colleagues focused their study on the NorR protein and the role it plays in reducing the levels of nitric oxide. In response to nitric oxide, NorR binds to DNA in order to regulate expression of an enzyme that reduces the amount of nitric oxide in the bacteria. Since nitric oxide binds to metals, the researchers suspected that there might be a metal in the protein.

Escherichia coli O157:H7 strain

It turns out that the protein NorR contains a single molecule of iron, said Spiro. Our study found that the nitric oxide binds to the iron, which in turn activates the protein.

Once activated, the protein controls expression of the norVW genes. These genes encode an enzyme that removes the nitric oxide, allowing the bacteria to fend off the bodys defenses.

The discovery of this mechanism is just the first step in what Spiro hopes will be a line of research aimed at disrupting the mechanism by which the bacteria rids itself of the poisonous nitric oxide.

If we can interfere with the mechanism, it could lead to better antibiotics and better treatments, said Spiro.

The research was funded by a grant from the Biotechnology and Biological Sciences Research Council.

Secrets to monoclonal antibody's success against West Nile Virus

Thu, 29 Sep 2005 20:53:38 PST

( from http://www.rxpgnews.com ) A monoclonal antibody that can effectively treat mice infected with West Nile virus has an intriguing secret: Contrary to scientists' expectations, it does not block the virus's ability to attach to host cells. Instead, the antibody somehow stops the infectious process at a later point.

"This was a complete surprise to us, but it gives us some very useful insights," says senior author Daved Fremont, Ph.D., associate professor of pathology & immunology and of biochemistry & molecular biophysics at Washington University School of Medicine in St. Louis. "Based on what we've learned, we are now developing therapeutic antibodies for related viruses that also are effective at stopping the process of infection after the virus attaches to host cells."

Detailed study of how the antibody physically binds to the virus has provided intriguing clues to how it may block infection. Scientists found evidence suggesting that the antibody prevents the virus from rearranging the protein envelope that surrounds its genetic material after it enters a host cell.

To reproduce, a virus must alter its envelope in order to inject its genetic material inside the cell. After that injection, the virus tricks the host cell into making more copies of the genetic material that can then be assembled into new viral particles or virions and sent out to infect other host cells and reproduce. But with the viral reproduction process blocked by the antibody, scientists suspect that the host cell eventually destroys the virion.

Fremont and colleagues, who publish their results in the Sept. 29 issue of Nature, hope to design a new diagnostic system that can determine whether vaccines for West Nile and related viruses undergoing clinical trials stimulate production of antibodies that stop infections at a similar point.

In 2004, West Nile virus, which is a mosquito-borne flavivirus, reportedly caused 2,470 infections and 88 deaths in the United States. First isolated in Africa in 1937, West Nile spread to the Middle East, Europe, and Asia before arriving in the United States in 1999. Most infections with the virus are mild or symptom-free, but infections in people with weakened immune systems and those over 50 sometimes lead to serious complications or death.

Like West Nile, dengue virus is a flavivirus spread by mosquito bites, but only in tropical regions of the world. The dengue virus is estimated by Centers for Disease Control and Prevention epidemiologists to cause100 million infections annually worldwide.

"Currently there are no effective and safe vaccines for pediatric dengue," says co-author Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, of pathology & immunology and of medicine. "Thanks to our data from the West Nile virus antibody, we believe we now have a much better idea of how to evaluate vaccines for dengue."

Fremont and Diamond led a team of researchers at Washington University and Macrogenics Inc., a private company, that announced the identification of the effective West Nile antibody earlier this year. In a line of mice genetically altered to increase vulnerability to the virus, they found injection of the new antibodies could boost survival rates of mice infected with the virus to greater than 90 percent.

Scientists at Macrogenics are working on the preliminary studies required before the West Nile antibody can be tested in humans. Meanwhile, researchers at Washington University wanted to know why the new antibody was so effective.

Antibodies normally work by binding to invaders to flag them for consumption and destruction by immune system cells known as macrophages. In the prior study, which screened several potential West Nile antibodies, scientists found that all the most potent antibodies bound to a particular section of a protein that makes up the exterior of the viral envelope. The envelope of a single viral particle or virion is comprised of 180 copies of this protein.

For the new study, scientists determined the detailed structure of a single antibody bound to its envelope protein target region using the technique of protein crystallography. Scientists were able to affirm in greater detail earlier observations suggesting that the antibody will be therapeutic for all strains of West Nile Virus.

Based on this data, they predicted how multiple copies of the successful antibody would bind to a virion.

"We were startled to find that the antibody only seemed to be able to attach to 120 of the 180 copies of the target region in the complete viral envelope," says Grant Nybakken, a Washington University M.D./Ph.D. student who was lead author of the study.

Further tests showed that virions covered in infection-stopping antibodies could still bind to host cells, while antibodies that were less effective at stopping infection could more effectively prevent the virion from binding to host cells.

How does an antibody that's better at preventing the virus from binding to host cells actually turn out to be worse at treating infection? The key may lie in a theory known as antibody-dependent enhancement (ADE) of infection, which has been observed in test tube studies of dengue virus and may be important to the onset of dengue hemorrhagic fever.

This theory suggests that dengue and other viruses may have developed tricks that let them reproduce inside macrophages, the immune cells that normally consume and destroy any object that they find covered in antibodies. In effect, these tricks turn antibodies that should be death warrants into passes into cells where invaders can reproduce.

Fremont cautions that this phenomenon has not been seen in West Nile virus, but notes that when he and his colleagues tested the ability of several antibodies to prevent West Nile from reproducing inside macrophages, they found that only the therapeutic antibodies blocked the virus' reproduction. The therapeutic antibodies' ability to stop reproduction in macrophages even worked when the virions were simultaneously exposed to antibodies known to enhance infection.

"Do the therapeutic antibodies also prevent the virus from properly injecting its genetic material into macrophages? It's a tempting possibility, but we don't have the evidence to prove it yet," he says.

Flaviviruses use a novel mechanism to evade host defenses

Thu, 29 Sep 2005 06:23:38 PST

( from http://www.rxpgnews.com ) Researchers from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, have made the surprising discovery that flaviviruses, which cause such serious diseases as West Nile fever, yellow fever and forms of encephalitis, evade immune system defenses in different ways depending on whether they are transmitted by mosquitoes or ticks. This finding could lead to new approaches to developing vaccines and treatments against these illnesses.

"Flaviviruses exact an enormous toll in terms of illness and death worldwide," notes NIAID Director Anthony S. Fauci, M.D. "Because this is a relatively new field of study, everything we learn about how these viruses operate is significant. This elegant work opens an array of new questions and research opportunities to pursue as we strive to better understand this family of viruses and develop countermeasures against them."

Mosquito-borne flaviviruses include West Nile virus, yellow fever virus, dengue virus and Japanese encephalitis virus; the less-familiar tick-borne flaviviruses are just as serious, causing tick-borne encephalitis or hemorrhagic fevers. Currently, a Japanese encephalitis outbreak is raging in India and Nepal and has killed more than 1,000 people. In Europe and Southeast Asia, tick-borne encephalitis typically results in more than 10,000 patient visits to hospitals annually and has a fatality rate of up to 25 percent in some regions. Viruses that cause encephalitis lead to inflammation of the brain. Hemorrhagic fevers are viral infections that cause capillaries to burst, leading to unusual bleeding on or under the skin or in various organs.

The study released this week online in the Journal of Virology describes how a single virus protein--NS5--from the tick-borne Langat flavivirus counteracts the natural ability of interferon to combat the virus. Langat virus was originally isolated in the 1950s in Malaysia and Thailand. Langat virus can infect people following a tick bite, but there are no cases of natural disease recorded. In the 1970s Langat was briefly used as a live vaccine against more virulent tick-borne encephalitis viruses in Russia but caused encephalitis complications in about 1 of every 10,000 people.

Interferon, the body's first defense against many viruses, triggers a cascade of immune defenses. According to researchers at NIAID's Rocky Mountain Laboratories (RML) in Hamilton, MT, NS5 blocks the body's attempt to signal for immune defenses, preventing the immune system from both stopping the spread of virus and helping the body recover from infection.

Interferon is so critical for recovery from these infections that it is being tested in clinical trials to treat infection with various flaviviruses. But the treatment appears to fail in about half of cases. Dengue virus, West Nile virus and yellow fever virus have a protein called NS4B that prevents interferon from functioning properly. It was thought that the tick-borne flaviviruses would use the same protein, so the NS5 finding was unexpected.

The RML group, directed by Marshall Bloom, M.D., chose Langat virus because it is spread by ticks--a trademark of RML expertise--and because it possesses the same survival mechanisms as the more serious tick-borne encephalitis, Omsk hemorrhagic fever (found in western Siberia) and the closely related Kyasanur forest disease (found in western India).

"These diseases are spread by the same tick that carries Lyme disease in the U.S.," says Dr. Bloom. "So, the fact that West Nile virus first appeared or emerged in the U.S. in 1999 should warn us about the potential for tick-borne flaviviruses to emerge on other continents." In preparation for such a development, Dr. Fauci notes that two other NIAID laboratories have similar flavivirus studies under way, and the three groups are building on the discoveries of each other.

Dr. Bloom says that all flaviviruses have a similar genomic structure, and many scientists thought they would use the same survival mechanism and respond to the same vaccines and therapies, but the RML work shows otherwise.

"NS5 prevents interferon from doing its sentry job and allows the virus to take over cells," says Dr. Bloom. "This is the first definitive study that dissects where the failure occurs in the signaling pathway, and then identifies some of the interacting partners in the cell and virus." Prior to this work, Dr. Bloom says, scientists knew only that NS5 helped tick-borne flaviviruses replicate.

RML's Sonja Best, Ph.D., who spearheaded the Langat virus work, says the group will continue to study tick-borne flaviviruses by examining the role and location of NS5 in Powassan virus. Powassan virus, found in North America, Russia, China and Southeast Asia, rarely infects people but is potentially fatal. If the research group can track the movement of NS5 in Powassan-infected cells and learn how it interacts with other proteins to block immune defenses, "that would provide a target for therapeutics to counteract tick-borne flaviviruses," says Dr. Best.

Adenovirus may deliver bird flu vaccine

Wed, 14 Sep 2005 01:45:38 PST

( from http://www.rxpgnews.com ) A harmless virus used as a delivery vehicle may help set a roadblock for a potentially catastrophic human outbreak of bird flu, according to researchers at Purdue University and the Centers for Disease Control and Prevention (CDC).

Purdue molecular virologist Suresh Mittal and his collaborators are investigating a new way to provide immunity against avian influenza viruses, or bird flu, the most lethal of which, H5N1, has a 50 percent fatality rate in humans. Under a $1.6 million grant from the National Institute of Allergy and Infectious Diseases (NIAID), the researchers are focusing on using a harmless virus, called adenovirus, as a transmitting agent for a vaccine to fight off highly virulent strains of the avian influenza viruses.

Current vaccines are designed for strains of flu found in local areas and are effective only as long as the virus doesn't change form. Existing vaccines will have limited success against new strains of avian influenza, he said. Every time a bird flu mutates, vaccines must be redesigned.

An additional important advantage to using an adenovirus as a vector, or transporter of vaccine into cells, is that is could be mass produced much more quickly than with current methods.

"Our approach is to use an adenovirus to deliver some components of the bird flu virus in a vaccine formulation," Mittal said. "We already know how to grow large amounts of adenovirus and how to purify it because adenoviruses already are used in clinical trials for gene therapy as vectors."

Mittal and collaborators Harm HogenEsch, co-principal investigator and head of Purdue's Department of Veterinary Pathobiology, and Jacqueline Katz and Suryaprakash Sambhara, both co-investigators from the CDC, are focused on creating better protection against the constantly mutating avian influenza viruses.

"The ultimate goal of our research is to develop an effective avian influenza virus vaccine that will provide long-lasting and broad immunity against multiple strains of this virus," Mittal said.

The proteins that form the basis for all of today's flu vaccines are grown in fertilized chicken eggs. It takes months to produce a new vaccine for a new virus strain using this method and limits vaccine supplies due to shortages in eggs for the purpose. The egg production method also creates difficulty in redesigning vaccine to keep pace with virus mutations.

"Do we still want to depend on the egg to make our flu vaccines?" Mittal said. "When these types of viruses strike humans, they also strike poultry. In that case, the availability of fertilized eggs to make enough vaccine will be compromised."

Even with a recently developed vaccine based on growing a single protein of H5N1, it would be difficult to rapidly produce enough protective medication to stem a pandemic, according to experts from the CDC, World Health Organization and NIAID. However, a large quantity of an adenovirus-based vaccine could easily be produced on short notice, Mittal said.

A "medium-level" bird flu pandemic in the United States would kill between 90,000 and 200,000 people with another 20 million to 47 million more sickened, CDC experts estimate. The economic impact on the United States alone would be between $71.3 billion and $166.5 billion.

Influenza viruses are respiratory pathogens responsible for widespread infections in humans, a variety of birds, marine mammals, pigs and horses. In Southeast Asia, millions of poultry have died or been euthanized because of H5N1 avian influenza.

Before the human cases of H5N1, there were no known cases of bird-to-human transmission that led to significant disease, Mittal said. However, this type of flu already has passed from poultry to people who had direct contact with ill birds or contact with contaminated bird feces, saliva or nasal mucous. A few cases have been documented of human-to-human infection, according to the World Health Organization.

In addition, migratory waterfowl at China's Qinghai Lake Nature Reserve have succumbed to H5N1. While migratory birds often can pass avian influenza to other animals, they generally don't become ill. Scientists from the U.S. Geological Survey National Wildlife Health Center believe this is further evidence that H5N1 could trigger a human flu pandemic. No cases of this type of bird flu have been found in the United States.

Since 2003 about 60 people have died in Southeast Asia from H5N1, about half of those afflicted. Outbreaks of H5N1 in birds have been confirmed in 11 countries, and the disease is spreading north into countries outside of Southeast Asia and already has been reported in Russia. The disease's spread, occurrences of other bird flu types and the already confirmed human cases, provide added impetus for finding more effective vaccine protection, Mittal said. The CDC reported two human cases of another bird flu in the United States in recent years - H7N2 in Virginia in 2002 and in New York in November 2003.

Viruses are classified according to the combination of two types of proteins found on the virus cell surface. The 15 types of hemagglutinin (H) protein and nine types of neuraminidase (N) protein form a large number of influenza viruses for which birds are the natural hosts.

New, often more dangerous, flu strains develop when the H and N combinations change. When the genes of a human or swine influenza mix with an avian variety, a highly pathogenic human flu likely will result, Mittal said.

These virus alterations work a bit like a jigsaw puzzle: As two viruses grow in the same animal, they replicate and reassemble their genome. If one virus takes a piece of genome from the other virus to fill an empty spot, then a new virus is born.

The potential for a pandemic exists when one of these new viruses is introduced into the human population. People with no previous exposure to this new flu strain have little or no immunity, making them highly susceptible to a virus that now can easily spread from person to person.

The last pandemic was 1968-69 when 34,000 Americans died of the Hong Kong flu (H3N2), a disease still circulating. In 1957-58 Asian flu (H2N2) killed 70,000 people in the United States. The worst flu pandemic was in 1918-19 when Spanish flu (H1N1) was fatal to 500,000 in the United States and as many as 50 million worldwide. Unlike other influenza outbreaks, the origin of that virus is still unknown.

Scientists believe that without the right vaccines and preparation, H5N1 has the potential to be one of the deadliest flu outbreaks if it mutates to easy human-to-human transmission. This flu strain was first identified in South Africa in 1961, with the first bird-to-human case recorded in 1997.

"Many people believe that this virus will mutate and become more widespread," Mittal said. "The question is when? Are we prepared? We hope that successful completion of this project will result in development of an adenovirus-based vaccine effective against pathogenic avian influenza viruses."

A fat-sugar complex that anchors LTA could be target to block bacterial CNS infection

Tue, 06 Sep 2005 20:21:38 PST

( from http://www.rxpgnews.com ) A single molecular anchor that allows bacteria to invade the nervous system may hold the key to treating many types of bacterial meningitis, a UCSD School of Medicine study has found.

By blocking the molecules anchoring ability, researchers may be able to find a way to stave off the most common serious infection of the central nervous system and a major cause of childhood death and disability. The researchers findings appear in the September 2005 issue of the Journal of Clinical Investigation.

Kelly Doran, Ph.D, assistant professor of pediatrics, Victor Nizet, M.D., associate professor of pediatrics, and their colleagues have identified a gene that produces a fat-sugar complex, which in turn anchors a molecule called LTA (short for lipoteichoic acid), found on the bacterial cell wall. This anchoring is a necessary first step for bacteria to cross from the bloodstream into the central nervous system through an anatomical obstacle called the blood-brain barrier.

Streptococcus, which can cause meningitis, has to penetrate the normally impermeable blood-brain barrier in order to enter the central nervous system and cause disease, said Doran. How this happens is not well known for bacteria. We wanted to see how bacteria interact with blood-brain barrier cells to begin the process of crossing over into the nervous system.

The team began by looking for new bacterial genes that allowed them to penetrate the barrier. Through a process that involved generating and screening thousands of Streptococcus mutants in a laboratory model of the human blood-brain barrier, the researchers found that a gene called iagA (short for invasion association gene-A) played a central role.

By producing a fat-sugar complex that anchors LTA, iagA establishes a link that allows bacteria to begin making its way into the nervous system. The researchers found that removing the iagA gene from the Streptococcus inhibited bacterial interactions with the blood-brain barrier, reducing mortality rates up to 90 percent in mice.

Mice that were infected with the normal, or wild-type, Streptococcus bacteria containing iagA died within days showing evidence of bacterial meningitis. In contrast, most of the mice survived when infected with bacteria missing the single iagA gene, Doran said. Blocking the anchoring of LTA on the bacterial cell surface could become new a therapeutic target for preventing bacterial meningitis.

Doran and Nizet noted that the study focused on how bacteria can begin the invasion process, and that additional Streptococcus toxins and the bodys own immune response also contribute to the development of meningitis. In their ongoing efforts, the researchers are looking at all of these factors in order to paint a complete picture of how the bacteria invade the brain and spinal cord to produce this potentially devastating infection.

Bacterial meningitis must be treated quickly and aggressively with antibiotics, since up to 25 percent of affected children may die or suffer permanent cognitive deficits, cerebral palsy, blindness, deafness or seizures. Therefore, an early acting treatment would help reduce the high rates of disability and death.

Previous studies have found that Streptococcus bacteria from infants with serious disease have significantly higher levels of LTA than bacterial strains in infants without symptoms, Nizet said. This underscores the importance of this anchor-LTA interaction, as well as its potential importance as a drug target.

Coronavirus HCoV-NL63 associated strongly with croup

Tue, 23 Aug 2005 21:06:38 PST

( from http://www.rxpgnews.com ) A forthcoming paper in the international, open-access journal PLoS Medicine makes the strongest association yet between a newly identified virus and the pediatric respiratory disease commonly known as croup. Following their recent description of the coronavirus HCoV-NL63, Lia van der Hoek and colleagues suggest this is one of the most frequently detected viruses in children with lower respiratory tract infections (LRTIs). These infections are estimated by the World Health Organization to be responsible for one fifth of all deaths in children under five years old.

The team, including researchers from University Medical Centres in Amsterdam, Bochum and Freiberg, determined the incidence of this novel virus in a sample of children under three years old with such respiratory infections. Nine hundred and forty nine samples of nasopharyngeal secretions were collected from both hospitalized patients and outpatients in four different regions of Germany. The study found that forty-nine samples (5.2%) were positive for the virus HCoV-NL63 overall, with a greater incidence in outpatients (7.9%) than hospitalized patients (3.2%). Co-infection with two other viruses also known to be prominent in the cause of LRTIs, was also frequently observed.

The researchers also investigated the occurrence of HCoV-NL63 in cases of respiratory disease where no other virus could be detected. Here, a strong relationship with the clinical symptoms associated with croup was apparent: 43% of the HCoV-NL63 positive patients with high HCoV-NL63 load and absence of co-infection had croup, compared with 6% of HCoV-NL63 negative patients. Previous studies have reported trends in croup, such as the relative susceptibility of boys to the disease, its peak occurrence in the second year of life and its predominance in late autumn and earlier winter, that are matched by patterns of HCoV-NL63 occurrence.

This systematic association of croup with HCoV-NL63 is particularly timely as the newly identified virus has spread worldwide, reported in Australia, Japan, the US and Canada. The study will also contribute towards the clarification of the viral causes of lower respiratory tract infections generally; causes that have not always been apparent despite the frequency of such infections amongst children.

Listeria monocytogenes uses receptor-mediated endocytosis to infect hosts

Mon, 22 Aug 2005 15:23:38 PST

( from http://www.rxpgnews.com ) French scientists have learned how Listeria monocytogenes, which causes a major food-borne illness, commandeers cellular transport machinery to invade cells and hide from the body's immune system. They believe that other infectious organisms may use the same mechanism.

The Listeria bacterium, found in soil and water, can be transmitted to humans via undercooked and unpasteurized food, causing flu-like symptoms or gastrointestinal distress. For individuals with weakened immune systems, listeriosis can be fatal, and infections during pregnancy can lead to miscarriage, stillbirth, premature delivery, or infection of the newborn.

The research was conducted by Pascale Cossart, a Howard Hughes Medical Institute international research scholar, and her colleague Esteban Veiga at the Institut Pasteur in Paris, and will be published in the August 21, 2005, issue of Nature Cell Biology. Cossart and Veiga detailed how Listeria invades cells by activating cellular machinery that transports viruses, small molecules, and proteins. Once it has safely entered a cell, the microbe can replicate and continue the process of infection.

The body usually deals with bacteria and other large, foreign microbes through a process called phagocytosis. Specialized cells engulf the invading microbe and destroy it. Scientists long believed that cells use a second process, called endocytosis, to deal with smaller molecules or viruses. In endocytsosis, a cell's outer membrane pinches inward around the target to form a pocket that's brought inside the cell, creating a structure called a vesicle.

"Phagocytosis and endocytosis may, in fact, be more similar than past research suggests. This is a totally new concept," Cossart says.

Cossart's lab had observed that Listeria which is 20 times the size of the largest particle scientists believed a cell could take in by endocytosis could invade non-phagocytic cells. Other labs had made similar observations with other bacteria. Cossart and Veiga investigated the underlying machinery behind this uncommon invasion strategy, which they knew depended on an interaction between a protein on the surface of the bacteria, known as InlB, and a protein called Met on the surface of the cell it was invading.

They discovered that when InlB interacts with Met, the cell responds by adding a chemical tag to Met that flags it for protein recycling or degradation. Since Met is on the outside surface of the cell and the recycling and degradation machineries are inside, the cell must bring Met inside through endocytosis in order to dispose of it. As the cell creates the vesicle that will transport tagged Met, Listeria stows away and invades the cell.

By manipulating the gene expression of the cells Listeria was invading, the researchers showed that specific molecules known to be involved in endocytosis were essential for successful invasion by Listeria. Similarly, they found that an enzyme that tags proteins for recycling was also required.

Listeria's use of receptor-mediated endocytosis to infect hosts, according to Cossart, suggests that other bacteria may exploit the same mechanism to gain entry into non-phagocytic cells. "This mechanism of cell entry may be used by several different kinds of bacteria, which is a major deviation from the belief that endocytosis is strictly for importing small molecules into cells," she says.

One bacterial cell can stop the growth of another on physical contact

Fri, 19 Aug 2005 22:28:38 PST

( from http://www.rxpgnews.com ) Scientists have discovered a new phenomenon in which one bacterial cell can stop the growth of another on physical contact. The bacteria that stop growing may go into a dormant state, rather than dying. The findings have implications for management of chronic diseases, such as urinary tract infections.

The discovery by a team of scientists working in the laboratory of David Low, professor of biology at the University of California, Santa Barbara, is reported in the August 19 issue of the journal Science. The findings indicate that Escherichia coli, one culprit in urinary tract infections, contains genes that when turned on block the growth of other E. coli bacteria that they touch. The finding was a complete surprise to the scientists, said Low.

The discovery may eventually lead to new antimicrobial agents to halt bacterial growth which would be an entirely new system to shut bacteria down, according to the scientists. "This has potential implications for new antibiotics," said Low. "If bacteria can do this, then maybe we can do it."

Doctoral student and first author Stephanie Aoki, and a team of scientists working in the Low lab, made the discovery while studying other aspects of E. coli. After working for two years, the team identified two genes required for this "stop on contact" phenomenon.

"We don't know if these 'stopped' cells are dead or alive," said Low. "They don't grow after they've been touched. They don't grow on plates, but laboratory stains show they may be alive. You might call them dead, but they don't break apart the way dead cells do. These cells appear to stay intact, perhaps in a quiescent mode, or dormant state."

Aoki explained, "We are currently exploring how contact between bacteria can inhibit cell growth and determining what this contact-dependent inhibition of growth (CDI) system is used for. These genes are present in E. coli, including uropathogenic E. coli that cause urinary tract infections, and similar genes may be present in other pathogens such as the plague bacillus, Yersinia pestis."

Low said that one possible interpretation is that bacteria use this system to eliminate competition in the environments they grow in. "Another possibility is that the bacteria use the CDI system to shut themselves off inside a host, going into a dormant state where they may go undetected by the immune system," he said.

Thousands of women in this country have chronic urinary tract infections, noted the scientists. The disease seems to go away for awhile, then something triggers recurrence of the disease.

Work by Scott Hultrgen at Washington University has indicated that E. coli cells may hide in the walls of the bladder and urinary tract in a dormant state, explained Low. It is possible that the newly discovered CDI system contributes to this process.

"By studying the CDI system, we hope to understand more about how bacteria interact with each other and with their hosts, and how these interactions contribute to disease," said Aoki.

The findings may have repercussions outside of better understanding of urinary tract infections. Other diseases may have similar mechanisms, according to the scientists. "This research is in its infancy, but opens the door for exploration of the roles of contact-dependent growth inhibition in urinary tract infections and possibly other diseases," said Low.

"Aoki has discovered an entirely new phenomenon," explained Low, who has studied E. coli for over 20 years. "It is fascinating that bacteria have developed a system by which one cell can contact another and inhibit its growth."

Oral Vaccine from Bacterial Ghosts May Protect Against E. coli

Thu, 18 Aug 2005 02:44:38 PST

( from http://www.rxpgnews.com ) Researchers from Austria and Russia have developed an oral vaccine comprised of bacterial ghosts, or empty bacterial envelopes, which may protect against E. coli in animals and humans. Their findings appear in the August 2005 issue of the journal Infection and Immunity.

Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen associated with several life threatening diseases in humans. O157:H7, one of the most harmful and frequently studied strains of the bacteria can cause intestinal inflammation ranging from diarrhea to hemorrhagic colitis, with more severe cases afflicting children and the elderly. EHEC O157:H7 has also been identified as a bioterrorism agent. There is currently no specific treatment against EHEC infection and antibiotics are not recommended as they prompt the liberation of toxins which can worsen the clinical course of the disease.

Because the major reservoir for EHEC O157:H7 is cattle, researchers are focusing on a vaccine that will prevent infection in both humans and animals. In order to mimic the bacteria's natural route of infection they developed an oral vaccine in hopes of eliciting local immunity in the gut.

In the study production of the protein E-mediated lysis was controlled to produce EHEC bacterial ghosts, or non-living bacterial cell envelopes. They have the same surface components of live cells and are capable of inducing strong immune responses, but the lack of genetic material inhibits transfer of resistance genes. An oral vaccine containing the bacterial ghosts was administered to mice that were challenged with a lethal dose of the EHEC strain 55 days later. A single dose of the vaccine resulted in an 86 percent protection rate and mice receiving a booster after 28 days showed a 93 percent survival rate. Non-immunized mice challenged with the bacteria had a 26 to 30 percent rate of survival.

"Bacterial ghosts as candidate vaccines and carriers of foreign viral and/or bacterial antigens are under development as multivalent vaccines against diarrheal diseases of humans and might represent new, improved nonliving bacterial vaccines with excellent safety properties and high immunological potential," say the researchers.

Olives May Successfully Transmit Beneficial Bacteria to Humans

Thu, 18 Aug 2005 02:42:38 PST

( from http://www.rxpgnews.com ) Table olives may serve as a carrier for delivering beneficial bacteria to humans, according to researchers from Italy. Their findings appear in the August 2005 issue of the journal Applied and Environmental Microbiology.

Probiotic foods contain healthy bacteria intended to promote microbial balance, inhibit pathogens and protect humans from gastrointestinal diseases. Researchers are also investigating their role in reducing risk of cancer, preventing food allergies, and alleviating symptoms of lactose intolerance.

The researchers studied survival rates of various strains of four probiotic bacteria, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium bifidum, and Bifidobacterium longum, on table olives at room temperature. L. paracasei was noted for its survival on olives throughout the three month experiment and was recovered from fecal samples in four out of five volunteers who consumed 10 to 15 olives per day for 10 days.

"The results reported here suggest that table olives are a suitable substrate for delivering probiotic species, since populations of L. paracasei, a strain selected for its potential probiotic characteristics assessed in vitro and for its lengthy survival on olives, were detected in the feces of human volunteers," say the researchers. "This result meets one of the aims of the current research, that of finding new delivery systems ensuring the stability and viability of strains."

New Test May Simultaneously Identify Herpesviruses, Enteroviruses, and Flaviviruses

Thu, 18 Aug 2005 02:39:38 PST

( from http://www.rxpgnews.com ) Researchers from France may have developed a new method of simultaneously detecting viruses from three different families that cause diseases of the central nervous system in humans. Their findings appear in the August 2005 issue of the Journal of Clinical Microbiology.

Viruses afflicting the central nervous system are mostly caused by herpesviruses, enteroviruses, and flaviviruses. Human herpesvirus can lead to serious diseases such as encephalitis, myelitis, and meningitis. Eighty to ninety-two percent of aseptic meningitis cases are caused by human enteroviruses as well as several poliovirus serotypes. Tick-borne encephalitis and West Nile encephalitis are two of many viruses belonging to the flavivirus family.

In the study a new diagnostic tool that uses reactive primers to detect for each family of viruses followed by DNA probe technology to differentiate between virus species within each family was tested. Researchers were able to accurately identify herpesviruses, specifically herpes simplex virus type 1 and 2, all serotypes of human enteroviruses and five flaviviruses including West Nile, Dengue, and Langat virus.

"This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis," say the researchers.

Research team isolates receptor for deadly viruses

Sun, 31 Jul 2005 14:12:38 PST

( from http://www.rxpgnews.com ) A collaborative research team from the Uniformed Services University of the Health Sciences (USU), the Australian Animal Health Laboratory (AAHL) and the National Cancer Institute (NCI) have made a major breakthrough in efforts to combat two deadly viruses that could be engineered for use as bioweapons. The team isolated the functional receptor for the Nipah and Hendra viruses--naturally occurring and highly pathogenic paramyxoviruses for which no treatments or vaccines are currently available.

Christopher C. Broder, Ph.D., associate professor in USU's Department of Microbiology, and his NIH-funded team of researchers and investigators demonstrated that a cell surface protein called Ephrin-B2 is a functional receptor for both the Hendra and Nipah viruses. Many animal species are vulnerable to these viruses, making the potential for amplification in intermediate hosts and transmission greater. Ephrin-B2 is highly conserved in animals, and this finding sheds light on how these viruses can infest such a wide range of hosts.

"In addition to our concern about Nipah and Hendra viruses as emerging global health and economic threats, we worry about their potential use as bioterror agents," stated Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases, the arm of NIH that funded the research, in an NIH news release. "This work, funded through our biodefense research program, is a major step towards developing countermeasures to prevent and treat Nipah and Hendra viruses."

"Now that we've identified the cell receptor, we have a new target for activity, hopefully blocking the viruses from infecting cells," Dr. Broder explained. Team members Matthew Bonaparte, Ph.D., and Anthony Dimitrov, Ph.D., both at USU, identified the cell receptor by analyzing a human cell line that was resistant to virus infection against two susceptible cell lines. The results of the research were published in the July 26 edition of the Proceedings of the National Academy of Sciences.

"We identified genes that are coded for known and predicted cell surface proteins that were missing from the resistant cell line," Dr. Broder said. "The genes were put into cells that were then exposed to a live virus at AAHL."

Hendra virus was first isolated in 1994 when an outbreak of respiratory and neurologic disease emerged among horses and humans in Hendra, Australia, killing two people. Hendra recently reemerged in Queensland, Australia, and researchers there isolated the virus at the biosafety level 4 facility.

Nipah virus, which is similar to and in the same genus as Hendra, was initially isolated in 1999, when a large outbreak of encephalitis and respiratory illness occurred in Malaysia and Singapore, killing more than 100 people. Last year, two further Nipah virus outbreaks occurred in Bangladesh, killing roughly 75% of those infected. Scientists are disturbed by the fact that many of these recent cases involved human-to-human transmission of Nipah, which originates in bats.

Ephrin-B2 is found on cells in the central nervous system, as well as in cells lining blood vessels. It is essential for central nervous system development and blood vessel growth in the embryos of humans and other mammals.

Broder and his team are among only a handful of scientists focusing on the viruses, which have been under investigation by USU researchers since 2000. Broder is a principal investigator on one of six projects from the Mid-Atlantic Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases funded by NIH.

The research has led to two inventions on which USU and the Henry M. Jackson Foundation for the Advancement of Military Medicine have filed patent applications.

The first patent application, "Soluble forms of Hendra Virus and Nipah Virus G glycoprotein," covers the production and use of the soluble G glycoprotein. This protein has utility as a vaccine, in the development of pharmaceutical compositions and in diagnostic assays. The second patent application, "Compositions and Methods for the Inhibition of Membrane Fusion by Paramyxoviruses," covers the use of a novel peptide sequence of the soluble F glycoprotein, to block fusion of the virus with the host cell. This peptide can be used as a prophylactic, and/or to treat infections, and antibodies developed using this peptide can be utilized in diagnostic assays.

Mechanism that lets herpes simplex virus infect is discovered

Mon, 25 Jul 2005 16:11:38 PST

( from http://www.rxpgnews.com ) It's one of the most common viruses in America, and one that causes the most guilt and shame. It can get inside almost any kind of human cell, reproduce in vast numbers, and linger for years in the body, causing everything from recurrent genital blisters to sores around the mouth. Its complications can kill, and it may increase susceptibility to many nerve and brain disorders.

But until now, scientists haven't fully understood how the herpes simplex virus (HSV) manages to do all of this. And that has stood in the way of developing more targeted, effective treatments against it to help those infected.

New research from the University of Michigan Medical School may help change that.

An estimated 45 million Americans have genital herpes and millions more have the more visible oral variety. Once someone is infected, they're infected for a lifetime. New medicines for herpes infection are badly needed; currently, antiviral drugs can quell symptoms of an outbreak, but not eliminate the virus. And, there's increasing evidence that HSV may damage the nerve cells in which it hides between outbreaks, possibly contributing to neurological disease.

In a presentation Sunday at the International Congress of Virology and in two new papers in the Journal of Virology, U-M researchers are reporting the discovery of a receptor that appears to function as one "lock" that HSV opens to allow it to enter human cells. They've also found the gene that controls the production of that receptor, deciphered some aspects of the receptor's structure, and developed a pig-cell system that could be used to test new anti-herpes drugs.

The findings may help explain why the oral and genital herpes virus has such a successful track record: The receptor, dubbed B5, is made by most cells for another purpose not yet understood. HSV appears to have evolved a way to latch onto it, and fool the cell into letting the virus in. And since most cells express the gene for the B5 receptor, this may be a reason HSV can get into most kinds of cells.

"This may be one central part of the Achilles' heel in interactions of herpes virus with a cell to start infection. We can use the receptor molecule to try to understand the process and perhaps combat infection at this vulnerable site," says A. Oveta Fuller, Ph.D. the leader of the U-M team, senior author on the two papers and an associate professor in the U-M Medical School's Microbiology and Immunology Department. "While we're still a few years away from being able to use this new knowledge to find effective drug candidates, this is a very exciting confluence of discoveries."

The U-M holds a patent on the system and methods that the team used to make the discoveries.

Coincidentally, the U-M team's findings about the B5 receptor are being published at about the same time as an Italian team's reports about a possible 'key' on the herpes simplex virus surface that may match the 'lock' found by the U-M team. The Italian team has identified a region of a viral surface protein that matches the U-M team's predictions of what the virus likely would use to bind and engage the B5 receptor.

"It appears that B5 is a new class of viral receptor. Unlike other viruses so far, HSV seems to have evolved to take advantage of a broadly present cellular protein that has properties like that of known cellular fusion machinery," says Fuller. "No other virus has been shown to use a cellular fusion protein for entry into cells."

She explains that the search for the mechanisms by which HSV enters cells has been hindered by the fact that the virus is very good at entering so many kinds of cells. The many possibilities for virus binding to cells make deciphering the entry process a difficult problem to solve.

The gene that encodes B5 had in fact been sequenced, but not characterized, as part of the Human Genome Project. Discovering its role and studying the HSV entry mechanism was tricky and near impossible until Fuller's team discovered a type of pig kidney cell that isn't vulnerable to infection by human herpes virus. They searched the genome library to find genes essential to HSV infection, isolated the B5-coding sequence, and figured out how to get pig cells to express the human B5 protein to allow the pig cells to be infected with human herpes virus.

For these studies, Fuller credits the persistence of research team members in working with the genomic library and culture of human and pig cells, especially U-M doctorate graduate Aleida Perez and postdoctoral fellows Qingxue Li and Pilar Perez-Romero. Perez-Romero is first author of one of the two new papers, and a co-author on the other.

The two new papers show that the B5 receptor has important features that could explain why it is important to HSV's ability to fuse with the fluid membrane that encloses every human cell. The researchers were able to show that by placing only the DNA sequence that encodes B5 into HSV-resistant pig cells, they could make the pig cells susceptible to HSV. They were also able to block viral infection of both human cells and susceptible pig cells by adding to cell cultures a synthetic peptide made to mimic the structure of a smaller region of the B5 receptor. This peptide looks like a functional region of B5 and apparently interferes with virus engaging of the cell receptor.

The papers detail how the team isolated and characterized the gene that encodes B5, called hfl-B5, and used the DNA sequence to find out more about the protein structure of the B5 receptor. In the presentation at the International Congress for Virology, Fuller will describe recent findings that further confirm B5's importance in HSV infection.

The virology team reports that the B5 molecule appears to form a shape called a coiled coil. This intricately wound structure, they believe, may be similar to the structure of some fusion proteins of viruses and also to cellular proteins called SNAREs. Typically, SNARE proteins help cells to manage the fusion of membranes of vesicles inside the cell with other specific vesicles. Vesicles are tiny membrane-encased packets that encapsulate neurotransmitters, enzymes or other important substances and allow them to be transported within and between cells.

The researchers were able to show that B5 sits in the cell membrane with one end of the protein exposed outside of the cell ready to link up with viruses -- or to serve the receptor's "real" function, which still remains to be discovered. They also showed that HSV does not enter into pig cells that have an altered human B5 protein that is changed by mutations that affect a functional region important to forming a coiled coil.

"If B5 is a SNARE-like cell fusion receptor", Fuller says, "it may turn out to be useful for more than HSV drug treatment. It could act as a way to link vesicles containing drugs with cells, and deliver them inside". She is currently collaborating with U-M nanotechnology researchers on this concept.

The findings suggest that B5 or its viral ligand could be a target for antiviral treatment, much like cell receptors for the entry of human immunodeficiency virus (HIV) into cells have become targets for new AIDS drugs.

Why 2003 Monkeypox outbreak in the US wasn't deadly

Sat, 16 Jul 2005 00:10:38 PST

( from http://www.rxpgnews.com ) An outbreak of 72 cases of monkeypox in the United States during the summer of 2003 didn't produce a single fatality, even though the disease usually kills 10 percent of those infected.

Why did none of the patients die? New research from Saint Louis University Health Sciences Center and several partner institutions may provide an answer.

In this month's issue of Virology, researcher and senior author Mark Buller, Ph.D., from Saint Louis University Health Sciences Center and colleagues conclude that some strains of monkeypox are more virulent than others, depending on where in Africa the virus came from.

"We have at least two biological strains of monkeypox virus - one on the west coast of Africa, and the other in the Congo basin," Buller said. "The 2003 outbreak in the United States was from West Africa. If it had come from Congo, we might have had a bigger problem on our hands and very well might have seen patient deaths."

Researchers from the University of Victoria, Washington University School of Medicine, U.S. Army Research Institute of Infectious Diseases, the University of Alabama, East Carolina University and the Centers for Disease Control and Prevention are co-authors of the research.

Buller said that recent studies suggest the incidence of monkeypox is increasing due to encroachment of human into habitats of animal reservoirs. Monkeypox is classified as a "zoonosis," which means that it is a disease of animals that can be transmitted to humans under natural conditions. The first cases of monkeypox reported in humans involved contact between humans and animals in Africa.

The first outbreak of monkeypox in the Western hemisphere occurred in the U.S. Midwest from April to June of 2003. The virus entered the U.S. in a shipment of African rodents from Ghana in West Africa destined for the pet trade. At a pet distribution center, prairie dogs became infected and were responsible for 72 confirmed or suspected cases of human monkeypox.

"Unlike African outbreaks, the U.S. outbreak resulted in no fatalities and there was no documented human-to-human transmission," Buller said.

Monkeypox is part of a family of viruses that cause human smallpox, cowpox, and camelpox as well as monkeypox. Monkeypox usually produces a less severe illness with fewer fatalities than smallpox. But its symptoms are similar: fever, pus-filled blisters all over the body, and respiratory problems.

"Our finding may explain the lack of case-fatalities in the 2003 monkeypox outbreak in the United States, which was caused by a West African virus," Buller said.

Biologists see combined structure of Coxsackievirus A21 and ICAM-1

Wed, 13 Jul 2005 12:29:38 PST

( from http://www.rxpgnews.com ) Biologists at Purdue University have determined the combined structure of a common-cold virus attached to a molecule that enables the virus to infect its host, information that ultimately may help researchers develop methods for treating certain viral infections.

Coxsackievirus A21 infects host cells first by recognizing a "receptor molecule" called ICAM-1, which is located on the cell's surface, and then by anchoring itself to the molecule. ICAM-1 stands for intracellular adhesion molecule 1.

"ICAM-1 is the same receptor molecule used by the vast majority of viruses that cause the common cold," said Chuan Xiao, a doctoral student who is leading the research in the laboratory of Michael Rossmann, the Hanley Distinguished Professor of Biological Sciences in Purdue's College of Science.

Findings will appear in the July issue of the journal Structure.

"The real objective of this work is to study the whole complex of ICAM-1 and the virus as a single entity," Rossmann said. "Being able to characterize the combined structure of the virus and ICAM-1 will teach us how the virus recognizes a particular kind of molecule and how it then anchors to the cell, which represents the initial stages of infection."

Ultimately, researchers are trying to learn more about the binding mechanisms because such knowledge might eventually lead to new treatments.

"One of the many different ways of inhibiting viral infection is to stop the virus from binding to cells," Rossmann said. "That has not been our objective in this case. We just want to learn how this virus infects its host cell. In other words, how does the virus get into the host?"

Coxsackievirus A21 is one of several viruses that cause the common cold.

The researchers used two methods to learn the structure of the virus-molecule complex. One method, a technique called X-ray crystallography, yielded images of the virus with a resolution of 2.5 angstroms, which is nearly fine enough to see individual atoms. Using this technique, researchers create crystals of a substance, in this case the virus. Then, X-rays are passed through the crystals, creating a "diffraction pattern" that can be interpreted with various computational procedures to produce an image.

The other method, a powerful imaging tool called cryo-electron microscopy, was used to determine the entire three-dimensional structure of the virus-molecule complex. With this technique, specimens are first frozen before they are studied with an electron microscope. Cryo-electron microscopy enables scientists to study details as small as 8 angstroms, resolution high enough to see groups of atoms. An angstrom is one ten-billionth of a meter, or roughly a millionth as wide as a human hair.

"The electron microscopy is necessary for studying the entire complex because you can't crystallize the complex of ICAM-1 and the virus," Rossmann said. "That's because crystallization often takes days, weeks or months, but the complex is only stable for hours, which means it doesn't stay together long enough to crystallize."

The researchers pieced together the overall structure of the virus and ICAM-1 by combining the high-resolution X-ray crystallography images of the virus with the lower resolution electron microscope view.

The findings represent the first time researchers have seen fine details of the complex's structure.

"It's important to see the shape of the complex because that could tell us how the virus recognizes the host cell," Rossmann said. "Knowing the structure might also reveal the initial stages of what happens after attachment, and indeed there probably are different steps in the attachment process.

"The receptor apparently binds into what we call a canyon, which is a surface depression on the virus, and it might do that in at least two different steps. Perhaps it binds once loosely on the surface, and then it might bind again deeply into the canyon to strengthen its attachment."

The research paper was written by Xiao; Carol M. Bator-Kelly, a technical assistant in Rossmann's lab; Elizabeth Rieder, a technical assistant for Eckard Wimmer, a virologist at the State University of New York at Stony Brook; Paul R. Chipman, an electron microscopist at Purdue; Alister Craig, a researcher from the Liverpool School of Tropical Medicine in the United Kingdom; Richard J. Kuhn, a professor of biological sciences at Purdue; Wimmer; and Rossmann.

The images are revealing new details about how amino acids, which are the building blocks of proteins, interact during the binding of Coxsackievirus A21 and ICAM-1.

"In general, some amino acids have a positive charge, and some have a negative charge, and these opposite charges can play a critical role in attracting a virus to a host cell," Rossmann said. "It turns out the attraction between negative and positive charges on the virus and on the host cell seem to be a dominating feature but not the entire story of the recognition process.

"There is also shape involved, and the canyon on the virus and features on the ICAM-1 molecule have to match each other, like a key going into a keyhole."

Xiao said the research is ongoing, and future work may delve into how Coxsackievirus A21 binds to another cell-receptor molecule called DAF, or decay acceleration factor. The virus binds to both ICAM-1 and DAF at the same time, so future work may result in finding the structure of a complex that includes both molecules attached to the virus.

How Nipah and Hendra viruses gain entry into cells

Thu, 07 Jul 2005 18:04:38 PST

( from http://www.rxpgnews.com ) Working independently, two research teams funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), have identified how Nipah and Hendra viruses, closely related viruses first identified in the mid-1990s, gain entry into human and animal cells.

Nipah and Hendra are emerging viruses that cause serious respiratory and neurological disease. People can acquire these deadly viruses from animals. Beginning in 1994, public health officials have recognized disease outbreaks in Malaysia, Singapore, Bangladesh and Australia.

Both viruses use a protein essential to embryonic development to enter cells and begin their often-fatal attack, report researchers at the University of California, Los Angeles (UCLA) and the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, MD.

The UCLA team, headed by Benhur Lee, M.D., describes its findings in a Nature paper posted online on July 6. The report by the USUHS researchers, led by Christopher Broder, Ph.D., is appearing online the week of July 4 in the Proceedings of the National Academy of Sciences.

The first reported outbreak of Nipah virus occurred in 1998-1999 in Malaysia, sickening 265 people and killing 105, according to the World Health Organization. This outbreak, which in this case spread from pigs to humans, was contained by culling more than a million pigs. Hendra virus, so far less of a threat to human health, was first identified in 1994 in Australia when it spread from horses to humans.

"In addition to our concern about Nipah and Hendra viruses as emerging global health and economic threats, we worry about their potential use as bioterror agents," says Anthony S. Fauci, M.D., director of NIAID. "This work, funded through our biodefense research program, is a major step towards developing countermeasures to prevent and treat Nipah and Hendra infection."

Using different methods, both teams identified a specific cell surface receptor, Ephrin-B2, as the doorway used by Nipah and Hendra viruses to get inside cells. This receptor is found on cells in the central nervous system and those lining blood vessels. It is crucial for the normal development of the nervous system and the growth of blood vessels in human and other animal embryos. Ephrin-B2 is found in humans, horses, pigs, bats and other mammals, which explains the unusually broad range of species susceptible to Nipah and Hendra virus infection.

Dr. Broder and his colleagues collaborated with researchers at the National Cancer Institute, also part of the NIH, and the Australian Animal Health Laboratory. The team narrowed the search for the Nipah/Hendra receptor by first sifting through the genetic sequences of 55,000 possible receptors using microarray technology as a molecular sieve.

The scientists compared microarray signals from the 55,000 genetic sequences in one set of Nipah virus-resistant human cells with microarray signals from three sets of human cells that the virus can infect. This enabled the research team to narrow the possible number of receptor proteins to 120 by identifying those present in the virus-susceptible cells but absent in the virus-resistant cells. They winnowed the possibilities further--to just 21--by selecting only those candidate receptors within the molecular weight range they expected. They selected 10 expressed at high levels in the susceptible cell lines and inserted them, one by one, into the cells that resisted Nipah virus to identify the receptor. When they inserted the gene for Ephrin-B2, the previously Nipah-resistant cells admitted the virus.

The UCLA team, with collaborators at the University of Pennsylvania, Philadelphia, took a different approach, using tools of advanced molecular biology as well as old-fashioned detective work to identify the Ephrin-B2 receptor. They knew the receptor would be abundant among the type of cells Nipah virus attacks, specifically, nerve cells and cells lining blood vessels.

To identify the human cell receptor, they created a bait: the Nipah protein that docks to the unknown receptor was attached to part of a human antibody, like a worm on a fishing hook. When they placed this bait onto cells susceptible to Nipah virus infection, it attached to a protein on the cell surface. When placed on Nipah-resistant cells, however, the antibody did not attach to the cells. The scientists used an instrument that sorts molecules by weight to identify that Ephrin-B2 was the cell receptor protein that bound to the antibody/Nipah protein "fishing pole" they had made.

They wanted to confirm their findings, but since they did not have access to a high-level biosafety laboratory as Dr. Broder's team did, the UCLA researchers engineered a harmless virus with Nipah virus proteins embedded in its coat. The UCLA team found that this artificial construct could infect cells vulnerable to Nipah virus but was unable to infect Nipah virus-resistant cells. They also showed that this engineered virus could infect nerve cells and cells lining blood vessels using Ephrin-B2 as the receptor, in the same way as actual Nipah virus would infect these cells.

Knowing the identity of the Nipah and Hendra receptor will not only help in developing vaccines and treatments, but also promises to lead to better understanding of how the viruses cause disease in people and a variety of animals, the researchers say.

Threadworms dependent on bacteria to survive

Sat, 25 Jun 2005 21:19:38 PST

( from http://www.rxpgnews.com ) The disease is triggered off by the bite of an infected mosquito: together with its anticoagulant the mosquito pumps threadworm larvae into its host's body. These gravitate towards the lymph nodes, where they grow into threadworms which may be up to ten centimetres long. The body reacts by producing inflammation which halts the flow of lymphatic fluid. The consequence of this is that arms, legs and genitals swell to monstrous proportions hence the name elephantiasis. More than 120 million people worldwide are infected with the pathogen wuchereria bancrofti.

Adult wuchereria worms have a lifespan of up to five years. During this time they produce millions of offspring, what are known as micro-filariae, each of them smaller than the full stop at the end of this sentence. If the host is bitten again by a mosquito, the micro-filariae are ingested together with the blood. Inside the insect they mature into infectious worm larvae, thereby completing the circle.

'Although the drugs currently in use kill the micro-filariae, they largely leave the adult worms unscathed,' Bonn parasitologist Professor Achim Hörauf explains. 'Due to the long lifespan of the wuchereria worms, therapy lasts several years, during which time the symptoms continue to persist.' What is more, the drugs may cause severe side-effects.

De-worming the roundabout way

Yet the threadworm, too, has a sub-tenant, and this may be its Achilles heel, since in each wuchereria worm there are specific bacteria which are absolutely indispensable to the parasite's survival. If these bacteria die, the parasite will also die sooner or later. 'This is why wuchereria is susceptible to antibiotics which are normally used against bacterial infections,' Professor Hörauf emphasises. One example is doxycyclin, which has been used for decades for infections of the respiratory tract and the gastro-intestinal tract.

In their study the medical experts in Tanzania treated 72 male patients for eight weeks with doxycyclin or a placebo. Initially the patients' blood was swarming with micro-filariae: the researchers counted up to 1,300 of them per millilitre of blood. Eight months later they had almost completely disappeared; only in one patient were sporadic micro-filariae still detected. However, the proportion of micro-filariae also dropped in the placebo group an effect which was probably due to the improved care given the test persons.

Unlike the drugs in use up to now the antibiotic also killed off the adult worms. Fourteen months after being treated with doxycyclin the doctors were only able to detect the typical movements of the worms ('the dance of the filariae') on ultrasound in one in five patients. In the placebo group the rate was 89%. In the doxycyclin group the concentration of specific worm proteins in the blood fell by over half.

Effective, cheap, few side-effects

'The importance of these findings for therapy should not be underestimated,' Professor Hörauf emphasises. 'The mature worms are after all responsible for such symptoms of the disease as the extreme swelling of the limbs. In the past there was no effective and reliable method of combating them.' The effectiveness of the antibiotic might be even greater than what was measured: 'We cannot exclude the possibility that several patients became re-infected in the months following treatment with doxycyclin. It is therefore quite possible that all the worms were killed and the remaining 20% are the result of re-infection which would no longer occur if infection was effectively prevented.

Doxycyclin has been used for many years and has only minor side-effects. However, in young children it may cause irreparable damage to the teeth and slow down growth of the bones. For this reason the antibiotic should not be used during pregnancy, either. For adolescents and adults, however, the drug is harmless. Moreover, it is comparatively cheap. 'Its biggest advantage is that it is already licensed for medical use,' Professor Hörauf points out. 'Elephantiasis hits the poor most of all. It is therefore not likely that the pharmaceuticals industry will develop completely new drugs.'

The Role of the Rab7 Protein

Wed, 22 Jun 2005 13:04:38 PST

( from http://www.rxpgnews.com ) When Robert Hooke first looked at cork bark with a light microscope in 1655, he saw small empty chambers, reminiscent of monastery cells. We now know that living cells are full of organellesspecialized subcompartments surrounded by membranes in which different cellular life functions occur. This complex organization raises major transport and sorting problems similar to those encountered in a large city in which trains and trucks carrying different cargos arrive at peripheral distribution centers.

The cargos must be sorted and transported to individual factories where goods are made for delivery to other city destinations or for export. At the same time, the different areas of the city produce waste products that also need to be sorted and transported correctly. Somehow, thousands of cargos must end up in exactly the right place in both the city and the cell.

One cellular system that sorts and transports cargos is the clathrin-mediated endocytic pathway. Endocytosisthe ingestion of materials into the cellis important for the interaction of cells with the environment because it allows the uptake of nutrients (the equivalent of the raw materials brought into the city) and signaling molecules (the letters brought in by the mail service). In clathrin-mediated endocytosis, materials arriving at the outside surface of the cell are engulfed in special areas of membrane known as coated pits, which pinch off to form intracellular vesicles. These lose their clathrin coat and other molecules involved in their formation to become early endosomes, a specific sort of intracellular vesicle. The cargos are then transferred to late endosomes, which have different proteins and functions than early endosomes. From endosomes, cargo can go either to lysosomes, where they are degraded, or to the Golgi apparatus, which sends cargo back to the cell surface.

Although many details of clathrin-mediated endocytosis have been uncovered, cell biologists still hotly debate whether early endosomes mature into late endosomes or whether transport vesicles take cargos from early to late endosomes. Unraveling such details will improve our understanding of normal cellular processes and should help in the design of intracellularly targeted drugs. Andreas Vonderheit and Ari Helenius now provide new insights into this controversy by examining how Semliki forest virus (SFV) is sorted and transported to late endosomes.

Like many animal viruses, SFV enters its host cells using clathrin-mediated endocytosis. One well-established way to study this process is to attach a fluorescent tag to individual virus particles and observe their travels through the cell. Vonderheit and Helenius now track this journey in greater detail than ever before by attaching different colored fluorescent tags to SFV and to protein markers of early and late endosomes. They then use video-enhanced triple-color microscopy to follow all the markers as they move through living cells. This analysis reveals that the virus is initially present in endosomes containing only proteins associated with early endosomes. Then, Rab7, a late endosome marker that is involved in transport of cargo from early to late endosomes, appears in distinct domains of these early endosomes. Finally, the viral cargo is transferred to a detached organelle that contains Rab7 but no early endosome markers. The researchers show that SFV transport to late endosomes requires Rab7 and the presence of intact microtubules, which often serve as a highway network along which vesicles travel.

The researchers conclude that, at least for SFV, the mechanism underlying sorting and transport from early to late endosomes falls somewhere in between the two existing models for clathrin-mediated endocytosis. Early endosomes, they postulate, have to acquire some characteristics of late endosomes before SFV can be transported to late endosomes in Rab7-positive vesicles. But other cargos, the authors point out, may follow different pathways through the cell.

Phages Affect Gene Expression and Fitness in E. coli

Wed, 22 Jun 2005 13:04:38 PST

( from http://www.rxpgnews.com ) Life is hard for bacteria. Not only must they constantly compete against their comrades for resources and living space, theyre also subject to infection by pathogensviruses called bacteriophageswhich can affect their ability to survive and prosper. Two types of bacteriophages threaten bacteria: lytic phages and lysogenic (or temperate) phages.

Acquisition of a lytic phage (for example, T2, T4, or T6) is an immediate death sentence for the bacterium; upon infection, a lytic phage subverts the bacteriums biochemical machinery to make copy after copy of itself until the bacterium bursts, or lyses, from the burden. In contrast, a temperate phage (for example, λ phage) can lie dormant for many generations before it co-opts the bacteriums machinery to reproduce, but eventually it, too, lyses the bacterial cell as it releases a host of new phages. From the perspective of the bacterium, it is better to be infected by a temperate phage than a lytic phage because infection with a lytic phage means instant death, while a temperate phage may lie dormant long enough for the bacterium to reproduce.

Temperate phages achieve dormancy by producing a phage gene product (in the case of λ phage, called cI) that represses the production of other phage genes; phage reproduction ceases as long as this repressor is produced. Once infected by a temperate phage, bacteria are protected from secondary infections by various other phages, because the temperate phage prevents the others from becoming established in the cell. But might temperate phage infection confer other advantages on bacterial survival?

Edward Coxs group at Princeton University examined this question by looking for evidence that temperate phage infection triggers changes in bacterial behavior. Working with λ phages, the authors studied how phage infection affects the regulation of genes that might impact the bacteriums survival by comparing the constellation of genes expressed in uninfected E. coli bacteria to those in E. coli carrying a dormant λ phage.

They found that λ phage caused reduced expression of the bacterial gene pckA, which codes for an enzyme that helps bacteria grow on carbon sources (fuels) other than glucose; without functioning pckA, bacteria grow normally in an environment containing glucose, but grow only slowly in an environment containing alternative carbon sources such as succinate. E. coli carrying λ phage fail to make the pckA gene product because the pckA gene is turned off by the virally encoded repressor cI. Interestingly, the researchers found evidence that the repressors made by other temperate phages may also be able to turn off pckA expression, and that the pckA genes of other bacteria related to E. coli might also be regulated by temperate phage repressors.

The fact that this relationship between temperate phage repressors and regulation of the pckA gene is so well conserved argues that the ability to turn off this gene might be positively selected; therefore, pckA repression must confer some sort of survival benefit to the bacterium. Its not clear what this benefit might be, but one explanation is that slowing bacterial growth in glucose-poor environments might help the bacterium elude detection by the immune system of any animal it invades, increasing its chances of survival. Alternatively, slower bacterial growth might slow down the onset of viral reproduction and eventual lysis. Regardless, it is clear that there is a strong relationship between the temperate phages and the bacteria they colonize. These results have significant implications for the evolution of fitness in these bacterial populations.

Three New Phases of Repairing DNA Damage in E. coli

Wed, 22 Jun 2005 13:04:38 PST

( from http://www.rxpgnews.com ) Any cell that receives a dose of radiation is placed in a dangerous situation. The DNA damage resulting from exposure to such radiation (or any other mutagen) can cause massive rearrangements to genetic information and potentially kill the cell. Bacteria have learned to cope with this threat by activating genes that repair DNA damage and by preventing a cell from dividing before these repairs are completed. In the bacteria Escherichia coli, these repair genes form what is known as the SOS response.

The E. coli SOS response has been used to study DNA repair for decades, and a great deal is known about how the more than 30 genes involved in the response function. Two proteins figure prominently in this response. The LexA protein acts as a repressor and inhibits the expression of SOS genes under normal conditions; in the event of DNA damage, the protein RecA inactivates the LexA repressor by enhancing its autocleavage into two fragments, which initiates the SOS response. While these initial stages are well understood, how all the SOS genes are coordinated, and ultimately turned off, is only beginning to be explored.

In a new study, Joel Stavans, Uri Alon, and colleagues have closely followed the SOS response in individual E. coli cells to investigate its dynamics. Previous studies, which monitored the temporal pattern of activation of entire populations of cells, found that SOS genes turned on in one peak upon DNA damage. But Friedman et al. found that SOS genes in individual bacteria respond to DNA damage in three precisely timed phases. This observation reveals the importance of examining complex processes at the level of single cells, while furthering our understanding of how the SOS response is structured in E. coli.

Friedman et al. monitored the SOS response by attaching a green fluorescent protein (GFP) to the promoters (the section of DNA responsible for activating a gene) of three SOS genes (lexA, recA, and umuDC). Bacteria expressing these promoter-GFP fusions became fluorescent within minutes of being exposed to UV radiation, visualized using time-lapse fluorescence microscopy. Since GFP fluorescence is directly correlated with the expression of each of the chosen genes (i.e., their promoter activity), the authors could gauge the SOS response rate upon DNA damage.

To induce the SOS response, the authors exposed E. coli cells to UV radiation. By monitoring individual cells at two-minute intervals after this dose, Friedman et al. found up to three peaks of promoter activity at roughly 30, 60, and 100 minutes. Although the amount of this activity and the average number of peaks varied between cells, the timing was always similar in different cells, suggesting a highly structured, timed response. When the authors averaged this response over the population, it washed out into a single peakwhich explains why the three peaks of expression were not previously detected.

A deeper look into the dynamics of the SOS response in single E. coli cells showed that it did not correlate with cell size, suggesting the SOS response is not synchronized with the cell cycle. In addition, Friedman et al. repeated their experiments in a bacterial strain lacking the SOS response gene umuDC. The peak pattern was altered in this mutant strain, and the precision in the appearance of the peaks was reduced. By re-examining the SOS response in single cells, Friedman et al. have visualized an accurately timed and synchronized DNA repair process. Modulations in response to DNA damage have also been observed recently in individual mammalian cells. Future experiments in E. colione of the most genetically tractable model systemsshould help explain how this timed response is related to the different pathways of DNA repair and shutoff of the response.

Virus Uses Tiny RNA to Evade the Immune System

Fri, 03 Jun 2005 16:52:38 PST

( from http://www.rxpgnews.com ) In the latest version of the hide-and-seek game between pathogens and the hosts they infect, researchers have found that a virus appears to cloak itself with a recently discovered gene silencing device to evade detection and destruction by immune cells.

The report by Howard Hughes Medical Institute (HHMI) researchers in an article published in the June 2, 2005, issue of Nature may be the first to show how a virus uses the gene silencing machinery for its own infectious purposes.

In people, plants, and worms, hundreds of tiny RNA molecules can silence specific genes by interfering with larger messenger RNAs (mRNAs). That interference prevents mRNAs from making proteins. Scientists do not know which genes are hushed by the microRNAs in people, but the new study bolsters growing evidence that the little molecules can play important roles not only in normal human cells but in infected cells as well.

A popular notion is that the whole system of generating small RNAs was designed to be a defense by cells against viruses. Our study shows that a virus can also adapt it to evade the immune response, said HHMI investigator Don Ganem, who is at University of California, San Francisco.

Ganem studies how viruses infect people and cause disease. When scientists found that RNA interference appeared to be a basic and widespread gene regulatory mechanism, it became clear that such a fundamental pathway could of course be pirated by a virus, said postdoctoral fellow Adam Grundhoff, co-first author of the paper.

Thomas Tuschl, a newly selected HHMI investigator at The Rockefeller University, had already reported the existence of several microRNAs encoded by Epstein-Barr virus, although their functions were unknown. Grundhoff and co-first author Christopher Sullivan, a postdoctoral fellow in Ganem's lab, started their search for viral microRNAs with a small virus, known as SV40, in the belief that its diminutive size would make it easier to understand the functions of any microRNAs they found.

SV40 is a relatively harmless monkey virus that can cause kidney infections in its natural simian host. In rodents, however, it can cause cancer. Although the SV40 genome has been found in some human tumors, its role in human cancer has been debated. The virus is better known as a model system that has greatly contributed to major scientific advances about how genes work.

To launch their study, Grundhoff wrote a computer program to screen the SV40 genome for possible microRNA precursors. MicroRNAs are made from messenger RNA molecules with distinctive hairpin folds. The hairpin structure is diced into a microRNA segment that works with another complex to disable other messenger RNAs with complementary sequences.

Among several dozen predicted microRNAs, the top candidate turned out to be abundantly expressed in human cells infected with SV40.

Sullivan soon found the target of the plentiful SV40 microRNA. It effectively targeted the messenger RNA for a protein known as T antigen, leading to its cleavage. SV40 may be the world's most studied virus, Sullivan said, and T antigen is its most studied part.

When SV40 enters a cell, it produces T antigen, which functions to trigger viral DNA replication. Unfortunately for the virus, T antigen also serves as a target for immune (T) cells, which can destroy infected cells and prevent the virus from spreading.

Conveniently, the microRNA that targets T antigen is made late in the infectious cycle, just when T antigen is no longer essential for virus replication. Further experiments showed that cytotoxic immune cells were more likely to kill cells infected with a mutant virus that cannot make the microRNA than the normal virus. Thus, microRNA-induced reductions in T antigen expression promote escape from antiviral T cells without affecting virus growth.

Viruses can use the host RNA inference machinery, which is often speculated to have evolved as an antiviral mechanism, to generate small RNAs that serve their own purposes the latest chapter in the long cat-and-mouse game known to virologists as host-virus coevolution, the researchers conclude in their Nature article.

Virus Subverts Cellular Defense for Reproduction and Escape

Wed, 27 Apr 2005 02:26:38 PST

( from http://www.rxpgnews.com ) Against the constant threat of infection by bacteria or viruses, one line of defense for the eukaryotic cell is the autophagosome. This double-membrane structure, which buds off from the endoplasmic reticulum, traps cytoplasmic intruders and, upon maturation, merges with a lysosome to destroy them. In this issue, however, Karla Kirkegaard and colleagues show that for one class of viruses, the autophagosome is not a holding cell but a breeding ground, and may even provide a novel escape route.

The viruses in question are picornaviruses, which include polioviruses and rhinoviruses. Infection of human cells with poliovirus is known to induce proliferation of double-membrane cytoplasmic vesicles that are morphologically similar to autophagosomes, but the origin and ultimate identity of these vesicles has not been resolved. To test whether these viral-laden vesicles are truly autophagosomes, the authors visualized two proteins: LC3, a specific marker for autophagosomes, and 3A, a part of the poliovirus RNA replication complex. After infection, these proteins colocalized, indicating the poliovirus was indeed within the autophagosome-like vesicles. LC3 also colocalized with LAMP1, a marker for lysosomes, indicating these vesicles mature in a manner similar to that of autophagosomes. This same effect could be induced simply by expressing two viral proteins.

All these results indicate that the virus stimulates production of vesicles that bear the traits of autophagosomes and contain the virus, but they dont indicate what the consequence is for viral replication. To determine that, the authors increased autophagosome production with two known stimulators of autophagy, tamoxifen and rapamycin. But rather than protecting the cell, this stimulation increased viral yield either 4-fold, in the case of tamoxifen, or 3-fold, for rapamycin. Conversely, inhibiting autophagosome production reduced viral yield. From these results, it seems the virus has subverted the components of the autophagy pathway for its own uses.

Inhibiting autophagosome production reduced viral yield inside the cell, but even more so outside. While they were not able to exclude other mechanisms, the authors argue that one possible explanation is that these vesicles are used by the virus to exit from the cell. Supporting this view, they produced electron micrographic images consistent with the fusion of the autophagosome with the plasma membrane and the extracellular release of its contents. This suggests that the virus, which is known to lyse cells to release new viral particles, has another, less lethal means of escape. This may increase the viruss chance of avoiding immune system detection as it infects new cells.

Monoclonal antibody cures West Nile Virus

Mon, 25 Apr 2005 19:58:38 PST

( from http://www.rxpgnews.com ) A newly developed monoclonal antibody can cure mice infected with the West Nile virus, scientists at Washington University School of Medicine in St. Louis report. If further studies confirm the effectiveness and safety of the antibody, it could become one of the first monoclonal antibodies used as a treatment for an infectious disease.

In a strain of mice that normally only has about a 10 percent survival rate after West Nile infection, scientists found that single doses of the antibodies given soon after infection could boost survival rates to 90 percent or higher.

"To our knowledge, these experiments are the first successful demonstration of the use of a humanized antibody as a post-exposure therapy against a viral disease," says senior investigator Michael Diamond, M.D., Ph.D., assistant professor of molecular microbiology, pathology and immunology and of medicine. "They also suggest antibody-based therapeutics may have a broader utility against other infectious diseases."

Diamond points out that Macrogenics Inc., of Rockville, Md., a company that contributed to the study and licensed the antibody from Washington University, must complete other preliminary studies before the antibody can be tested in humans. But he and his colleagues are excited both by the apparent potency of the antibody and its potential to help them explore new possibilities for treating related viruses that are more prolific causes of human disease and death.

"We could give a single dose of this antibody to mice as long as five days after infection, when West Nile virus had entered the brain, and it could still cure them," says Diamond. "It also completely protected against death from the disease."

Diamond and his colleagues will report their results in the May issue of Nature Medicine.

In 2004, West Nile virus reportedly caused 2,470 infections and 88 deaths in the United States. The mosquito-borne virus, first isolated in Africa in 1937, spread to the Middle East, Europe, and Asia before arriving in the United States in 1999. Most infections with the virus are mild or symptom-free, but infections in people with weakened immune systems and those over 50 sometimes lead to serious complications or death.

Scientists initially produced a panel of many West Nile virus antibodies from mouse cells. The human immune system would clear out these foreign antibodies quickly, so when they had identified a potent antibody, scientists at Macrogenics clipped out the genetic material that controls the antibody's targeting and cloned it into a human antibody. The "humanized" antibody should be less likely to induce an adverse human immune system response. A second round of tests in mice confirmed that the new antibodies retained their ability to stop West Nile virus.

Other monoclonal antibodies are currently in development or use as anti-cancer and anti-inflammatory treatments. An antibody against respiratory syncytial virus (RSV) is approved for use as a prophylactic treatment in children at risk of the disease in hospitals. Unlike the West Nile virus antibody, though, the RSV antibody has to be given prior to infection.

West Nile virus belongs to a family of viruses known as flaviviruses, several of which are spread by mosquito bites. Other flaviviruses include the virus that causes dengue fever, a potentially life-threatening infection prevalent in tropical cities. Centers for Disease Control and Prevention epidemiologists estimate that there are100 million cases of dengue worldwide every year.

"A lot of what we're learning from the West Nile virus antibody will be of consequence for the development of a pediatric dengue vaccine," says co-author Daved Fremont, Ph.D., associate professor of biochemistry and molecular biophysics and of pathology and immunology. "Currently there are no safe vaccines for dengue infections."

Important insights from the production and selection of the new antibody include a close fix on where the antibody binds to West Nile virus. Antibodies typically work by attaching to a piece of a foreign cell or substance, which causes immune system cells known as macrophages to pick up the substance and clear it from the body.

Binding to the invader is just the beginning of the battle, though. Some antibodies can bind to an invader but do so in a way that fails to slow the invader down or trigger a response from macrophages. In one rare case that involves the dengue fever virus, antibodies can adhere to the virus in a way that accelerates the infection.

From their initial pool of West Nile virus antibodies, researchers identified 46 that could bind to the West Nile virus' envelope (E) protein. Further testing showed that 12 could bind to the virus in a way that consistently neutralized it, shutting down infections in cell cultures and in mice.

To determine where these potently neutralizing antibodies were binding to the envelope protein, a task known as epitope mapping, researchers modified a yeast-based screening system. The system let them test individual antibodies for their ability to bind to many versions of the E protein, each with slight alterations. By analyzing the changes in the versions of the protein that antibodies had difficulty binding to, they isolated first a region of the E protein, known as domain III, and then a group of amino acids in that domain.

"The big surprise for us was that all of the potently neutralizing antibodies appear to recognize the same general region of this domain," says Fremont. "It was very consistentall the neutralizing antibodies that bind this domain adhere to that area; all the non-neutralizing antibodies that bind this domain adhere to different areas."

Fremont notes that while the E proteins of various flaviviruses are generally very similar, domain III can vary significantly. He and others are working to detail the precise mechanisms that allow the new West Nile antibody to block viral infection.

Diamond and Fremont are looking for other areas of the West Nile virus E protein that antibodies can bind to and neutralize the virus. Diamond is also using the yeast screening system to epitope map the sites on the dengue fever virus where antibodies can bind and inadvertently enhance infection instead of fighting it.

"We don't really understand on a molecular level what's happening in these cases, which are called enhancing antibodies," Diamond explains. "Epitope mapping may help us better understand this potentially dangerous interaction."

Genomes Offer Ecological Clues to Viruses

Tue, 19 Apr 2005 17:11:38 PST

( from http://www.rxpgnews.com ) Cyanobacteria have a long and checkered past. When their ancestors first appeared some 3 billion years ago, earths atmosphere likely contained mostly carbon dioxide, along with hydrogen sulfide, ammonia, nitrogen, and water vapor. Thought to be the first photosynthesizers, cyanobacteria forebears used water from their ocean habitat, carbon dioxide, and sunlight to make sugar, and produced oxygen as wastethe kiss of death for most ancient microorganisms, which eventually died from oxygen poisoning.

Modern cyanobacteria continue to exert disproportionate influence for their size. The Prochlorococcus group of cyanobacteriawhich measure in at less than a micron in diameter, allowing 500-plus individuals to fit comfortably on the head of a pinaccount for a significant fraction of global photosynthesis by virtue of their ubiquitous presence in nutrient-depleted ocean waters. Even tinier agentsthe viruses that infect these bacteria, called cyanophagesappear capable of wielding equally surprising influence on global cycles by affecting the population dynamics and evolutionary path of Prochlorococcus.

To better understand the nature of virushost interactions at sea, Sallie Chisholm and colleagues investigated the genetic makeup of three cyanophages. The marine phages resemble two terrestrial phagescalled T4 and T7that infect Escherichia coli but carry genes that appear specially adapted to infecting photosynthetic bacteria in nutrient-poor oceans.

Of over 430 completed phage genomes, only one (P60) infects cyanobacteria. Since marine phages likely face different selection pressures than their terrestrial equivalents, the authors explain, genome analysis can shed light on the agents of selection, besides providing a survey of marine phage types. Chisholm and colleagues chose to sequence three marine phagesone podovirus (P-SSP7) and two myoviruses (P-SSM2 and P-SSM4)based on their morphology and host range, and characterized their genomes. The P-SSP7 virus has genes that closely match many of T7s so-called core genessignature genes required for that viruss mode of infection, which involves killing its host. P-SSP7 also has the same genome structure as other T7-like phages, though it appears capable of coexisting with its host (based on the presence of an integrating enzyme) while T7 kills as it infects. Chisholm and colleagues go on to characterize the two myoviruses and find that both viruses share most of the core genes found in T4-like phages. And like T4 phages, both myoviruses lack the integrating enzymes, suggesting they share T4 phages homicidal approach to infection.

Beyond the core phage genes, Chisholm and colleagues also present a survey of genes likely derived from cyanobacteria that could play defining functional roles in marine phagehost interactions. All three cyanophages contain photosynthesis-related genes, some of which, the authors propose, may mean the virus helps the host maintain photosynthesis during infection. The podovirus also has a candidate gene involved in DNA synthesis, which the authors speculate might help the virus reproduce in nutrient-poor environments, and all three cyanophages carry genes involved in metabolizing carbon. The absence of such genes in terrestrial phages, the authors argue, lends support to the notion that marine phages have evolved different adaptive mechanisms in response to the ocean environment.

Given the intimate relation between virus and host, the effects of gene swapping between virus and host is likely to be a two-way street. Just as cyanophages may help shape the fate of their hosts, its likely that cyanobacterial genes influence phage ecology and perhaps even its range. The cyanophages characterized here take after two phages that were central to many fundamental breakthroughs in molecular biology, including the discovery that genes are made of DNA. It remains to be seen how the marine versions of these legendary laboratory viruses contribute to our understanding of phage infections in one of the most abundant, ecologically diverse primary producers in the open seas. See also the related Primer The Third Age of Phage (DOI: 10.1371/journal.pbio.0030182).

Tegument Proteins Help Route Herpesvirus

Tue, 29 Mar 2005 17:49:38 PST

( from http://www.rxpgnews.com ) Tegument proteins, which lie between the viral capsid and membrane envelope, route herpesviruses to either the cell bodies or axon terminals of neurons, according to Gant Luxton et al.

The α-herpesviruses, which cause cold sores and shingles, enter sensory neurons, where they take up lifelong residence. When the viruses become reactivated, the progeny virus particles travel down axons to the periphery, resulting in physical symptoms.

The viral proteins associated with the microtubule motors that allow this transport remain unknown. To determine which viral proteins were involved in trafficking, Luxton et al. used correlative motion analysis to simultaneously track fluorescently labeled capsid and tegument proteins in living neurons.

The researchers found that the tegument proteins are the key components of the capsid transport complex; when tegument proteins were associated with the capsid, the viral particles moved toward the axon (anterograde motion). Conversely, when tegument proteins were removed, viral particles moved toward the cell body (retrograde motion). Identifying tegument proteins as an important component of the capsid transport complex reveals a key mechanistic step in the infectious cycle of human herpesvirus.

Clues to a Parasitic Nematodes Bacterial Partnership

Tue, 29 Mar 2005 16:31:38 PST

( from http://www.rxpgnews.com ) More than a billion people are at risk for infection with filarial nematodes, parasites that cause elephantiasis, African river blindness, and other debilitating diseases in more than 150 million people worldwide. The nematodes themselves play host to bacteria that live within their cells, but in this case, the relationship is classic mutualism, with each benefiting from the other. Indeed, the Wolbachia bacterium is so crucial to its host nematode that apparently eradicating it with antibiotics severely compromises the nematodes ability to complete its life cycle within its human host. Thus, understanding the details of this symbiosis may help identify new strategies for controlling diseases caused by filarial nematodes. In a new study, Barton Slatko and colleagues present the complete DNA sequence of the Wolbachia pipientis strain within Brugia malayi, a parasitic nematode responsible for lymphatic filariasis, and analyze its genome for clues to the interdependence of the two species.

This Wolbachia genome is small, only about a million base pairs, and many metabolically critical genes have degraded through mutation to the point of uselessness. This phenomenon, called reductive evolution, is typical of long-term symbioses, as the two partners increasingly complement one anothers biochemical activities, reducing the selection pressure on otherwise lethal mutations. Wolbachias translational machinery and DNA repair equipment are largely intact. The bacterium appears to supply nucleotides to its host, as it contains complete pathways for biosynthesis of both purine and pyrimidine nucleotides. This is in contrast to Rickettsia, a close relative of Wolbachia and a mammalian parasite. Slatko and colleagues enumerate a variety of other pathways that have either been degraded or preserved, and highlight patterns in the genome structure through comparisons with both Rickettsia and another Wolbachia strain, found in fruit flies. For example, the two Wolbachia strains appear to have different membrane structures, possibly reflecting their different lifestyles (mutualistic versus parasitic).

Wolbachia can manufacture riboflavin and FAD, which are essential metabolic coenzymes and which do not appear to be made by its host. Conversely, it cannot synthesize amino acids and a variety of other vitamins and cofactors, and probably depends on the nematode to supply them. One discovery of possible significance is the presence in the bacterium of the synthetic pathway for hemethe oxygen-carrying iron component of hemoglobin. The nematode may require heme for synthesis of developmental hormones, so Wolbachias heme pathway may be an inviting target for therapy against nematode infection. Since no new antifilarial has been developed in two decades, these results may quickly lead to new therapeutic strategies against these parasites.

The Bacterias Guide to Survival

Tue, 22 Mar 2005 20:50:38 PST

( from http://www.rxpgnews.com ) From The Worst Case Scenario Survival Handbookwith handy entries like How to escape from killer bees and How to escape from quicksandto The Zombie Survival Guide: Complete Protection from the Living Dead, survival guides are one of the latest publishing fads.

If there was a market for it, a survival guide for bacteria might include topics like How to use your pili to keep your host from going apoptotic. A hosts cells can respond to a bacterial infection with apoptosis, or programmed cell death. For bacteria that pass directly from host to host, this can pose a problem. If the bacteria are highly virulent and induce too much cell death, they could take down their host before theyre able to jump ship, thus hurting the bacterias chances of survival in the long run.

Earlier studies suggested that bacteria can use their pili, finger-like appendages that many bear on their surface, to pull on a hosts cell membranes and thus influence the cells behavior. But these studies, which looked at mutant bacteria that could not retract their pili, did not examine the matter of how the bacteria coax their hosts to stay alive.

Now, in PLoS Biology, a group of researchers present more direct evidence that bacteria can induce changes in hosts gene expressionand possibly keep the host cells alive longerthrough tiny tugs on cell membranes. The study, led by Magdalene So, examined gene activity in human epithelial cells infected with Neisseria gonorrhoeae, the bacteria responsible for the sexually transmitted disease gonorrhea.

By comparing cells infected with normal N. gonorrhoeae to those infected with a mutant strain with defective pili, the researchers found a subset of 52 host genes that had higher activity when the host was infected with the normal bacteria, suggesting that the pulls of the pili were responsible. They also ran a key control experiment with an artificial mechanical pull on the host cell membrane. By coating magnetic beads with a preparation of bacterial pili, the beads attached themselves to the cell membranes. Then, in the presence of a magnetic field, the beads tugged on the cell membrane, approximating the effects on gene expression during infection with normal bacteria.

Thus, the mechanical tugs seem responsible for triggering a signaling cascade in the host cells, which ultimately affects the hosts gene expression. Many of the genes that increased in activity due to the tugs were already known to regulate apoptosis and cellular response to stress, including mechanical strain on the membrane. Also, a majority of these genes were known to be induced by a family of proteins called mitogen-activated protein kinases, or MAPKs. The researchers showed that blocking MAPKs reduced the activity of several of the genes that are usually enhanced by infection with the normal bacteria. Also, they found that cells infected with the bacteria tended to survive treatment with staurosporine, a chemical that normally induces apoptosis.

Overall, the groups findings support previous speculations that some bacteria influence gene expression and the fate of cells in their hosts by tugging on the host cells membranes with their pili. For bacteria like N. gonorrhoeae that pass directly from host to host, the researchers argue, it would be in a bacteriums interest to help keep its host alive. And bacteria appear to do this with the help of their pili.

How a Latent Virus Eludes Immune Defenses

Tue, 22 Mar 2005 20:50:38 PST

( from http://www.rxpgnews.com ) For a virus to survive, it must elude the ever vigilant immune sentinels of its host. A latent virus can escape immune detection if it resides in nondividing cells and doesnt produce any proteins. No viral proteins means no red flags for immune cells. If the virus targets one of the many cell types that rarely divide, its relatively safe while latent. But some viruses, like the gamma-herpesvirus, infect B cells of the immune system, which occasionally divide. The gamma-herpesvirus genome persists as circular pieces of DNA called episomes. When an infected B cell divides, the latent gamma-herpes virus episome must replicate and segregate into daughter cells along with the cells genome. Viral replication and segregation requires the services of a protein called the episome maintenance proteina potentially recognizable target for immune cells.

Gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposis sarcomaassociated herpesvirus (KSHV), can induce uncontrolled lymphocyte (immune cell) proliferation and result in lymphoma, Hodgkins disease, and Kaposis sarcoma. These diseases arise from the persistent latent infections that take hold after initial infections are controlled by immune defenses. The episome maintenance protein produced by EBV, called EBNA-1, harbors an amino acid element in its epitopethe region that binds to a T cell and triggers an immune responsethat helps the viral protein evade the killer T cells that could destroy it. Lab studies show that the amino acid element limits EBNA-1s interaction with T cells by inhibiting synthesis and, to a lesser degree, degradation of the protein. How this evasive action works or helps the virus in a living organism is not entirely clear. But if T cells arent presented with bits of viral protein, they have no way of knowing the virus is present.

In a new study, Neil Bennett, Janet May, and Philip Stevenson explore this question by studying virushost interactions in mice infected with the murine gamma-herpesvirus-68 (MHV-68). Though MHV-68 infects mice, it behaves similarly to EBV and KSHV infections in humans, producing an acute mononucleosis-like illness and a pervasive pool of latently infected B cells. The episome maintenance protein in MHV-68 and KSHV is called ORF73. None of the viruses can maintain latent infections with deficient episome maintenance proteins.

Stevenson and colleagues first demonstrated that ORF73 limits T cell recognition and then identified a key region responsible for immune evasion by modifying different regions of the viral protein. In the next round of experiments, the authors asked how the viral protein manages this feat. They discovered that ORF73 limits T cell recognition much like EBNA-1 does, by reducing synthesis and degradation of the protein. One region strongly associated with inhibiting epitope presentation to killer T cells corresponded to reduced protein synthesis. When the authors modified the ORF73 transcript to circumvent T cell evasion, the T cells wiped out latent virus. These results indicate that avoiding epitope presentation during episome maintenance is key to the viruss survival.

Interestingly, the MHV-68 episome maintenance protein mediates immune evasion even though it lacks the amino acid element that does the job for EBV. Future studies will have to determine the responsible MHV-68 epitope and the mechanisms that engineer immune avoidance. Since a majority of epitopes that killer T cells recognize come from aborted translation events, it may be that evasive action is taken at the RNA transcript stage, before RNA is translated into protein. Evading killer T cells, the authors argue, is key to the survival of the gamma-herpesvirus. By figuring out just how evasion occurs, scientists can identify a promising target for controlling infection.

Host Cell Lipids Facilitate Listeria monocytogenes Movement

Tue, 22 Mar 2005 20:44:38 PST

( from http://www.rxpgnews.com ) When the bacterium Listeria monocytogenes invades the body, it commandeers its host cell's actin cytoskeleton to invade other cells. In a report published in the Journal of Biological Chemistry, a group of scientists provide insight into the molecular mechanisms behind this infection technique.

Listeria causes a variety of diseases, the most severe being meningoencephalitis, an inflammation of the brain and the membranes that envelop the brain and spinal cord. Infection begins when the bacterium binds to a receptor on the surface of a cell, causing the cell to ingest it. The bacterium multiplies inside the cell and then uses a cellular protein called ActA to stimulate the host cell's actin to form filaments at one end of the bacterium.

"As these filaments lengthen, they drive the bacterium through the cell until it reaches the peripheral or outer cell membrane," explains Dr. Frederick Southwick of the University of Florida College of Medicine. "Here the growing actin filaments push the bacterium against the membrane, forming long membrane projections called filopodia. These filopodia push into adjacent cells and are ingested by them. The bacteria then enter the new cell and begin the cycle anew. Essentially Listeria takes over or hijacks the host cell's actin cytoskeleton to move within cells, and to spread from cell to cell."

In most cells, two membrane lipids, PIP2 and PIP3, are associated with the formation of new actin filaments. PIP3 is synthesized from PIP2 by an enzyme called PI3-kinase. The lipids attract and modify the functions of proteins involved in regulating actin assembly. PIP2 and PIP3 also prevent capping proteins from binding to the ends of actin filaments, allowing new actin filament assembly.

Because Listeria is capable of stimulating actin assembly and PIP2 and PIP3 are known to localize to regions of new actin assembly, Dr. Southwick and his colleagues decided to explore the roles these lipids play in Listeria infection.

"We had expected to see PIP2 and PIP3 only at the very back of Listeria where new actin assembly was taking place," recalls Dr. Southwick. "To our surprise these lipids also localized to the front of the moving bacteria." The researchers also noticed that Listeria movement slowed down when the bacteria were treated with molecules that inhibited PI3-kinase, proving that Listeria depend on PI3-kinase to move.

"Our studies show that Listeria is capable of inside-out signaling," explains Dr. Southwick. "Most signals arise from molecules binding receptors on the outside of the cell. In the case of Listeria, we find that this intracellular pathogen can harness signals from the inner rather than the outer surface of the cell membrane.

"The most exciting and surprising finding is that an intracellular bacteria is able to attract host cell membrane lipids to its surface and these membrane lipids facilitate the ability of the bacterium to move within cells. This capability is unique to Listeria and is not found in another intracellular bacteria, Shigella. Our experiments show that Listeria is a simplified model system for studying how phosphoinositides regulate the actin cytoskeleton, and this model promises to yield additional insights into how these phospholipids control the cell's actin cytoskeleton. Our discoveries provide additional fundamental clues as how cells move."

These findings may also open the door to using PI3-kinase inhibitors or other agents that lower PIP2 and PIP3 levels to slow the spread of Listeria and control infection in patients who are not responding to antibiotics, although that application is a long way off, says Dr. Southwick.

Norovirus Prevalent in Those Suffering from Traveler's Diarrhea

Sat, 19 Mar 2005 15:06:38 PST

( from http://www.rxpgnews.com ) Norovirus may be the most common cause of travelers' diarrhea for United States citizens returning from Mexico and Guatamala say researchers from the U.S., Guatemala, Mexico and Sweden.

It is estimated that 20 to 50 percent of the people traveling to tropical areas of the world will experience traveler's diarrhea (TD). TD, defined as three or more loose stool movements in a 24-hour period, frequently results from exposure to bacterial or viral pathogens. Noroviruses (NoV's) are considered to be one of the leading causes of nonbacterial gastro-related illnesses resulting in over 23 million cases annually.

In the study stool samples were collected from 34 patients with traveler's diarrhea and tested for the presence of norovirus. The virus was detected in 65 percent of the samples tested, with time spent at travel destinations playing an important role in determining infection frequency.

"This study is the first of its kind to indicate that NoVs may be a major cause of illness among United States travelers who experience TD during extended stays in developing countries," say the researchers. The high frequency of NoV infection among TD cases examined in this study suggests that further investigations concerning the role of these viruses in TD are warranted."

Oysters in US showed high prevalence of Salmonella

Fri, 18 Feb 2005 16:38:38 PST

( from http://www.rxpgnews.com ) Oysters harvested from thirty-six bays around the United States showed high prevalence of Salmonella according to a report that appears in the February 2005 journal Applied and Environmental Microbiology.

Known carriers of viral and bacterial pathogens, seafood and shellfish accounted for 7.42% of food poisoning related deaths attributed to Salmonella between 1990 and 1998. Characterized by fever, abdominal cramps, and diarrhea, salmonellosis is responsible for approximately 500 deaths annually in the U.S. alone. Current guidelines require the shellfish industry to test for evidence of bacterial contamination, however previous studies indicate that Salmonella could be present in oysters appearing otherwise healthy, indicating the need for testing specific to Salmonella.

"There are no current requirements for U.S. states to test harvesting waters for the presence of human pathogens, such as Salmonella spp.," say the researchers.

In the study oysters were harvested from thirty-six U.S. bays, twelve from the West, East, and Gulf coasts during the summer of 2002 and four bays per coast in the winter of 2002, and tested for the presence of Salmonella. Results showed that 7.4% of the oysters tested were positive for Salmonella and they came from all three U.S. coasts.

"Potential pathogenic serotypes of Salmonella were isolated from oysters harvested on all three U.S. coasts," say the researchers. "The testing of the oyster meat specifically for Salmonella spp. on a regular basis throughout the year, in each bay open for harvesting, would appear to be the only mechanism to remedy this oversight."

New Test May Differentiate Between Poultry Vaccinated Against or Infected with Avian Flu

Fri, 18 Feb 2005 16:34:38 PST

( from http://www.rxpgnews.com ) Avian influenza (AI), a viral disease of poultry, causes a wide range of diseases affecting multiple organs and often resulting in death. In recent years, new strains of the virus have continued to emerge and cross over from the wild bird reservoir to domestic poultry. A new diagnostic test monitoring antibody response to the NS1 virus protein may allow for differentiation between poultry vaccinated or infected with avian influenza say researchers from Georgia.

"Over the last decade, AI viruses circulating in live-bird markets have provided a secondary reservoir from which influenza viruses have crossed over to infect commercial chicken and turkey operations," say the researchers.

A vaccine incorporating the inactivated whole-virus has been used in an attempt to prevent AI infection. While these traditional vaccines can protect against clinical infection and death, they can interfere with surveillance programs. Vaccination produces the same antibodies as an actual AI infection, and can commonly cause false positives with traditional diagnostic tests.

In the study, the researchers designated the NS1 virus protein, typically found in large amounts in virus-infected cells but not the virus itself, as a potential differential marker. They monitored both infected and vaccinated poultry for antibodies reactive to NS1. While live virus infection induced high levels of the NS1 antibody, commercial vaccines induced little or no antibody response.

"These results demonstrate the potential benefit of a simple, specific enzyme-linked immunosorbent assay (ELISA) for anti-NS1 antibodies that may have diagnostic value for the poultry industries," say the researchers.

Bat-CoV - New Coronavirus in Bats

Fri, 18 Feb 2005 16:30:38 PST

( from http://www.rxpgnews.com ) Transmission of animal viruses to humans poses a growing threat worldwide. The recent emergence of SARS, a coronavirus transmitted to humans from wild animals in live animal markets, reinforces the need for virus surveillance in exotic wildlife. Chinese researchers have identified a novel coronavirus found in bats.

Most coronaviruses are disease-causing agents associated with respiratory and gastrointestinal illness in humans and respiratory and neurological symptoms in animals. In the study respiratory and fecal samples were collected from twelve bat species of which three tested positive for the novel virus (Bat-CoV). All bats testing positive for the virus were healthy when physically examined so it remains unclear as to whether or not the virus is pathogenic in bats.

"Here, we report the identification of a novel bat coronavirus," say the researchers. "It is not known whether this virus would cause zoonotic disease in humans or other animals, but given the catastrophic consequences of SARS, further surveillance work on viruses in wildlife should be encouraged."

Household Dust May Be Source of Infant Botulism

Sun, 16 Jan 2005 13:28:38 PST

( from http://www.rxpgnews.com ) Household Dust May Be Source of Infant Botulism

A fatal case of infant botulism may have been contracted from household dust, say researchers from Finland and California. The case study appears in the January 2005 issue of the Journal of Clinical Microbiology.

Clostridium botulinum, the bacterium that produces the toxin responsible for botulism, is typically harmless in adults because it cannot grow in the presence of oxygen. But the bacteria have been known to grow in the intestines of infants under the age of 1, often resulting in weakness, paralysis and even death. The sudden onset of infant botulism followed by unexplained death bears a resemblance to circumstances associated with Sudden Infant Death Syndrome (SIDS).

In the case study the intestinal contents of an eleven-week old infant who died suddenly were tested for the presence of C. botulinum. Vacuum cleaner dust from the patient's house was also collected and tested for the bacterium. Researchers found genetically similar isolates of C. botulinum in both. The study noted that the child had been healthy since birth and no environmental factors that could predispose an infant to botulism were identified.

"For the first time the C. botulinum isolates from an intestinally colonized infant and from household dust from the infant's home were demonstrated to be genetically similar," say the researchers. "This genetic similarity suggests that airborne spores of C. botulinum in an infant's surroundings may cause infant botulism and, as in this case, also result in the fulminant form of the illness that resembles SIDS."

New Coronavirus Identified in Pneumonia Patients

Sun, 16 Jan 2005 13:26:38 PST

( from http://www.rxpgnews.com ) Researchers from Hong Kong have identified a novel coronavirus in patients suffering from pneumonia. Their findings appear in the January 2005 issue of the Journal of Virology. Coronaviruses are responsible for 5 to 30 percent of human respiratory tract infections largely due to their unique ability to replicate. Because so many cases of respiratory tract infections are reported each year, researchers are actively trying to identify new causative agents.

The new virus, labeled CoV-HKU1, was first identified in a 71-year old pneumonia patient that had just returned from China. Following the discovery, nasal samples were taken from patients suffering from respiratory illness, but negative for SARS and screened for the presence of CoV-HKU1. Samples taken from a 35-year old woman suffering from pneumonia were positive for the virus, supporting the identification of a new group 2 coronavirus.

"Our data support the existence of a novel group 2 coronavirus associated with pneumonia in humans," say the researchers. "Further clinical, seroepidemiological and phylogenetic studies would be required to determine the relative importance of CoV-HKU1 compared to other respiratory tract viruses in causing upper and lower respiratory tract infections, its seroprevalence, and the origin of the virus."

Llama Antibodies May Help Prevent Dandruff

Sun, 16 Jan 2005 13:24:38 PST

( from http://www.rxpgnews.com ) The addition of llama antibodies to shampoo could be a new strategy for fighting dandruff, say European researchers. Their findings appear in the January 2005 issue of the journal Applied and Environmental Microbiology.

Malassezia furfur, a fungus frequently found on the human scalp, is often associated with the formation of dandruff. Current methods of treatment consist of shampoos containing antifungal compounds.

In the study researchers immunized a llama with M. furfur three times over a period of five weeks. They then screened blood samples and found antibodies that targeted a specific protein on the surface of the organism even in the harsh chemical conditions of shampoo.

"Here we describe a novel approach for preventing the formation of dandruff by inhibition of M. furfur with antibodies," say the researchers.

Bacteria can use a Sonar-like system to spot other cells

Fri, 24 Dec 2004 15:56:38 PST

( from http://www.rxpgnews.com ) For the first time, scientists have found that bacteria can use a Sonar-like system to spot other cells (either normal body cells or other bacteria) and target them for destruction. Reported in the December 24 issue of Science, this finding explains how some bacteria know when to produce a toxin that makes infection more severe. It may lead to the design of new toxin inhibitors. "Blocking or interfering with a bacterium's "detection" mechanism, should prevent toxin production and limit the severity of infection," says Michael Gilmore, PhD, lead author of the study, and currently director of research at the Schepens Eye Research Institute and professor of ophthalmology at Harvard Medical School.

Gilmore and his team have spent years studying the bacterium known as Enterococcus faecalis, one of the leading causes of hospital-acquired infections, to find new ways to treat them. These infections are frequently resistant to many, and sometimes all, antibiotics. Tens of thousands of deaths due to antibiotic resistant infection occur each year in the US, adding an estimated $ 4 Billion to health care costs. Scientist have known since 1934 that especially harmful strains of Enterococcus produce a toxin that destroys other cells, including human cells and even other types of bacteria. They also knew that this toxin was made only under some conditions. Until Gilmore's study, scientists were unable to explain how the Enterococcus knew when to make it.

In the Science study, Gilmore and his team found that this toxin is made whenever there is another cell type in the environment near the bacterium, such as a human blood cell. They discovered how these bacteria know when other cells are present, and respond accordingly.

In the laboratory, the team found that Enterococcus releases two substances into the environment. One substance sticks to foreign cells. The second substance reports back and tells the Enterococcus to make the toxin. If no cells are in the area, the first substance sticks to the second, preventing it from reporting back to the Enterococcus, and as a result, no toxin is made. According to Gilmore, "These bacteria are actively probing their environment for enemies or food. Based on whether or not they 'see' other cells, they make the toxin appropriately."

Gilmore says this discovery has several significant implications for the future. "This is a new mechanism that nature devised to 'see' the environment, and based on that information, respond accordingly. We may be able to learn from nature and adapt a similar strategy to help the aging population cope with loss of vision," says Gilmore.

"Secondly, this discovery will help us to develop new ways to treat infections that are resistant to antibiotics, making them less severe. Based on an understanding of how this toxin system works, we hope to develop toxin inhibitors," says Gilmore.

The third area of interest is currently science fiction, says Gilmore. "If bacteria can see cells in the environment, maybe we can tame these bacteria and engineer this system so that it can be used to see other things in the environment, such as minerals or possibly other disease-causing bacteria," says Gilmore.

C. jejuni May Survive Refrigeration and Frozen Storage Combined

Thu, 23 Dec 2004 12:58:38 PST

( from http://www.rxpgnews.com ) A common cause of foodborne disease from poultry products can survive refrigeration and freezing say researchers from Pennsylvania. Their findings appear in the December 2004 issue of the journal Applied and Environmental Microbiology.

Campylobacter bacteria are estimated to be responsible for 2.5 million cases of infection in the United States each year and 50% of those cases are attributed to contaminated poultry. Campylobacters are believed to achieve optimal growth in extremely warm temperatures while failing to thrive in temperatures below 86 degrees. Campylobacter jejuni appears to be the exception. Previous studies have shown a small portion able to withstand refrigeration and freezing independently, but the combined effect of both has yet to be tested.

In the study samples of ground chicken and chicken skin infected with C. jejuni were refrigerated, frozen or exposed to a combination of both. A significant portion of the bacteria were able to survive refrigerated and frozen temperatures in both ground chicken and chicken skin.

"A significant portion of C. jejuni on the poultry samples studied survived during refrigerated, frozen, and combined refrigerated and frozen storage," say the researchers. "The present study indicates that these treatments alone will not add a significant margin of safety with respect to this pathogen and cannot replace sanitary production and handling."

Houseflies on cattle farms may contribute to the spread of Escherichia coli

Thu, 23 Dec 2004 12:57:38 PST

( from http://www.rxpgnews.com ) Houseflies on cattle farms may contribute to the spread of Escherichia coli O157:H7 among animals, their food supply and potentially humans say researchers from Kansas. Their findings appear in the December 2004 issue of the journal Applied and Environmental Microbiology.

E. coli, one of the leading causes of food-borne diseases throughout the world, is responsible for more than 73,000 cases annually in the United States alone. E. coli O157:H7 can be life-threatening to children, the elderly and immuno-compromised patients. The intestinal tracts of cattle serve as the main reservoir for E. coli O157:H7 and the environment in which they are housed frequently attracts large populations of houseflies (HF).

"One of the potential modes of dissemination of this pathogen in the environment is by insects that are associated with animal feces and manure, primarily houseflies," say the researchers.

In the study houseflies were gathered from the feed bunks of a cattle farm in Kansas from June through October 2003. E.coli O157:H7 was found in every batch of houseflies collected, with 30% of the positive houseflies coming from a flaked corn shed. Ninety percent of the isolates contained genes indicating highly virulent strains.

"Our study demonstrated that houseflies carry virulent E. coli O157:H7 in the farm environment primarily during the summer and may play an important role in the ecology and transmission of this pathogen among individual cattle and potentially to the surrounding farm and urban environment," say the researchers. "Information on the association of E. coli O157:H7 with houseflies will assist in developing more comprehensive and quantitative risk assessments, as well as formulating E. coli O157:H7 intervention strategies that should include an effective HF management program."

New Herpes Vaccine May be Ready for Human Trials

Thu, 23 Dec 2004 12:50:38 PST

( from http://www.rxpgnews.com ) New research suggests that a promising herpes vaccine may be ready for testing in humans say researchers from the National Institutes of Health and Harvard Medical School. Their findings appear in the January 2005 issue of the Journal of Virology.

Herpes simplex virus type 2 (HSV-2), or genital herpes, is a virus that infects approximately 22% of adult Americans. Bearing physical, psychological, and social effects on those who acquire it, it can pose an even more severe risk for immuno-compromised patients further emphasizing the need for an effective vaccine.

"In the aggregate, the burden of genital herpes has made development of more effective prevention strategies a health priority," say the researchers.

The study compared three different vaccines, a DNA vaccine, an antigenic vaccine and a live mutant strain of the type 2 virus, d15-29, in mice and guinea pigs. The live mutant strain, d15-29, showed minimal risk of causing disease as it is missing two of the genes necessary for replication and it stimulated a stronger immune response in both animals.

"Given its efficacy, its defectiveness for latency, and its ability to induce rapid, virus-specific CD8+-T-cell responses, the dl5-29 vaccine may be a good candidate for early-phase human trials," say the researchers.

(Y. Hoshino, S.K. Dalai, K. Wang, L. Pesnicak, T.Y. Lau, D.M. Knipe, J.I. Cohen, S.E. Straus. 2004. Comparative efficacy and immunogenicity of replication-defective, recombinant glycoprotein, and DNA vaccines for herpes simplex virus 2 infections in mice and guinea pigs.)

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