Understanding A Cell's Split Personality Aids Synthetic Circuits.

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ScienceDaily (Oct. 5, 2009) — As scientists work toward making genetically altered bacteria create living "circuits" to produce a myriad of useful proteins and chemicals, they have logically assumed that the single-celled organisms would always respond to an external command in the same way.

Alas, some bacteria apparently have an individualistic streak that makes them zig when the others zag.
A new set of experiments by Duke University bioengineers has uncovered the existence of "bistability," in which an individual cell has the potential to live in either of two states, depending on which state it was in when stimulated.
Taking into account the effects of this phenomenon should greatly enhance the future efficiency of synthetic circuits, said biomedical engineer Lingchong You of Duke's Pratt School of Engineering and the Duke Institute for Genome Sciences & Policy.
In principle, re-programmed bacteria in a synthetic circuit can be useful for producing proteins, enzymes or chemicals in a coordinated way, or even delivering different types of drugs or selectively killing cancer cells, the scientists said.
Researchers in this new field of synthetic biology "program" populations of genetically altered bacteria to direct their actions in much the same way that a computer program directs a computer. In this analogy, the genetic alteration is the software, the cell the computer. The Duke researchers found that not only does the software drive the computer's actions, but the computer in turn influences the running of the software.
"In the past, synthetic biologists have often assumed that the components of the circuit would act in a predictable fashion every time and that the cells carrying the circuit would just serve as a passive reactor," You said. "In essence, they have taken a circuit-centric view for the design and optimization process. This notion is helpful in making the design process more convenient."
But it's not that simple, say You and his graduate student Cheemeng Tan, who published the results of their latest experiments early online in the journal Nature Chemical Biology.
"We found that there can be unintended consequences that haven't been appreciated before," said You. "In a population of identical cells, some can act one way while others act in another. However, this process appears to occur in a predictable manner, which allows us to take into account this effect when we design circuits."
Bistability is not unique to biology. In electrical engineering, for example, bistability describes the functioning of a toggle switch, a hinged switch that can assume either one of two positions – on or off.
"The prevailing wisdom underestimated the complexity of these synthetic circuits by assuming that the genetic changes would not affect the operation of the cell itself, as if the cell were a passive chassis," said Tan. "The expression of the genetic alteration can drastically impact the cell, and therefore the circuit.
"We now know that when the circuit is activated, it affects the cell, which in turn acts as an additional feedback loop influencing the circuit," Tan said. "The consequences of this interplay have been theorized but not demonstrated experimentally."
The scientists conducted their experiments using a genetically altered colony of the bacteria Escherichia coli (E.coli) in a simple synthetic circuit. When the colony of bacteria was stimulated by external cues, some of the cells went to the "on" position and grew more slowly, while the rest went to the "off" position and grew faster.
"It is as if the colony received the command not to expand too fast when the circuit is on," Tan explained. "Now that we know that this occurs, we used computer modeling to predict how many of the cells will go to the 'on' or 'off' state, which turns out to be consistent with experimental measurements"
The experiments were supported by the National Science Foundation, the National Institutes of Health and a David and Lucille Packard Fellowship. Duke's Philippe Marguet was also a member of the research team.
Adapted from materials provided by Duke University, via EurekAlert!, a service of AAAS.

New Method For Improving The Functional Characteristics Of Enzymes.

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ScienceDaily (Sep. 28, 2009) — An international team of scientists from the Czech Republic, Germany and Japan have developed a new method for improving the properties of enzymes. The method has potential for wide application in the chemical, medicinal and food industries. The research has been published in Nature Chemical Biology.

The modified enzymes can be used, for example, for disposal of highly harmful chemical substances which enter into the environment as a result of human activity and can have a very negative influence on human and animal health. Nature cannot degrade many of these chemicals but, in this work, the scientists have developed an approach that can be applied to remove them efficiently from the environment.
The principle of the discovery is based on genetic manipulation of the enzyme which is starting and accelerating the chemical reaction. „Now we can use genetic modifications for changing the properties of the enzymes so they can faster and more easily dispose of harmful substances in the environment,” says Jiri Damborsky, leader of the Protein Engineering Group at the Institute of Experimental Biology, Faculty of Science, Masaryk University.
Up to now, the scientists had focused during the modification of an enzyme’s properties on the site in its structure where the chemical reaction happens, the active site. The new method is based on the modification of so-called access tunnels that connect the active site with the surface of the enzyme. “Specialized computational techniques guided the experimental work to engineer these tunnels to alter their accessibility to the degraded substances,” notes Rebecca Wade, leader of the Molecular and Cellular Modeling Group at EML Research in Heidelberg.
The scientists applied the approach by modifying an enzyme to degrade the highly toxic substance, trichloropropane (TCP). This colourless liquid is a secondary product of chemical production. It can reside in the soil and groundwater for over 100 years, can contaminate drinking water and is a carcinogen. Using the new approach, the protein engineers developed a modified enzyme capable of degrading this substance 32 times faster than the original enzyme.
But the method has much wider scope for application than just in the fight against harmful substances and in environmental protection. The targeted modification of the tunnels in enzymes can be utilized in different application areas, including biomedicine, and the chemical and food industries.
Journal reference:
Martina Pavlova et al. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nature Chemical Biology, 5, 727 - 733 (2009); Published online 23 August 2009 DOI: 10.1038/nchembio.205
Adapted from materials provided by European Media Laboratory (EML), via AlphaGalileo.

Yale engineers have for the first time observed and tracked E. coli bacteria moving in a liquid medium with a particular motion.

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ScienceDaily (Sep. 26, 2009) — Yale engineers have for the first time observed and tracked E. coli bacteria moving in a liquid medium with a motion similar to that of a kayak paddle.

Their findings, which appear online September 29 in the journal Physical Review Letters, will help lead to a better understanding of how bacteria move from place to place and, potentially, how to keep them from spreading.
Scientists have long theorized that the cigar-shaped cell bodies of E. coli and other microorganisms would follow periodic orbits that resemble the motion of a kayak paddle as they drift downstream in a current. Until now, no one had managed to directly observe or track those movements.
Hur Koser, associate professor at Yale's School of Engineering & Applied Science, previously discovered that hydrodynamic interactions between the bacteria and the current align the bacteria in a way that allows them to swim upstream. "They find the most efficient route to migrate upstream, and we ultimately want to understand the mechanism that allows them to do that," Koser said.
In the new study, Koser, along with postdoctoral associate and lead author of the paper, Tolga Kaya, devised a method to see this motion in progress. They used advanced computer and imaging technology, along with sophisticated new algorithms, that allowed them to take millions of high-resolution images of tens of thousands of individual, non-flagellated E. coli drifting in a water and glycerin solution, which amplified the bacteria's paddle-like movements.
The team characterized the bacteria's motion as a function of both their length and distance from the surface. The team found that the longer and closer to the surface they were, the slower the E. coli "paddled."
It took the engineers months to perfect the intricate camera and computer system that allowed them to take 60 to 100 sequential images per second, then automatically and efficiently analyze the huge amount of resulting data.
E. coli and other bacteria can colonize wherever there is water and sufficient nutrients, including the human digestive tract. They encounter currents in many settings, from riverbeds to home plumbing to irrigation systems for large-scale agriculture.
"Understanding the physics of bacterial movement could potentially lead to breakthroughs in the prevention of bacterial migration and sickness," Koser said. "This might be possible through mechanical means that make it more difficult for bacteria to swim upstream and contaminate water supplies, without resorting to antibiotics or other chemicals."
Adapted from materials provided by Yale University, via EurekAlert!, a service of AAAS.

Mechanism Related To Onset Of Various Genetic Diseases Revealed

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ScienceDaily (Sep. 17, 2009) — Researchers at the Department of Biochemistry and Molecular Biology of Universitat Autònoma de Barcelona (UAB) have revealed the process by which proteins with a tendency to cause conformational diseases such as amyotrophic lateral sclerosis, familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, etc. finally end up causing them.

Researchers have carried out an analysis of their 3D structure and studied why these proteins finally become toxic although they are correctly folded, an indicator that they are functioning correctly. The answer can be found in the separation of the proteins, which under normal conditions are found in groups of two or more, caused by a genetic mutation in their composition. Researchers believe this discovery, published recently in the journal PLoS Computational Biology, could also be the cause of other diseases of unknown origins.
Every day cells produce thousands of new proteins which renew themselves every second and which, by obeying the orders prescribed in our genetic code, work towards the proper functioning of our body. However, these proteins occasionally suffer genetic mutations which can cause changes in their composition, thus preventing them from carrying out their functions and the activities they are assigned. In many cases this gives way to the formation of toxic macromolecular aggregates - amyloid fibrils - which block our body's protein quality control system and finally provoke cell death.
Protein aggregation and the misfolding of proteins can be linked to the origin of many conformational diseases which can be either genetic or spontaneous. The proteins involved can either have an unstructured or lineal unfolded form such as in Alzheimer's and Parkinson's disease or Type II Diabetes, or can be globular, showing a folded 3D-structure. The former have been widely characterised by scientists and the process by which they unfold is known. The process leaves regions uncovered which are in the risk of becoming aggregated and these eventually form toxic assemblies. Globular proteins are known to be linked to hepatic, cardiac, renal and neurological disorders. However scientists do not know exactly how they manage to aggregate despite the fact that they are correctly folded within the body.
Through computational analysis, researchers Salvador Ventura and Virgínia Castillo, from the UAB Department of Biochemistry and Molecular Biology, have discovered that, in non-disease conditions, globular proteins related to conformational diseases are found associated in pairs to other proteins or in complex subunits, in a way that one protein covers the aggregation-prone region of the other and thus prevents the onset of this process. Therefore these regions remain obscured in the interior of the structure and are inoffensive to the organism as long as the two proteins are joined together. Researchers have found that genetic mutations produced in the interaction sites of the protein pair prevents their association, leaving aggregation-prone regions uncovered and favouring the formation of toxic aggregates. According to researchers, this would explain why out of two people with the same globular proteins and the same risk regions, only the one who suffers a genetic mutation would finally develop a disease.
The conclusions obtained have led researchers to contemplate the possibility that dissociation is a general mechanism, which not only affects globular proteins with a clearly defined structure, but also others which have not yet been characterised and which could be the cause of diseases of unknown origin.
As possible strategies to prevent the dissociation of proteins, the authors propose introducing genetic mutations into the proteins to strengthen their association and developing specific molecules to block the risk regions of already dissociated proteins.
The results of the study carried out by UAB researchers coincides with those obtained by researchers at Cambridge University, who also published similar data in the journal Proceedings of the National Academic of Sciences.
In the future UAB researchers are planning to expand their computational analysis to cover the whole set of human proteins with a defined 3D-structure. With this objective they seek to discover the proteins responsible for different genetic diseases of unknown origins and offer a series of new therapeutic targets for these disorders.
Journal reference:
Castillo V, Ventura S. Amyloidogenic Regions and Interaction Surfaces Overlap in Globular Proteins Related to Conformational Diseases. PLoS Computational Biology, 2009; 5 (8): e1000476 DOI: 10.1371/journal.pcbi.1000476
Adapted from materials provided by Universitat Autonoma de Barcelona, via EurekAlert!, a service of AAAS.

Frontiers of Biomedical Engineering with Prof. Mark Saltzman (Yale University)

Growing Sea Lamprey Embryos Dramatically Alter Genomes, Discard Millions Of Units Of DNA

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ScienceDaily (July 21, 2009) — Researchers have discovered that the sea lamprey, which emerged from jawless fish first appearing 500 million years ago, dramatically remodels its genome. Shortly after a fertilized lamprey egg divides into several cells, the growing embryo discards millions of units of its DNA.

The findings were published this month in the Proceedings of the National Academy of Sciences. The lead author is Jeramiah Smith, a postdoctoral fellow in genome sciences at the University of Washington (UW) working in the Benaroya Research Institute laboratory of Chris Amemiya, who is also a UW affiliate professor of iology.
Theirs is believed to be the first recorded observation of a vertebrate -- an animal with a spinal column -- extensively reorganizing its genome as a normal part of development. A few invertebrate species, like some roundworms, have been shown to undergo extensive genome remodeling. However, stability was thought to be vital in vertebrates' genomes to assure their highly precise, normal functioning. Only slight modifications to allow for immune response were believed to occur in the vertebrate genome, not broad-scale rearrangements.
Smith, Amemiya and their research team inadvertently discovered the dynamic transformations in the sea lamprey genome while studying the genetic origins of its immune system. The researchers were trying to deduce how the sea lamprey employs a copy-and-paste mechanism to generate diverse receptors for detecting a variety of pathogens.
The researchers were surprised to notice a difference between the genome structure in the germline -- the cells that become eggs and the sperm that fertilize them -- and the genome structure in the resulting embryonic cells. The DNA in the early embryonic cells had myriad breaks that resembled those in dying cells …but the cells weren't dying. The embryonic cells had considerably fewer repeat DNA sequences than did the sperm cells and their precursors.
"The remodeling begins at the point when the embryo turns on its own genes and no longer relies on its mom's store of mRNA," said Smith.
The restructuring doesn't occur all at once, but continues for a long while during embryonic development. It took at lot of work for the scientists to see what was lost and when. They learned, among other findings, that the remodeled genome had fewer repeats and specific gene-encoding sequences. Deletions along the strands of DNA are also thought to move certain regulatory switches in the genome closer to previously distant segments.
The scientists don't know how this happens, or why. Smith said that his favorite hypothesis, yet unproven, is that the extra genetic material might play a role in the proliferation of precursor cells for sperm and eggs, and in early embryonic development. The genetic material might then be discarded either when it is no longer needed or to prevent abnormal growth.
The alteration of the sea lamprey genome and of invertebrates that restructure their genome appears to be tightly regulated, according to Smith, yet the resulting structural changes seem almost like the DNA errors that give rise to cancers or other genomic disorders in higher animals. Learning how sea lamprey DNA rearrangements are regulated during development might provide information on what stabilizes or changes the genome, he said, as well the role of restructuring in helping form different types of body cells, like fin, muscle, or liver cells.
If 20 percent of their genome disappears, how do sea lampreys pass along the full complement of their genes to their offspring?
"The germline -- those precursor cells for sperm and eggs -- is a continuous lineage through time," Smith explained. "The precursor cells for sperm and egg are set apart early in lamprey development. The genome in that cell population should never change." Genetic material is assumed to be lost only in the early embryonic cells destined to become body parts and not in cells that give rise to the next generation. The researchers have been looking for the primordial stem cells for sperm and eggs hidden away in the lamprey, but they are difficult to find.
Researchers do not yet know how the sea lamprey's genome guides the morphing it undergoes during its life. Sea lampreys have a long juvenile life as larvae in fresh water, where they eat on their own. Their short adult lives are normally spent in the sea as blood-sucking parasites. Their round, jawless mouths stick like suction cups to other fish. Several circular rows of teeth rasp through the skin of their unlucky hosts. Their appetite is voracious.
Later, as they return to streams and rivers along the northern Atlantic seaboard, sea lampreys atrophy until they are little more than vehicles for reproduction. After mating, they perish. Populations of sea lamprey were landlocked in the Great Lakes and other nearby large lakes after canals and dams were built in the early 1900's. They thrive by parasitizing (and killing) commercially important fish species and are considered a nuisance in the Great Lakes region.
Biologists are interested in the sea lamprey partly because of its alternating lifestyles, but largely because it represents a living fossil from around the time vertebrates originated. Close relatives of sea lampreys were on earth before the dinosaurs. It's possible that the sea lamprey's dynamic genome biology might someday be traced back in evolutionary history to a point near, and perhaps including, a common ancestor of all vertebrates living today, the authors of the study noted.
"Sea lampreys have a half billion years of evolutionary history," Smith said. "Evolutionary biologists and geneticists can compare their genomes to other vertebrates and humans to see what parts of the lamprey genome might have been present in our primitive ancestors. We might begin to understand how changes in the sea lamprey genome led to their distinct body structure and how fishes evolved from jawless to jawed."
Amemiya added, "We don't really know where this discovery about the sea lamprey's remodeling of its genome will take us. It's common in science for the implications of a finding not to be realized for several decades. It's less about connecting the dots to a specific application, and more about obtaining a broad understanding of how living things are put together."
In addition to Smith and Amemiya, the other researchers on this study were Francesca Antonacci and Evan E. Eichler of the UW Department of Genome Sciences. Research grants from the National Institutes of Health,the National Science Foundation and the Howard Hughes Medical Institute funded the project. Smith also received National Research Service Awards, including an Institutional Ruth L. Krischstein Award through the University of Washington Department of Genome Sciences and an individual Ruth L. Krischstein Award through the National Institute of General Medical Sciences.
Adapted from materials provided by University of Washington.

How Staph Infections Alter Immune System

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ScienceDaily (July 17, 2009) — Infectious disease specialists at UT Southwestern Medical Center have mapped the gene profiles of children with severe Staphylococcus aureus infections, providing crucial insight into how the human immune system is programmed to respond to this pathogen and opening new doors for improved therapeutic interventions.

In recent years, much research has focused on understanding precisely what the bacterium S. aureus does within the host to disrupt the immune system. Despite considerable advances, however, it remained unclear how the host's immune system responded to the infection and why some people are apt to get more severe staphylococcal infections than others.
By using gene expression profiling, a process that summarizes how individual genes are being activated or suppressed in response to the infection, UT Southwestern researchers pinpointed how an individual's immune system responds to a S. aureus infection at the genetic level.
"The beauty of our study is that we were able to use existing technology to understand in a real clinical setting what's going on in actual humans – not models, not cells, not mice, but humans," said Dr. Monica Ardura, instructor of pediatrics at UT Southwestern and lead author of the study available online in PLoS One. "We have provided the first description of a pattern of response within an individual's immune system that is very consistent, very reproducible and very intense."
The immune system consists of two components: the innate system, which provides immediate defense against infection; and the adaptive system, whose memory cells are called into action to fight off subsequent infections.
In this study, researchers extracted ribonucleic acid from a drop of blood and placed it on a special gene chip called a microarray, which probes the entire human genome to determine which genes are turned on or off. They found that in children with invasive staphylococcal infections, the genes involved in the body's innate immune response are overactivated while those associated with the adaptive immune system are suppressed.
"It's a very sophisticated and complex dysregulation of the immune system, but our findings prove that there's consistency in the immune response to the staphylococcus bacterium," Dr. Ardura said. "Now that we know how the immune system responds, the question is whether we can use this to predict patient outcomes or differentiate the sickest patients from the less sick ones. How can we use this knowledge to develop better therapies?"
Researchers used blood samples collected between 2001 and 2005 from 77 children – 53 hospitalized at Children's Medical Center Dallas with invasive S. aureus infections and 24 controls. The control samples were collected from healthy children attending either well-child clinic or undergoing elective surgical procedures. Children with underlying chronic diseases, immunodeficiency, multiple infections, and those who received steroids or other immunomodulatory therapies were excluded from the study.
The children ranged in age from a few months to 15 years and included 43 boys and 34 girls. Those with S. aureus infections – both methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) – were matched with healthy controls for age, sex and race. The researchers also characterized the extent as well as the type of infection in each patient to make sure that the strain of bacteria didn't influence the results.
Dr. Ardura stressed that more research is needed because the results represent a one-time snapshot of what's going on in the cell during an invasive staphylococcal infection.
"The median time to get the blood sample was day four because we wanted to make sure the hospitalized children had a S. aureus infection, and its takes four days to have final identification of the bacterial pathogen," she said.
The next step, Dr. Ardura said, is to study those dynamics in patients before, during and after infection. They also hope to understand better how various staph-infection therapies affect treatment.
"This is a very important proof-of-concept that the information is there for us to grab," Dr. Ardura said. "Now we have to begin to understand what that data tells us."
The work was supported by the National Institutes of Health, the Center for Lupus Research and the Baylor Health Care System Foundation.
Adapted from materials provided by UT Southwestern Medical Center, via EurekAlert!, a service of AAAS.

Male Sex Chromosome Losing Genes By Rapid Evolution, Study Reveals

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ScienceDaily (July 17, 2009) — Scientists have long suspected that the sex chromosome that only males carry is deteriorating and could disappear entirely within a few million years, but until now, no one has understood the evolutionary processes that control this chromosome's demise. Now, a pair of Penn State scientists has discovered that this sex chromosome, the Y chromosome, has evolved at a much more rapid pace than its partner chromosome, the X chromosome, which both males and females carry.

This rapid evolution of the Y chromosome has led to a dramatic loss of genes on the Y chromosome at a rate that, if maintained, eventually could lead to the Y chromosome's complete disappearance. The research team, which includes Associate Professor of Biology Kateryna Makova, the team's leader, and National Science Foundation Graduate Research Fellow Melissa Wilson, will publish its results in the 17 July 2009 issue of the journal PLoS Genetics.
"There are three classes of mammals," said Makova, "egg-laying mammals, like the platypus and the echidna; marsupials, like the opossum and the wallaby; and all other mammals -- called eutherians -- which include humans, dogs, mice, and giraffes. The X and Y chromosomes of marsupials and eutherians evolved from a pair of non-sex chromosomes to become sex chromosomes."
Humans have 23 pairs of chromosomes, which are the structures that hold our DNA, but just one pair of these chromosomes are sex chromosomes, while the others are referred to as non-sex chromosomes. "In eutherian mammals, the sex chromosomes contain an additional region of DNA whereas, in the egg-laying mammals and marsupials, this additional region of DNA is located on the non-sex chromosomes," said Makova. "At first, bits of DNA within this additional region were readily swapped between the X and Y chromosomes, but some time between 80 and 130 million years ago, the region became two completely separate entities that no longer swapped DNA. One of the regions became specifically associated with the X chromosome and the other became specifically associated with the Y chromosome."
By comparing the DNA of the X and Y chromosomes in eutherian mammals to the DNA of the non-sex chromosomes in the opossum and platypus, the team was able to go back in time to the point when the X and Y chromosomes were still swapping DNA, just like the non-sex chromosomes in the opossum and platypus. The scientists then were able to observe how the DNA of the X and Y chromosomes changed over time relative to the DNA of the non-sex chromosomes. "Our research revealed that the Y-specific DNA began to evolve rapidly at the time that the DNA region split into two entities, while the X-specific DNA maintained the same evolutionary rate as the non-sex chromosomes," said Makova.
Once the biologists determined that the Y chromosome has been evolving more rapidly and has been losing more genes as a result, they wanted to find out why the Y chromosome has not already disappeared entirely. "Today, the human Y chromosome contains less than 200 genes, while the human X chromosome contains around 1,100 genes," said Wilson. "We know that a few of the genes on the Y chromosome are important, such as the ones involved in the formation of sperm, but we also know that most of the genes were not important for survival because they were lost, which led to the very different numbers of genes we observe between the once-identical X and Y. Although there is evidence that the Y chromosome is still degrading, some of the surviving genes on the Y chromosome may be essential, which can be inferred because these genes have been maintained for so long."
The team then decided to test the hypothesis that some of the genes on the Y chromosome are being maintained because they are essential. The team's approach was to compare the expression and function of genes on the Y chromosome with analogous genes on the X chromosome. "If the genes' expressions and/or functions were different, then it would make sense that the genes on the Y chromosome would be maintained because they are doing something that the genes on the X chromosome can't do," said Makova. "This hypothesis turned out to be correct."
Although some of the genes on the Y chromosome have been maintained, most of them have died, and the team found evidence that some others are on track to disappear, as well. "Even though some of the genes appear to be important, we still think there is a chance that the Y chromosome eventually could disappear," said Makova. "If this happens, it won't be the end of males. Instead, a new pair of non-sex chromosomes likely will start on the path to becoming sex chromosomes."
In the future, the team plans to use its newly generated data to create a computer model that tracks the degeneration of the Y chromosome. The scientists hope to determine how long it will take for the Y chromosome to disappear. They also hope to identify the processes that are most important for degeneration of the Y chromosome.
This research was funded by the National Institutes of Health, Penn State, and the National Science Foundation.
Adapted from materials provided by Penn State, via EurekAlert!, a service of AAAS.

Handle With Care: Telomeres Resemble DNA Fragile Sites

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ScienceDaily (July 17, 2009) — Telomeres, the repetitive sequences of DNA at the ends of linear chromosomes, have an important function: They protect vulnerable chromosome ends from molecular attack. Researchers at Rockefeller University now show that telomeres have their own weakness. They resemble unstable parts of the genome called fragile sites where DNA replication can stall and go awry. But what keeps our fragile telomeres from falling apart is a protein that ensures the smooth progression of DNA replication to the end of a chromosome.

The research, led by Titia de Lange, head of the Laboratory of Cell Biology and Genetics, and first author Agnel Sfeir, a postdoctoral associate in the lab, suggests a striking similarity between telomeres and common fragile sites, parts of the genome where breaks tend to occur, albeit infrequently. (Humans have 80 common fragile sites, many of which have been linked to cancer.) De Lange and Sfeir found that these newly discovered fragile sites make it difficult for DNA replication to proceed, a discovery that unveils a new replication problem posed by telomeres.
At the center of the discovery is a protein known as TRF1, which de Lange, in an effort to understand how telomeres protect chromosome ends, discovered in 1995. Using a conditional mouse knockout, de Lange and Sfeir have now revealed that TRF1, which is part of a six-protein complex called shelterin, enables DNA replication to drive smoothly through telomeres with the aid of two other proteins.
“Telomeric DNA has a repetitive sequence that can form unusual DNA structures when the DNA is unwound during DNA replication,” says de Lange. “Our data suggest that TRF1 brings in two proteins that can take out these structures in the telomeric DNA. In other words, TRF1 and its helpers remove the bumps in the road so that the replication fork can drive through.”
The work, published in the July 10 issue of Cell, began when Sfeir deleted TRF1 and saw that the telomeres resembled common fragile sites, suggesting that TRF1 protects telomeres from becoming fragile. Instead of a continuous string of DNA, the telomeres were broken into fragments of twos and threes. To see if the replication fork stalls at telomeres, de Lange and Sfeir joined forces with Carl L. Schildkraut, a researcher at Albert Einstein College of Medicine in New York City. Using a technique called SMARD, the researchers observed the dynamics of replication across individual DNA molecules — the first time this technique has been used to study telomeres. In the absence of TRF1, the fork often stalled for a considerable amount of time.
The only other known replication problem posed by telomeres was solved in 1985 when it was shown that the enzyme telomerase elongates telomeres, which shorten during every cell division. The second problem posed by telomeres, the so-called end-protection problem, was solved by de Lange and her colleagues when they found that shelterin protects the ends of linear chromosomes, which look like damaged DNA, from unnecessary repair. Working with TRF1, the very first shelterin protein ever to be identified, de Lange and Sfeir have not only unveiled a completely unanticipated replication problem at telomeres, they have also shown how it is solved.
The research lays new groundwork for the study of common fragile sites throughout the genome, explains de Lange. “Fragile sites have always been hard to study because no specific DNA sequence preceeds or follows them,” she says. “In constrast, telomeres represent fragile sites with a known sequence, which may help us understand how common fragile sites break throughout the genome — and why.”
Journal reference:
Agnel Sfeir, Settapong T. Kosiyatrakul, Dirk Hockemeyer, Sheila L. MacRae, Jan Karlseder, Carl L. Schildkraut and Titia de Lange. Mammalian Telomeres Resemble Fragile Sites and Require TRF1 for Efficient Replication. Cell, 138(1): 90%u2014103 (July 10, 2009) [link]
Adapted from materials provided by Rockefeller University.

By Manipulating Oxygen, Scientists Coax Bacteria Into Never-Before-Seen Solitary Wave

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ScienceDaily (July 17, 2009) — Bacteria know that they are too small to make an impact individually. So they wait, they multiply, and then they engage in behaviors that are only successful when all cells participate in unison. There are hundreds of behaviors that bacteria carry out in such communities. Now researchers at Rockefeller University have discovered one that has never been observed or described before in a living system.

In research published in the May 12 issue of Physical Review Letters, Albert J. Libchaber, head of the Laboratory of Experimental Condensed Matter Physics, and his colleagues, including first author Carine Douarche, a postdoctoral associate in the lab, show that when oxygen penetrates a sample of oxygen-deprived Escherichia coli bacteria, they do something that no living community had been seen to do before: The bacteria accumulate and form a solitary propagating wave that moves with constant velocity and without changing shape. But while the front is moving, each bacterium in it isn’t moving at all.
“It’s like a soliton,” says Douarche. “A self-reinforcing solitary wave.”
Unlike the undulating pattern of an ocean wave, which flattens or topples over as it approaches the shore, a soliton is a solitary, self-sustaining wave that behaves like a particle. For example, when two solitons collide, they merge into one and then separate into two with the same shape and velocity as before the collision. The first soliton was observed in 1834 at a canal in Scotland by John Scott Russell, a scientist who was so fascinated with what he saw that he followed it on horseback for miles and then set up a 30-foot water tank in his yard where he successfully simulated it, sparking considerable controversy.
The work began when Libchaber, Douarche and their colleagues placed E. coli bacteria in a sealed square chamber and measured the oxygen concentration and the density of bacteria every two hours until the bacteria consumed all the oxygen. (Bacteria, unlike humans, don’t die when starved for oxygen, but switch to a nonmotile state from which they can be revived.) The researchers then cracked the seals of the chamber, allowing oxygen to flow in.
The result: The motionless bacteria, which had spread out uniformly, began to move; first those around the perimeter, nearest to the seals, and then those further away. A few hours later, the bacteria began to spatially segregate into two domains of moving and nonmoving bacteria and pile up into a ring at the border of low-oxygen and no-oxygen. There they formed a solitary wave that propagated slowly but steadily toward the center of the chamber without changing its shape.
The effect, which lasted for more than 15 hours and covered a considerable distance (for bacteria), could not be explained by the expression of new proteins or by the addition of energy in the system. Instead, the creation of the front depends on the dispersion of the active bacteria and on the time it takes for oxygen-starved bacteria to completely stop moving, 15 minutes. The former allows the bacteria to propagate at a constant velocity, while the latter keeps the front from changing shape.
However, a propagating front of bacteria wasn’t all that was created. “To me, the biggest surprise was that the bacteria control the flow of oxygen in the regime,” says Libchaber. “There’s a propagating front of bacteria, but there is a propagating front of oxygen, too. And the bacteria, by absorbing the oxygen, control it very precisely.”
Oxygen, Libchaber explains, is one of the fastest-diffusing molecules, moving from regions of high concentration to low concentration such that the greater the distance it needs to travel, the faster it will diffuse there. But that is not what they observed. Rather, oxygen penetrated the chamber very slowly in a linear manner. Equal time, equal distance. “This pattern is not due to biology,” says Libchaber. “It has to do with the laws of physics. And it is organized in such an elegant way that the only thing it tells us is that we have a lot to learn from bacteria.”
Journal reference:
Douarche et al. E. Coli and Oxygen: A Motility Transition. Physical Review Letters, 2009; 102 (19): 198101 DOI: 10.1103/PhysRevLett.102.198101
Adapted from materials provided by Rockefeller University.

DNA Not The Same In Every Cell Of Body

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ScienceDaily (July 16, 2009) — Research by a group of Montreal scientists calls into question one of the most basic assumptions of human genetics: that when it comes to DNA, every cell in the body is essentially identical to every other cell. Their results appear in the July issue of the journal Human Mutation.

This discovery may undercut the rationale behind numerous large-scale genetic studies conducted over the last 15 years, studies which were supposed to isolate the causes of scores of human diseases.
Except for cancer, samples of diseased tissue are difficult or even impossible to take from living patients. Thus, the vast majority of genetic samples used in large-scale studies come in the form of blood. However, if it turns out that blood and tissue cells do not match genetically, these ambitious and expensive genome-wide association studies may prove to have been essentially flawed from the outset.
This discovery sprang from an investigation into the underlying genetic causes of abdominal aortic aneurysms (AAA) led by Dr. Morris Schweitzer, Dr. Bruce Gottlieb, Dr. Lorraine Chalifour and colleagues at McGill University and the affiliated Lady Davis Institute for Medical Research at Montreal's Jewish General Hospital. The researchers focused on BAK, a gene that controls cell death.
What they found surprised them.
AAA is one of the rare vascular diseases where tissue samples are removed as part of patient therapy. When they compared them, the researchers discovered major differences between BAK genes in blood cells and tissue cells coming from the same individuals, with the suspected disease "trigger" residing only in the tissue. Moreover, the same differences were later evident in samples derived from healthy individuals.
"In multi-factorial diseases other than cancer, usually we can only look at the blood," explained Gottlieb, a geneticist with McGill's Centre for Translational Research in Cancer. "Traditionally when we have looked for genetic risk factors for, say, heart disease, we have assumed that the blood will tell us what's happening in the tissue. It now seems this is simply not the case."
"From a genetic perspective, therapeutic implications aside, the observation that not all cells are the same is extremely important. That's the bottom line," he added. "Genome-wide association studies were introduced with enormous hype several years ago, and people expected tremendous breakthroughs. They were going to draw blood samples from thousands or hundreds of thousands of individuals, and find the genes responsible for disease.
"Unfortunately, the reality of these studies has been very disappointing, and our discovery certainly could explain at least one of the reasons why."
AAA is a localized widening and weakening of the abdominal aorta, and primarily affects elderly Caucasian men who smoke, have high blood pressure and high cholesterol levels. It often has no symptoms, but can lead to aortic ruptures which are fatal in 90 per cent of cases.
If the mutations discovered in the tissue cells actually predispose for AAA, they present an ideal target for new therapies, and may have even wider therapeutic implications.
"This will probably have repercussions for vascular disease in general," said Schweitzer, of McGill's Department of Medicine. "We have not yet looked at coronary or cerebral arteries, but I would suspect that this mutation may be present across the board."
Schweitzer is optimistic that this discovery may lead to new treatments for vascular disease in the near to medium term.
"The timeline might be five to 10 years," he said. "We have to do in-vitro cell culture experiments first, prove it in an animal model, and then develop a molecule or protein which will affect the mutated gene product. This is the first step, but it's an important step."
Adapted from materials provided by McGill University.

Genomes Of Parasitic Flatworms Decoded

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ScienceDaily (July 16, 2009) — Two international research teams have determined the complete genetic sequences of two species of parasitic flatworms that cause schistosomiasis, a debilitating condition also known as snail fever. Schistosoma mansoni and Schistosoma japonicum are the first sequenced genomes of any organism in the large group called Lophotrochozoa, which includes other free-living and parasitic flatworms as well as segmented roundworms, such as the earthworm.

The research was supported in part by the National Institute of Allergy and Infectious Diseases (NIAID), one of the National Institutes of Health (NIH), and is published in the current issue of Nature. The genomic information obtained through these sequencing projects suggests ways to design drugs or other compounds targeted specifically at proteins or other gene products required by the parasite to find or survive in its human or snail host.
"Chronic infection with Schistosoma parasites makes life miserable for millions of people in tropical countries around the globe, and can lead to death," says NIAID Director Anthony S. Fauci, M.D. Anemia, fever, fatigue and other symptoms can make it difficult for sufferers to work or go to school, he adds. "New drugs and other interventions are badly needed to reduce the impact of a disease that lowers quality of life and slows economic development."
People become infected with Schistosoma when they wade or bathe in water inhabited by tiny snails that are the parasite's intermediate hosts. Microscopic fork-tailed parasites released into the water by the snails burrow into a bather's skin and travel to blood vessels that supply urinary and intestinal organs, including the liver, where they mature. Female worms, which live inside the thicker males, release many thousands of eggs each day. Eggs shed in urine and feces may make their way into snail-inhabited water, where they hatch to release parasites that seek out snails to begin the cycle again.
Schistosomiasis cases top 200 million every year, and some 20 million people are seriously disabled by severe anemia, chronic diarrhea, internal bleeding and organ damage caused by the worms and their eggs, or the immune system reactions they provoke. Though best known for causing chronic illness, schistosomiasis also kills: In sub-Saharan Africa alone it kills some 280,000 people each year.
Since the 1980s, the inexpensive anti-worm medication praziquantel has been administered to people in nationwide schistosomiasis control programs in dozens of tropical countries where the disease is common. While the drug is effective, it does not prevent a person from becoming re-infected through exposure to infested waters.
"The mass administration of a single drug increases the chance the parasites will become resistant to it," notes Martin John Rogers, Ph.D., a program officer in NIAID's Parasitology and International Programs branch. "Reliance on one drug is not a satisfactory long-term solution to the problem of schistosomiasis."
Finding new drug targets was a key goal of the team that sequenced the S. masoni genome. Led by NIAID grantee Najib M. El-Sayed, Ph.D., of University of Maryland, College Park, the group determined the sequence of 363 million nucleotides, encoding 11,809 genes. Analysis of the genes and the proteins they encode revealed the loss of some types of genes (and proteins) and expansion of other gene families relative to corresponding genes found in non-parasitic worms.
These genetic gains and losses are tied to the parasitic lifestyle of Schistosoma. For example, the researchers detected a large percentage of genes encoding proteases (enzymes that break down proteins.) Parasites, like Schistosoma, that must bore through skin and other tissues to invade their hosts require many such enzymes. Befitting a parasite that must navigate murky waters to find its intermediate host and later must travel through several tissue types in its human host, Schistosoma flatworms have sophisticated neurosensory systems that allow them to, for example, detect chemical, light and temperature levels in water or inside their hosts. Genes that encode signaling proteins involved in these neurosensory processes made up a significant proportion of both S. masoni and S. japonicum genomes.
The team responsible for the S. masoni genome also used bioinformatic computational techniques to translate genetic sequence information into maps of over 600 enzymatic reactions arrayed in multiple metabolic pathways. The analysis revealed 120 flatworm enzymes that could potentially be targeted with drugs that would disable the enzyme and inhibit the parasite's metabolism.
Finally, in an effort to find currently marketed drugs (such as protein or enzyme inhibitors) that might also be deployed against schistosomiasis, the researchers compared information about parasite proteins to a database of drugs directed at other human diseases. They found 66 instances of currently marketed drugs that might also be effective against schistosomiasis. "This list represents a good starting point, but, of course, more research is needed to determine whether any of the compounds could also be used to treat schistosomiasis," says Dr. Rogers.
NIAID provided major funding for the S. masoni genome sequencing. Additional support was provided by the Wellcome Trust of Great Britain and through grants from the Fogarty International Center and the National Institute of General Medical Sciences (both components of NIH.)
The S. japonicum genome was produced by an international team of researchers led by Zhu Chen, Ph.D., Ze-Guang Han, Ph.D., and Shengyue Wang, Ph.D., of the Chinese National Human Genome Center, Shanghai. NIAID grantee Zheng Feng, M.D., of the Chinese Center for Disease Control and Prevention, is a coauthor on the paper.
Journal references:
M Berriman et al. The genome of the blood fluke Schistosoma mansoni. Nature, DOI: 10.1038/nature08160
Zhou et al. The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature, 2009; 460 (7253): 345 DOI: 10.1038/nature08140
Adapted from materials provided by NIH/National Institute of Allergy and Infectious Diseases.

Avian Bacterium More Dangerous Than Believed

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ScienceDaily (July 15, 2009) — Bordetella hinzii just may be the Eddie Haskell of avian bacteria. Like the notoriously sneaky character from the iconic 1950s television show "Leave It to Beaver," B. hinzii has been causing trouble and dodging the blame.

Until recently, B. hinzii was believed to be nonpathogenic in poultry. But Agricultural Research Service (ARS) scientists have shown that the bacterium caused severe disease in turkeys that was attributed to another Bordetella species.
B. avium is a pathogenic bacterium that causes upper respiratory disease in poultry and wild birds. It is very similar to B. hinzii, and the two species are difficult to distinguish without using highly specific, DNA-based tests.
Scientists at the ARS National Animal Disease Center (NADC) in Ames, Iowa, used these tests to examine several Bordetella isolates, including some that had caused 100 percent morbidity in turkey poults. Although the isolates had been labeled as B. avium, the scientists found that they were actually B. hinzii, flouting conventional wisdom that the bacterium could not cause disease in poultry.
B. hinzii has been found in poultry with respiratory disease, but was believed to be nonpathogenic because previous attempts to cause disease in chickens and turkeys with the bacterium have failed.
To test the bacterium's pathogenicity, NADC microbiologist Karen Register and veterinary medical officer Robert Kunkle selected six genetically distinct strains of B. hinzii and attempted to infect turkeys with them. Four of the strains were able to grow and persist in the trachea and also caused clinical disease. The strains varied in severity, although none demonstrated 100 percent morbidity.
This study showed for the first time that some strains of B. hinzii can cause disease in turkeys. The results of the study were published in the March 2009 issue of Avian Diseases.
In a related study with chickens, no birds developed clinical disease, suggesting that the pathogenicity of B. hinzii does not extend to chickens.
Now, NADC scientists are examining how the disease-causing strains of the bacterium differ. They are also working to identify virulence factors that influence disease development in turkey poults.
Adapted from materials provided by USDA/Agricultural Research Service.

DNA Is Dynamic And Has High Energy; Not Stiff Or Static As First Envisioned

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ScienceDaily (July 14, 2009) — The interaction represented produced the famous explanation of the structure of DNA, but the model pictured is a stiff snapshot of idealized DNA. As researchers from Baylor College of Medicine and the University of Houston note in a report that appears online in the journal Nucleic Acids Research, DNA is not a stiff or static. It is dynamic with high energy. It exists naturally in a slightly underwound state and its status changes in waves generated by normal cell functions such as DNA replication, transcription, repair and recombination.

DNA is also accompanied by a cloud of counterions (charged particles that neutralize the genetic material's very negative charge) and, of course, the protein macromolecules that affect DNA activity.
"Many models and experiments have been interpreted with the static model," said Dr. Lynn Zechiedrich, associate professor of molecular virology and microbiology at BCM and a senior author of the report. "But this model does not allow for the fact that DNA in real life is transiently underwound and overwound in its natural state."
DNA appears a perfect spring that can be stretched and then spring back to its original conformation. How far can you stretch it before something happens to the structure and it cannot bounce back? What happens when it is exposed to normal cellular stresses involved in doing its job? That was the problem that Zechiedrich and her colleagues tackled.
Their results also addresses a question posed by another Nobel laureate, the late Dr. Linus Pauling, who asked how the information encoded by the bases could be read if it is sequestered inside the DNA molecular with phosphate molecules on the outside.
It's easy to explain when the cell divides because the double-stranded DNA also divides at the behest of a special enzyme, making its genetic code readily readable.
"Many cellular activities, however, do not involve the separation of the two strands of DNA," said Zechiedrich.
To unravel the problem, former graduate student, Dr. Graham L. Randall, mentored jointly by Zechiedrich and Dr. B. Montgomery Pettitt of UH, simulated 19 independent DNA systems with fixed degrees of underwinding or overwinding, using a special computer analysis started by Petttitt.
They found that when DNA is underwound in the same manner that you might underwind a spring, the forces induce one of two bases – adenine or thymine – to "flip out" of the sequence, thus relieving the stress that the molecule experiences.
"It always happens in the underwound state," said Zechiedrich. "We wanted to know if torsional stress was the force that accounted for the base flipping that others have seen occur, but for which we had no idea where the energy was supplied to do this very big job."
When the base flips out, it relieves the stress on the DNA, which then relaxes the rest of the DNA not involved in the base flipping back to its "perfect spring" state.
When the molecule is overwound, it assumes a "Pauling-like DNA" state in which the DNA turns itself inside out to expose the bases -- much in the way Pauling had predicted.
Zechiedrich and her colleagues theorize that the base flipping, denaturation, and Pauling-like DNA caused by under- and overwinding allows DNA to interact with proteins during processes such as replication, transcription and recombination and allows the code to be read. And back to the idea of the "perfect spring" behavior of the DNA helix - "This notion is entirely wrong," said Zechiedrich. "Underwinding is not equal and opposite to overwinding, as predicted, not by a long shot, that's really a cool result that Graham got."
Support for this work came from the Robert A. Welch Foundation, the National Institutes of Health and the Keck Center for Interdisciplinary Bioscience Training of the Gulf Coast Consortia. The computations were performed in part using the Teragrid and the Molecular Science Computing 85 Facility in the William R. Wiley Environmental Molecular Sciences Laboratory, sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at the Pacific Northwest National Laboratory.
Adapted from materials provided by Baylor College of Medicine, via EurekAlert!, a service of AAAS.

New Drugs Faster From Natural Compounds

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ScienceDaily (July 13, 2009) — Researchers have invented computational tools to decode and rapidly determine whether natural compounds collected in oceans and forests are new—or if these pharmaceutically promising compounds have already been described and are therefore not patentable.

This University of California, San Diego advance will finally enable scientists to rapidly characterize ring-shaped nonribosomal peptides (NRPs)—a class of natural compounds of intense interest due to their potential to yield or inspire new pharmaceuticals.
"These advances will speed the process by which we discover and describe new and biologically active molecules from organisms such as marine cyanobacteria, also known as blue-green algae. This, in turn, will accelerate the timeline for bringing new experimental therapies into clinical application," said William Gerwick, an author on the paper and a professor with the UC San Diego Scripps Institution of Oceanography Center for Marine Biotechnology and Biomedicine and the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences.
Nonribosomal peptides (NRPs) often serve as chemical defenses for the bacteria that manufacture them. Starting from penicillin, NRPs have an unparalleled track record in pharmacology: most anti-cancer and anti-microbial agents are natural products or their derivatives. However, it is currently difficult, time-consuming and costly to determine the molecular structure of NRPs which, by definition, are not directly inscribed in the genomes of the organisms that produce them.
"NRPs are one of the last bastions of pharmacologically important biological compounds that remain virtually untouched by computational research. As a result, it is currently one of the most painfully slow processes, it is a real bottleneck that we have now removed," said Pavel Pevzner, a computer science professor at UC San Diego's Jacobs School of Engineering and the corresponding author on the Nature Methods paper.
Researchers can now separate known compounds from those that are unknown.
"If I collect 1,000 ocean compounds, why waste time with compounds that are already known or patented?" added Nuno Bandeira, co-lead author on the paper, director of UC San Diego's Center for Computational Mass Spectrometry (CCMS) and a researcher at the UC San Diego division of Calit2, the California Institute of Telecommunications and Information Technology.
"Our algorithms can tell natural product researchers what their compounds are. Manual annotations should be something of the past," said Julio Ng, a co-lead author on the Nature Methods paper and a doctoral student in Bioinformatics at UC San Diego.
"Compound 879," for example, is a cyclic NRP discussed in the Nature Methods paper that was thought to be novel when it was isolated. A lengthy and expensive patenting process, however, uncovered that compound 879 had already been described as an antibiotic and named neoviridogrisen. The new UC San Diego algorithms would have quickly identified this fact. These algorithms make sense of the flood of tiny peptide fragments that are generated by machines called mass spectrometers that blast nonribosomal peptides apart and determine their sizes.
Two complementary processes are used to glean insights from data generated from the mass spectrometers that break the cyclic peptides into smaller and smaller linear pieces.
First, the authors present new algorithms that computers use to piece these peptide fragments back together in order to determine the chemical structure of a cyclic NRP. This is called "De Novo sequencing of NRPs."
Second, the researchers created "dereplication" tools for moving the other direction: taking the chemical structures of known NRPs and other related information and determining what the data signature would look like if a mass spectrometer had blown the compound part.
"Natural products have a long history in therapeutic development and many were discovered before the digital recording of mass spectrometry data. Therefore, we do not have an extensive mass spectrometry database for natural products as we do for proteomics. Our new tools enable dereplication without an experimental database to compare to," said Pieter Dorrestein, assistant professor in the UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences and the Departments of Pharmacology, Chemistry and Biochemistry.
By using these two approaches, the researchers have created tools that enable researchers to both characterize the compound they have isolated and check to see if it, or something similar, has been previously described. With dereplication, researchers can leverage known information and are not forced to start from scratch each time a new compound needs to be identified.
"As long as the structure of the therapeutic or a related therapeutic or natural product is in the library, we can accurately dereplicate the molecule. This is the first generation of algorithms that can accomplish this and is a glimpse into the future of modern drug discovery."
Performing de novo sequencing without knowing amino acid masses is completely novel, according to Bandeira. "Until we created them, there were no algorithmic approaches available to do this from mass spectrometry data and it was generally thought to be impossible," said Bandeira, who earned his Ph.D. in computer science from the UC San Diego Jacobs School of Engineering.
The work allows mass spectrometry to go into the natural products field and actually do the identification and characterization of natural products in a high throughput fashion, explained Ng, a bioinformatics PhD student advised by Pavel Pevzner in computer science and Pieter Dorrestein in the Skaggs School of Pharmacy.
The researchers note that currently there is no one place to look for known NRPs, a situation they are trying to change with a new data repository effort.
The UC San Diego web-based tools for sequencing nonribosomal peptides (at not cost to researchers) are available at: bix.ucsd.edu/nrp
"This new study has shown that marine cyanobacteria are incredible sources of new molecules that may have medical value, especially in cancer, infectious diseases and neurological disorders," said Gerwick.
This project was supported by US National Institutes of Health grants 1-P41-RR024851-01, GM086283 and cA10u851, and by the PhRMA foundation.
Journal reference:
Julio Ng et al. Dereplication and De Novo Sequencing of Nonribosomal Peptides. Nature Methods, July 13, 2009
Adapted from materials provided by University of California - San Diego, via EurekAlert!, a service of AAAS.

'Rosetta Stone' Of Bacterial Communication Discovered

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ScienceDaily (July 13, 2009) — The Rosetta Stone of bacterial communication may have been found. Although they have no sensory organs, bacteria can get a good idea about what's going on in their neighborhood and communicate with each other, mainly by secreting and taking in chemicals from their surrounding environment. Even though there are millions of different kinds of bacteria with their own ways of sensing the world around them, Duke University bioengineers believe they have found a principle common to all of them.

The researchers said that a more complete understanding of communication between cells and bacteria is essential to the advancement of the new field of synthetic biology, where populations of genetically altered bacteria are "programmed" to do certain things. Such re-programmed bacterial gene circuits could see a wide variety of applications in medicine, environmental cleanup and biocomputing.
It is already known that a process known as "quorum sensing" underlies communication between bacteria. However, each type of bacteria seems to have its own quorum-sensing abilities, with tremendous variations, the researchers said.
"Quorum sensing is a cell-to-cell communication mechanism that enables bacteria to sense and respond to changes in the density of the bacteria in a given environment," said Anand Pai, graduate student in bioengineering at Duke's Pratt School of Engineering. "It regulates a wide variety of biological functions such as bioluminescence, virulence, nutrient foraging and cellular suicide."
The researchers found that the total volume of bacteria in relation to the volume of their environment is a key to quorum sensing, no matter what kind of microbe is involved.
"If there are only a few cells in an area, nothing will happen," Pai said. "If there are a lot of cells, the secreted chemicals are high in concentration, causing the cells to perform a specific action. We wanted to find out how these cells know when they have reached a quorum."
Pai and scientist Lingchong You, assistant professor of biomedical engineering and a member of Duke's Institute for Genome Sciences & Policy and Center for Systems Biology, have discovered what they believe is a common root among the different forms of quorum sensing. In an article in the July 2009 issue of the journal Molecular Systems Biology, they term this process "sensing potential."
"Sensing potential is essentially the linking of an action to the number of cells and the size of their environment," You said. "For example, a small number of cells would act differently than the same number of cells in a much larger space. No matter what type of cell or their own quorum sensing abilities, the relationship between the size of a cell and the size of its environment is the common thread we see in all quorum sensing systems.
"This analysis provides novel insights into the fundamental design of quorum sensing systems," You said. "Also, the overall framework we defined can serve as a foundation for studying the dynamics and the evolution of quorum sensing, as well as for engineering synthetic gene circuits based on cell-to-cell communications."
Synthetic gene circuits are carefully designed combinations of genes that can be "loaded" into bacteria or other cells to direct their actions in much the same way that a basic computer program directs a computer. Such re-programmed bacteria would exist as a synthetic ecosystem.
"Each population will synthesize a subset of enzymes that are required for the population as a whole to produce desired proteins or chemicals in a coordinated way," You said. "We may even be able to re-engineer bacteria to deliver different types of drugs or selectively kill cancer cells"
For example, You has already gained insights into the relationship between predators and prey by creating a synthetic circuit involving two genetically altered lines of bacteria. The findings from that work helped define the effects of relative changes in populations.
The research was supported by National Institutes of Health, a David and Lucile Packard Fellowship, and a DuPont Young Professor Award.
Adapted from materials provided by Duke University.

DNA Patterns Of Microbes

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ScienceDaily (July 13, 2009) — The genomes or DNA of microbes contain defined DNA patterns called genome signatures. Such signatures may be used to establish relationships and to search for DNA from viruses or other organisms in the microbes' genomes. Foreign DNA in bacteria has often been associated with disease-causing abilities.

In his doctorate, Jon Bohlin studied methods for examining the genome signatures of microbes. Since foreign DNA in the genomes of bacteria often give the bacteria disease-causing abilities, part of his work was aimed at developing fast and simple methods for finding foreign DNA.
The explosive development in technology for sequencing DNA molecules has made enormous amounts of genetic information available for analysis. This has both led to an upheaval in biological research and simultaneously created a great need for fast and effective methods of interpreting the steadily increasing amounts of information.
To solve the challenges that these large amounts of information present, bioinformational research is utilising techniques taken from statistical, mathematical and information technologies. Most of the methods that were used in this project were originally established in the field of theoretical bioinformation. However, because of insufficient information, it was not previously possible to investigate the methods properly.
The increasing number of sequenced genomes that has become available during recent years has made it possible to test the methods' advantages and disadvantages, possibilities and limitations. This has given us more reliable information on how different microbes' DNA composition is influenced by environment and lifestyle.
The methods can also be used to deepen our understanding of the evolutionary development that follows natural selection at the DNA level. Such knowledge is absolutely necessary to understand mechanisms leading to bacteria becoming pathogenic (disease-producing) and resistant to antibiotics.
This doctorate comprises analyses of the genome signatures of microbes, and describes how genome signatures vary in the genomes of both closely-related microbes and among different microbial genomes. One of the central questions is how environment influences the genome signatures and if this influence may be may be linked to different characteristics of the microbes, such as size, DNA composition, lifestyle and niche.
Cand. scient. Jon Bohlin defended his Ph. D. thesis, entitled "Genomic signatures in prokaryotic genomes", at the Norwegian School of Veterinary Science, on June 5, 2009.
Adapted from materials provided by Norwegian School of Veterinary Science.

Key To Maintaining Embryonic Stem Cells In Lab

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ScienceDaily (July 13, 2009) — In a new study that could transform embryonic stem cell (ES cell) research, scientists at UT Southwestern Medical Center have discovered why mouse ES cells can be easily grown in a laboratory while other mammalian ES cells are difficult, if not impossible, to maintain.

If the findings in mice can be applied to other animals, scientists could have an entirely new palette of research tools to work with, said Dr. Steven McKnight, chairman of biochemistry at UT Southwestern and senior author of the study appearing in the July 9 issue of Science Express.
"This might change the way medical research is done. But it's still a big 'if,'" he said.
According to the research, the activation of a gene called TDH in mouse ES cells results in the cells entering a unique metabolic state that is similar to that of rapidly growing bacterial cells. The gene controls the production of the threonine dehydrogenase (TDH) enzyme in mouse ES cells. This enzyme breaks down an amino acid called threonine into two products. One of the two products goes on to control a cellular process called one carbon metabolism; the other provides ES cells with an essential metabolic fuel.
Both of the threonine breakdown products are necessary to keep the ES cells growing and dividing rapidly in a petri dish without differentiating into specific tissues.
The various substances currently used by scientists to keep mouse ES cells alive in the laboratory were found by trying many different combinations until something worked, Dr. McKnight said. But until now, it wasn't known that these culture conditions keyed into keeping the TDH gene actively expressed.
"Scientists added this and that until they got the right 'soup,' one that works in the mouse ES cells to somehow activate the TDH gene," he said, adding that exactly how that gene is regulated is still unknown.
Other mammalian species have a functional version of the TDH gene, suggesting the possibility that the process could also be activated in them.
"You would think that the 'mouse soup' would then work for all species, but it doesn't. Researchers have been trying for 20 years to get the right formula for maintaining ES cells from other species. With few exceptions, however, they still haven't gotten it right," Dr. McKnight said.
The research was funded by a National Institutes of Health Director's Pioneer Award, which Dr. McKnight received in 2004. The program encourages investigators to take on creative, unexplored avenues of research that carry a relatively high potential for failure but that also possess a greater chance for truly groundbreaking discoveries.
"By applying a highly innovative technique to manipulate the TDH gene, McKnight's work could be an important breakthrough with a profound impact on future research," said Dr. Raynard S. Kington, acting director of the NIH. "This research, which was partially funded by our Pioneer Award program, shows the value of supporting exceptionally creative approaches to major challenges in biomedical and behavioral research."
Embryonic stem cells are "blank slate" cells – derived from embryos – that go on to develop into any of the more than 200 types of cells in the adult body.
Because mouse ES cells are easily maintained in the lab, they can be manipulated genetically to produce adult mice in which various genes are either modified or eliminated. So-called "knockout mice" allow scientists to study the genetic aspects of many diseases and conditions, including cancer, Alzheimer's, Parkinson's and paralysis.
In the living mouse, and in other species, ES cells exist for only a short time. In that time, they need to grow rapidly in order to accumulate enough cells to begin the process of differentiating into all the body's cell types. Dr. McKnight hypothesizes that the TDH gene tightly controls this process in the animal, allowing the ES cells to grow, but then it shuts off when it's time to differentiate.
"If we can tweak conditions and determine how to keep the gene turned on in other animals, we might be able to grow and maintain ES cells for study in many species. It's still speculative at this point whether it will work, but if it does, then this may prove to represent a transformational discovery," Dr. McKnight said.
Interestingly, although humans carry a form of the TDH gene, it contains three inactivating mutations. As such, human ES cells do not produce the TDH enzyme.
"In the human embryo, something else is taking the place of this TDH-mediated form of rapid cell growth," Dr. McKnight said. "Human ES cells may exist in a unique metabolic state, but it would not appear to involve threonine breakdown."
Human ES cells grow slowly and are difficult to maintain in the laboratory, which is a huge impediment to this field of study, Dr. McKnight said.
"If scientists could repair the mutated human TDH gene and replace it into human ES cells, could they make those cells grow faster in culture? I don't know whether this will work or not – it's highly speculative. But if so, it would be profound," he said.
Other UT Southwestern researchers involved in the study were lead author Dr. Jian Wang, postdoctoral researcher in biochemistry; Peter Alexander, graduate student in biochemistry; Leeju Wu, senior research scientist in biochemistry; Dr. Robert Hammer, professor of biochemistry; and Dr. Ondine Cleaver, assistant professor of molecular biology.
Adapted from materials provided by UT Southwestern Medical Center.

New Insights Into Formation Of The Centromere, A Key Cellular Structure In Powering And Controlling Chromosome Segregation

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ScienceDaily (July 13, 2009) — Lars Jansen* has described the formation of the centromere, a key cellular structure in powering and controlling chromosome segregation and accurate cell division.

A new Nature Cell Biology paper, published in collaboration with a group at Stanford University School of Medicine, provides insights into the scaffold of proteins that ensures accurate segregation of chromosomes during cell division - a fundamental step to ensure that daughter cells have the same genetic information as their mother, with reduced risk of cancer.
When segregating, chromosomes attach and move along proteins tracks (the mitotic spindle), from the centre of the cell to the poles. The centromere is the area of the chromosome that directs this attachment by controlling the assembly of a scaffold of proteins (called the kinetochore), which tether the chromosome to the spindle, and power its movement along the protein track. The location of the centromere on the chromosome is marked by the presence of a protein, called CENP-A, but how this protein is recognised by the other components of the cell to orchestrate the assembly of the centromere was not understood - until now.
Using a newly developed assay, Lars and his colleagues were able to identify the protein that triggers the assembly of the centromere. It's called CENP-N. According to Mariana Silva, a PhD student in the lab, 'When we depleted CENP-N in cells, the centromere did not assemble correctly and chromosomes segregated abnormally, leading to situations similar to cancer'.
This study shows the applicability of this new assay and open doors to future studies into centromere assembly and structure.
*Lars Jansen moved from California to the Instituto Gulbenkian de Ciência (IGC), in Portugal, last year to head the Epigenetic Mechanisms group.
An EMBO installation grant, of 50,000 euro per year, for a maximum of five years has been awarded.
Adapted from materials provided by Instituto Gulbenkian de Ciencia, via EurekAlert!, a service of AAAS.

Using nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

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ScienceDaily (July 12, 2009) — In a landmark technical achievement, investigators in the Vanderbilt Center for Structural Biology have used nuclear magnetic resonance (NMR) methods to determine the structure of the largest membrane-spanning protein to date.

Although NMR methods are routinely used to "take molecular pictures" of small proteins, large proteins – and particularly those that reside within the cell membrane – have been reluctant to smile for the camera.
In the June 26 issue of Science, Charles Sanders, Ph.D., professor of Biochemistry, and colleagues report the NMR structure of the large bacterial protein diacylglycerol kinase (DAGK), a complex of three subunits that each cross the membrane three times (for a total of nine membrane spans).
The group's ability to determine the NMR structure of DAGK suggests that similar methods can now be used to study the structures of other membrane proteins.
"We're taking the methods that we used for diacylglycerol kinase and applying them to high value targets such as G protein-coupled receptors," Sanders said.
G protein-coupled receptors – the largest family of cell signaling proteins – are targets for about half of all pharmaceuticals. Sanders is collaborating with other Vanderbilt investigators to tackle G protein-coupled receptor structure using both NMR and a complementary structural approach, X-ray crystallography.
DAGK may be a therapeutic target for certain types of bacterial infections. It is a virulence factor in the bacteria Streptococcus mutans, which causes tooth decay.
Sanders selected DAGK as a model for studying membrane enzymes when he started his own research lab 17 years ago. DAGK is the smallest known kinase (a protein that adds chemical groups called phosphates onto other molecules), and it is not similar to any other known proteins.
The DAGK structure, Sanders said, "confirmed that this is a really strange kinase." The enzyme has a porch-like structure, with a wide opening for its substrate diacylglycerol and the active site at the top of the porch.
"The active site looks nothing like any other kinase active site – it's a unique architecture," Sanders said.
The researchers also performed exhaustive mutagenesis studies in which they characterized mutations at each amino acid in DAGK and used the data to map the active site of the enzyme onto the structure. They identified two sets of mutations that resulted in non-functional DAGK. One set altered the active site so that it no longer did its job, and the second set caused the protein to fold incorrectly (misfolding).
Sanders said the team was surprised to find that nearly all of the mutations that caused misfolding were in the active site. The expectation, he explained, is that mutations in the active site would cause a loss of function but would not usually affect protein folding, whereas key residues for folding would be located elsewhere in the protein to underpin the scaffold for the active site.
"Our study shows that you can't make that assumption," he said.
Sanders cautions that investigators cannot simply predict the impact of a mutation based on it being located in the active site. The finding has implications for personalized medicine, which aims to use the predicted impact of disease-causing mutations to make therapy decisions.
"The therapeutic strategy for addressing catastrophic misfolding versus simple loss of function may be very different," Sanders said.
Sanders and his team, who got interested in protein folding because of their work with DAGK, are now pursuing structural studies of misfolded membrane proteins that cause diseases including peripheral neuropathy (Charcot-Marie-Tooth Disease), diabetes insipidus and Alzheimer's disease.
"For proteins that misfold because of mutations, we're using NMR tools to understand exactly what the mutations do to the proteins in terms of structure and stability," Sanders said. "We believe that understanding will lead to predictions about how to intervene and avoid misfolding."
Co-authors include Wade Van Horn, Ph.D., Hak-Jun Kim, Ph.D., Charles Ellis, Ph.D., Arina Hadziselimovic, Endah Sulistijo, Ph.D., Murthy Karra, Ph.D., and Changlin Tian, Ph.D., at Vanderbilt and Frank Sönnichsen, Ph.D., at Christian Albrechts University in Kiel, Germany.
Adapted from materials provided by Vanderbilt University Medical Center, via EurekAlert!, a service of AAAS.