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.